Dissertations / Theses on the topic 'Viral fusion protein'

Enveloped viruses, such as HIV, influenza, and Ebola, utilize surface glycoproteins to bind and fuse with a target cell membrane. This fusion event is necessary for release of viral genomic material so the virus can ultimately reproduce and spread. The recently emerged Hendra virus (HeV) is a negative-sense, single-stranded RNA paramyxovirus that presents a considerable threat to human health as there are currently no human vaccines or antivirals available. The HeV utilizes two surface glycoproteins, the fusion protein (F) and the attachment protein (G), to drive membrane fusion. Through this process, the F protein undergoes an irreversible conformational change, transitioning from a meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements which control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Studies that replace or mutate the TM domain of the F protein of several viruses implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was used to demonstrate that isolated TM domains of HeV F protein associate in a monomer-trimer equilibrium. To determine factors driving this association, we analyzed the sequence of several paramyxovirus F protein TM domains and found a heptad repeat of β-branched residues. Analysis of the HeV F TM domain specifically revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein expression and pre-fusion conformation. To further understand the role of the TM domain, the TM domain was targeted as a potential modulator of F protein stability and function. Exogenous HeV F TM constructs were co-expressed with the full length F protein in Vero cells to analyze the effects on protein expression. Co-expression of the exogenous HeV F TM constructs dramatically reduced the expression of HeV F. However, the co-expression of exogenous HeV F TM constructs with a different paramyxovirus F protein, PIV5 F, did not strongly affect PIV5 F expression levels, suggesting that the interaction of the exogenous TM constructs is specific. Fusion assays revealed that HeV F TM constructs dramatically reduced HeV F, but not PIV5 F fusion activity. We hypothesize that the short exogenous HeV TM constructs associate with the TM domain from full-length HeV F, resulting in pre-mature triggering or protein misfolding. The work presented here demonstrates that specific elements in the TM domain contribute to TM association and pre-fusion protein stability. Furthermore, targeting these interactions may be a viable approach for antiviral development against this important pathogen.

Thesis (M.S.)--University of Tennessee Health Science Center, 2008.
Title from title page screen (viewed on January 6, 2009). Research advisor: Charles J. Russell, Ph.D. Document formatted into pages (ix, 41p. : ill.). Vita. Abstract. Includes bibliographical references (p. 38-41).

Measles (MV) virions, like those of other enveloped viruses, enter cells by fusing their lipid membranes with those of the target host cells. Additionally, infected tissues often possess giant multinucleate cells, known as syncytia, which are formed by fusion of infected cells with uninfected neighbors. Expression of both the MV attachment (H) and fusion (F) proteins is required for membrane fusion. MV H mediates receptor binding in order to bring the two membranes into close proximity prior to F activation and is thought to trigger F activation through a specific interaction between the two proteins. Although measles H and F are efficiently transported to the cell surface when expressed independently, evidence has been reported in support of an intracellular interaction between the two proteins that can be detected using an ER co-retention approach. However, it was not determined if the putative co-retention was specific to the two measles glycoproteins, as is their ability to complement each other for efficient fusion promotion. Thus, in this thesis, the formation of an intracellular complex between MV H and F was re-examined. Consistent with the formation of an intracellular complex, cell surface expression and receptor binding of untagged wt MV H is slightly reduced by co-expression of an excess of ER-tagged MV F compared to co-expression with wt F. However, the reduction in surface expression is non-specific in that it can also be induced with heterologous proteins of NDV, which lack significant homology with those of MV. Although this approach did not detect a specific intracellular interaction between MV H and F, it cannot be ruled out that there is a weak association of the proteins that is undetectable by this method. This led to the use of an alternative approach to investigate the cellular site(s) of interaction between the measles H and F proteins. Consistent with a cell surface interaction between MV H and F, the combination of surface biotinylation and co-immunoprecipitation detects formation of a virus-specific H-F complex. Approximately, 21% of the total amount of MV H at the cell surface can be captured with MV F using an antibody against the latter protein. Two complementary approaches were used to address the relationship between this cell surface interaction and receptor recognition by MV H. First, the proteins were co-immunoprecipitated from the surface of Chinese hamster ovary (CHO) cells, which do not express either MV receptor, CD46 or CD150. Similar levels of MV H can be co-immunoprecipitated with F from the surfaces of parental CHO cells and stably transfected cells that express, human CD46 (CHO-CD46), indicating that binding to CD46 is not the trigger for the H-F interaction. Second, MV H proteins, carrying mutations that dramatically reduce CD46 binding, were shown to co-immunoprecipitate efficiently with F from the surface of HeLa cells. Significantly, these results indicate that MV H and F interact in the absence of, and thus prior to, receptor binding. This is in direct contrast to the NDV HN-F cell surface interaction, which is thought to be triggered by receptor binding. Identification of the domains of the para myxovirus attachment and fusion proteins that mediate membrane fusion activities is an essential part of understanding the mechanism of fusion. As a result of the H-F interaction prior to receptor binding, MV H attachment to its cellular receptor must result in conformational changes that trigger activation of the F protein. Site-directed mutagenesis analyses of two regions of MV H indicate that a HR domain in the stalk of the attachment protein is essential to the ability of H to activate F. However, either it is not the only region of H that interacts with F or it is indirectly involved in F activation because mutations in the HR do not disrupt MV H-F complex formation at the cell surface. Additionally, the functional interaction between MV H and F may be mediated, at least in part, by Loop 1 of the amino terminus of the C-rich region of the fusion protein. However, the exact role of this region of the F protein in fusion promotion remains to be determined. Importantly, the cell surface interaction between MV H and F proteins appears to be mediated by more that one region of each protein. In contrast to NDV, in no case has a definitive link between any single amino acid difference in MV H or F and an inability to form the cell surface H-F complex been established. In conclusion, the data presented in this dissertation support a model of measles membrane fusion in which the Hand F proteins form a complex prior to receptor recognition. This complex may hold F in its meta-stable pre-fusion state until binding of H to receptors at the cell surface triggers dissociation of the complex, releasing F to assume its fusogenic form. Importantly, these data also indicate that, although paramyxoviruses may all use the same general process. for promotion of membrane fusion, the mechanism may vary in multiple aspects. A more complete understanding of the means by which measles promotes membrane fusion may direct the development of specific strategies aimed at interfering with the early stages of infection.

Human metapneumovirus (HMPV) is a worldwide respiratory pathogen that belongs to the paramyxovirus family of enveloped viruses and affects primarily the pediatric, geriatric, and immunocompromised populations. Despite its prevalence and importance to human health, no therapies are available against this pathogen. For paramyxoviruses, it is believed that infection starts by attachment of the virus to the surface of the cell through the viral attachment protein followed by fusion between the viral and cellular membranes, a process mediated by the fusion (F) protein at the plasma membrane and at neutral pH. Previous work showed that HMPV infection can occur in the absence of the attachment protein and membrane fusion triggered by the F protein can be promoted by low pH. The work presented here are significant advances in our understanding of the entry process of HMPV. We confirmed that the F protein has receptorbinding functions and identified the cellular binding partner to be heparan sulfate proteoglycans (HSPGs). Additionally, we provide evidence that electrostatic interactions at two different regions play important roles for the proper folding, stability, and low pH triggering of the HMPV F protein. We confirmed the hypothesis that protonation of H435 is important for HMPV F triggering and provide additional evidence that the entry of HMPV may be occurring through endocytosis. Therefore, we hypothesize that HMPV entry occurs through endocytosis after viral binding to HSPGs through the F protein and membrane fusion occurs in an acidified compartment.

The first step of viral infection requires the binding of the viral attachment protein to cell surface receptors. Following binding, viruses penetrate the cellular membrane to deliver their genome into the host cell. For enveloped viruses, which have a lipid bilayer that surrounds their nucleocapsids, entry into the host cell requires the fusion of viral and cellular membranes. This process is mediated by viral glycoproteins located on the surface of the virus. For many enveloped viruses, such as influenza, Ebola, and human immunodeficiency virus, the fusion protein is responsible for mediating both attachment to cellular receptors and membrane fusion. However, paramyxoviruses are unique among fusion promoting viruses because their receptor binding and fusion activities reside on two separate proteins. This unique distribution of functions necessitates a mechanism by which the two proteins can transmit the juxtaposition of the viral and host cell membranes, mediated by the attachment protein (HN/H), into membrane fusion, mediated by the fusion (F) protein. This mechanism allows for paramyxoviruses to gain entry into and spread between cells, and therefore, is an important aspect of virus infection and disease progression. Despite the conservation of receptor binding activity among members of the Paramyxovirinaesubfamily, for most of these viruses, including Newcastle disease virus (NDV), heterologous HN proteins cannot complement F in the promotion of fusion; both the HN and F proteins must originate from the same virus. This is consistent with the existence of a virus-specific interaction between the two glycoproteins. Thus, one or more domains on the HN and F proteins is thought to mediate a specific interaction between them that is an integral part of the fusion process. Therefore, the primary focus of this thesis is the identification of the site(s) on HN that directly contacts F in the HN-F interaction. The ectodomain of the HN protein consists of a stalk and a terminal globular head. Analysis of the fusion activity of chimeric paramyxovirus HN proteins indicates that the stalk region of HN determines its F protein specificity. The first goal of this research was to address the question of whether the stalk not only determines F-specificity, but does so by directly mediating the interaction with F. To establish a correlation between the amount of fusion and the extent of the HN-F interaction, a specific and quantitative co-immunoprecipitation assay was used that detects the HN-F complex at the cell surface. As an initial probe of the role of the HN stalk in mediating the interaction with F, N-glycans were individually added at several positions in the region. N-glycan addition at positions 69 and 77 in the stalk specifically and completely block both fusion and the HN-F interaction without affecting either HN structure or its other activities. However, though they also prevent fusion, N-glycans added at other positions in the stalk also modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein as indicated by sucrose gradient sedimentation analyses. These additional N-glycans likely indirectly affect fusion, perhaps by interfering with changes in the conformation of HN that link receptor binding to the fusion activation of F. To address the issue of whether N-glycan addition at any position in HN would abolish fusion, an N-glycan was added in another region at the base of the globular head of HN (residues 124-152), which was previously predicted by a peptide-based analysis to mediate the interaction with F. HN carrying this additional N-glycan exhibits significant fusion promoting activity, arguing against this site being part of the F-interactive domain in HN. These data support the idea that the F-interactive site on HN is defined by the stalk region of the protein. Site-directed mutagenesis was used to begin to explore the role of individual residues in the stalk in the interaction with F. The characteristics of the F-interactive domain in the stalk of HN are that it is a conserved motif with enough sequence heterogeneity to account for the specificity of the interaction. One such region that meets these requirements is the intervening region (IR) (residues 89-95); a non-helical domain situated between two conserved heptad repeats. Several amino acid substitutions for a completely conserved proline residue in this region impair not only fusion and the HN-F interaction, but also decrease neuraminidase activity in the globular domain and alter the structure of the protein, suggesting that the substitutions indirectly affect the HN-F interaction. Substitutions for L94 also interfere with fusion, but have no significant effect on any other HN function or its structure. Amino acid substitutions at two other positions in the IR (A89 and L90) also modulate only fusion. In all cases, diminished fusion correlates with a decreased ability of the mutated HN protein to interact with F at the cell surface. These findings indicate that the IR is critical to the role of HN in the promotion of fusion and are consistent with its direct involvement in the interaction with the homologous F protein. These are the first point mutations in the HN protein for which a correlation has been demonstrated between the extent of the HN-F interaction and the amount of fusion. This argues strongly that the co-IP assay is an accurate reflection of the HN-F interaction. The second goal of this research was to address the HN-F interaction from the perspective of the F protein by investigating the relationship between receptor binding, the HN-F interaction, and fusion using a highly fusogenic form of the F protein. It has previously been shown that an L289A substitution in NDV F eliminates the requirement for HN in the promotion of fusion and enhances HN-dependent fusion above wild-type (wt) levels. Here, it was shown that the HN-independent fusion exhibited by L289A-F in Cos-7 cells cannot be duplicated in BHK cells. However, when L289A-F is co-expressed with wt HN, enhanced fusion above wt levels is observed in BHK cells. Additionally, when L289A-F is co-expressed with IR-mutated HN proteins previously shown to promote low levels of fusion with wt F, a 2.5-fold increase in fusion was observed. However, similar to wt F, an interaction between L289A-F and the IR-mutated HN proteins was not detected. These results imply that the attachment function of HN, as well as the conformational change in L289A-F, are necessary for the enhanced level of fusion exhibited by HN proteins co-expressed with L289A-F. Indeed, two MAbs detected a conformational difference between L289A-F and the wt F protein. These findings support the idea that the L289A substitution converts F to a form that is less dependent on an interaction with HN for conversion to the fusion-active form. The last goal of this research was to address the cellular site of the HN-F interaction, still a controversial issue based on conflicting data from studies of different paramyxoviruses, using various approaches. This is a particular point of interest, as it speaks to the mechanism by which the HN-F interaction regulates fusion. Thus, NDV HN and F were successfully retained intracellularly with a multiple arginine or KK motif, respectively. The results of Endoglycosidase H resistance and F cleavage studies indicate that the mutated proteins, HN-ER and F-ER, are retained in a compartment prior to the medial-Golgi apparatus and that they are unable to interact with a high enough affinity to co-retain or even cause reduced transport of their wt partner glycoproteins. This is consistent with the HN-F interaction occurring at the cell surface, possibly triggered by receptor binding. In conclusion, this thesis presents evidence to argue that the IR in the stalk of the NDV HN protein directly mediates the interaction with the F protein that is necessary for fusion. Overall, the data presented in this thesis extend the current knowledge of the mechanism by which the paramyxovirus attachment protein can trigger the F protein to initiate membrane fusion. A clear understanding of this process has the potential to identify new anti-viral strategies, such as small molecule inhibitors, aimed at controlling paramyxovirus infection by interfering with early steps in the virus infection cycle.

Newcastle disease virus (NDV) is a member of the genus Avulavirus of the Paramyxoviridaefamily of enveloped negative-stranded RNA viruses. The virus causes respiratory, neurological, or enteric disease in many species of birds, resulting in significant losses to the poultry industry worldwide. Strains of the virus are classified into three pathotypes based on the severity of disease in chickens. Avirulent strains that produce mild or asymptomatic infections are termed lentogenic, whereas virulent strains are termed velogenic. Strains of intermediate virulence are termed mesogenic. The envelope of NDV virions contains two types of glycoproteins, the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. HN mediates three functions: 1) virus attachment to sialic acid-containing receptors; 2) neuraminidase activity that cleaves sialic acid from progeny virions to prevent self-aggregation; and, 3) complementation of the F protein in the promotion of fusion. Though it is widely accepted that cleavage of a fusion protein precursor is the primary determinant of NDV virulence, it is not the sole determinant. At least two other proteins, HN and the V protein, contribute to virulence. The V protein possesses interferon (IFN) antagonistic activity. The long-range goal of these studies is to understand the roles of HN and V in the differential virulence patterns exhibited by members of the NDV serotype. The first aim is to compare the IFN antagonistic activity of the V protein from a lentogenic and a mesogenic strain of the virus. The results of this study demonstrate that the V protein of the mesogenic strain Beaudette C (BC) exhibits greater IFN antagonistic activity than that of the lentogenic strain La Sota. Hence, the IFN antagonistic activities of the two V proteins correlate with their known virulence properties. Comparison of the C-terminal regions of La Sota and BC V proteins revealed four amino acid differences. The results demonstrate that the IFN antagonistic activity of La Sota V increases when any one of these residues is mutated to the corresponding residue in BC V. Conversely, the IFN antagonistic activity of BC V decreases when any one of these four residues is mutated to the corresponding residue in La Sota V. However, no single residue accounts for the difference in IFN antagonistic activity between the two V proteins. Also, analysis of La Sota V and BC V proteins with multiple mutations in these positions revealed that the four residues are collectively responsible for the difference in the IFN antagonistic activity of the two V proteins. Finally, characterization of chimeric La Sota/BC V proteins showed that the N-terminal region also contributes to the IFN antagonistic activity of V. Contrary to an earlier report, results described here demonstrate that the NDV V protein does not target STAT1 for degradation. However, both La Sota and BC V proteins target interferon regulatory factor (IRF)-7 for degradation and promote the conversion of full-length IRF-7 to a lower molecular weight form (IRF-7*). This is the first demonstration that IRF-7 is targeted by a paramyxovirus V protein. The amount of IRF-7* decreases in a dose-dependent manner in the presence of a proteasome inhibitor, suggesting that IRF-7* is a degradation product of IRF-7. Furthermore, the BC V protein promotes complete conversion of IRF-7 to IRF7*, whereas the La Sota V protein does so less efficiently. Again, this is consistent with the difference in IFN antagonistic activity of the two V proteins, and in turn, with their virulence. The second aim is to characterize an HN-specific monoclonal antibody called AVS-I. A previous study suggested that AVS-I recognizes an epitope that is conserved in lentogenic strains and raises the possibility that this epitope may colocalize with a determinant of virulence in HN. To further characterize antibody AVS-I and the epitope it recognizes, we (i) determined its specificity for several additional strains of the virus, (ii) mapped its binding to HN in competition with our own antibodies, (iii) determined its functional inhibition profile, and (iv) isolated and sequenced an AVS-I escape mutant. The results demonstrate that AVS-I binds to a conformational epitope at the carboxy terminus of HN. This suggests that this region of HN may define a determinant of virulence. However, it was also shown that AVS-I, which was previously thought to be specific for avirulent strains of NDV, actually recognizes individual mesogenic and velogenic strains. In conclusion, the data presented in this dissertation contributes to a greater understanding of the molecular basis for NDV virulence and may aid in development of antiviral strategies and generation of recombinant NDVs suitable for use in cancer and gene therapy.

Orientador: Fátima Pereira de Souza
Banca: Karina Alves de Toledo
Banca: José Roberto Ruggiero
Resumo: O Vírus Sincicial Respiratório Humano (hRSV) foi identificado em 1957 e mesmo após vários anos de investigação, nenhuma vacina foi desenvolvida. Acredita-se que a chave de inibição da ação viral são suas glicoproteínas de membrana, em especial a proteína de fusão (F), que com auxílio da proteína de ligação (G), é responsável pela instalação do hRSV na célula hospedeira. Há evidências experimentais de que compostos como flavonóides e glicosaminoglicanos podem diminuir a infecção viral, sendo então a proteína F um bom alvo para a ação destes compostos. O presente estudo utilizou de ferramentas de bioinformática para verificar as possíveis regiões de interação da proteína F com a Heparina Sulfatada e Flavonóides. Os programas de bioinformática foram utilizados para: modelagem dos compostos, caracterização e previsão da estrutura secundária da proteína, modelagem da estrutura terciária e docking molecular entre o modelo da proteína F e as estruturas tridimensionais dos Flavonóides e da Heparina Sulfatada. Modelos válidos foram obtidos para as estruturas tridimensionais dos flavonóides e para o modelo completo da proteína F. As características da proteína incluem um alto nível de conservação na seqüência de aminoácidos e, especialmente, em seus sítios de ligação. O docking da proteína com a Heparina, e o virtual screening da biblioteca de Flavonóides e a estrutura da proteína, resultaram em sítios de interação com grande potencial de inibição, uma vez que concordam com evidências experimentais descritos na literatura. A Heparina liga-se ao sítio de clivagem II, importante região para obtenção da atividade de fusão da proteína. Os Flavonóides podem se ligar a região hidrofóbica que desestabiliza... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Human Respiratory Syncytial Virus (hRSV) was identified in 1957 and even after several years of research, no vaccine has been developed yet. It is believed that the key to the inhibition of viral action is its membrane glycoproteins, including the Fusion Protein (F), responsible for the installation of the hRSV in the host cell. There are evidences that compounds such as flavonoids and glycosaminoglycans can decrease the viral infection, and F protein can be a good target for the action of these compounds. The present study checked the possible sites of interaction between F protein and heparin and flavonoids, using computational tools. Bioinformatics programs were used for: modeling compounds, characterization and prediction of protein secondary structure, tertiary structure modeling and the docking between the protein model and the structures of flavonoids and sulfated heparin. Valid models were obtained for flavonoids structures and the complete model of F protein. The characteristics of the protein include a high level of conservation in amino acid sequence and especially in its binding sites. The heparin docking and virtual screening of flavonoids resulted in interaction sites with great potential for inhibition, since they agree with other studies and experimental evidence of F protein inhibition. This study shows that compounds such as sulfated heparin and flavonoids interact in important sites of F protein. Heparin binds to the cleavage site II and flavonoids can bind to the hydrophobic site that destabilizes the formation of the six-helix-bundle region. Both regions are important for conformational changes that F protein undergoes to get its fusion activity. Docking showed that molecular interactions are likely to occur and selected the best candidates for a possible inhibitor. These evidences... (Complete abstract click electronic access below)
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O Vírus Sincicial Respiratório Humano (hRSV) foi identificado em 1957 e mesmo após vários anos de investigação, nenhuma vacina foi desenvolvida. Acredita-se que a chave de inibição da ação viral são suas glicoproteínas de membrana, em especial a proteína de fusão (F), que com auxílio da proteína de ligação (G), é responsável pela instalação do hRSV na célula hospedeira. Há evidências experimentais de que compostos como flavonóides e glicosaminoglicanos podem diminuir a infecção viral, sendo então a proteína F um bom alvo para a ação destes compostos. O presente estudo utilizou de ferramentas de bioinformática para verificar as possíveis regiões de interação da proteína F com a Heparina Sulfatada e Flavonóides. Os programas de bioinformática foram utilizados para: modelagem dos compostos, caracterização e previsão da estrutura secundária da proteína, modelagem da estrutura terciária e docking molecular entre o modelo da proteína F e as estruturas tridimensionais dos Flavonóides e da Heparina Sulfatada. Modelos válidos foram obtidos para as estruturas tridimensionais dos flavonóides e para o modelo completo da proteína F. As características da proteína incluem um alto nível de conservação na seqüência de aminoácidos e, especialmente, em seus sítios de ligação. O docking da proteína com a Heparina, e o virtual screening da biblioteca de Flavonóides e a estrutura da proteína, resultaram em sítios de interação com grande potencial de inibição, uma vez que concordam com evidências experimentais descritos na literatura. A Heparina liga-se ao sítio de clivagem II, importante região para obtenção da atividade de fusão da proteína. Os Flavonóides podem se ligar a região hidrofóbica que desestabiliza...
Human Respiratory Syncytial Virus (hRSV) was identified in 1957 and even after several years of research, no vaccine has been developed yet. It is believed that the key to the inhibition of viral action is its membrane glycoproteins, including the Fusion Protein (F), responsible for the installation of the hRSV in the host cell. There are evidences that compounds such as flavonoids and glycosaminoglycans can decrease the viral infection, and F protein can be a good target for the action of these compounds. The present study checked the possible sites of interaction between F protein and heparin and flavonoids, using computational tools. Bioinformatics programs were used for: modeling compounds, characterization and prediction of protein secondary structure, tertiary structure modeling and the docking between the protein model and the structures of flavonoids and sulfated heparin. Valid models were obtained for flavonoids structures and the complete model of F protein. The characteristics of the protein include a high level of conservation in amino acid sequence and especially in its binding sites. The heparin docking and virtual screening of flavonoids resulted in interaction sites with great potential for inhibition, since they agree with other studies and experimental evidence of F protein inhibition. This study shows that compounds such as sulfated heparin and flavonoids interact in important sites of F protein. Heparin binds to the cleavage site II and flavonoids can bind to the hydrophobic site that destabilizes the formation of the six-helix-bundle region. Both regions are important for conformational changes that F protein undergoes to get its fusion activity. Docking showed that molecular interactions are likely to occur and selected the best candidates for a possible inhibitor. These evidences... (Complete abstract click electronic access below)

The fusion protein of the Newcastle Disease Virus (NDV) contains three hydrophobic domains. To explore the topogenic signals of these domains, mutants were constructed in which each of the hydrophobic domains was deleted. The membrane insertion and topology of these proteins was characterized in a wheat germ cell-free translation system supplemented with canine microsomal membranes. The results indicated that the first 13 amino acids of the fusion protein are necessary to confer translation inhibition by SRP. Translocation of the nascent chains containing all or part of the first hydrophobic sequence resulted in the appearance of a species of higher molecular weight consistent with glycosylation of at least four of the five potential N-linked glycosylation sites. When glycosylation was inhibited with a glycosylation competitor peptide, signal sequence cleavage was detected. Protease digestion of mutants missing the C-terminal hydrophobic domain indicated that the C-terminus has stop transfer activity. A comparison of membrane insertion of the wild-type fusion protein to that of a mutant missing the second hydrophobic domain, the fusion sequence, indicated that the fusion domain has stop-transfer activity when synthesized in vitro. Furthermore, the fusion domain shows little signal sequence activity when positioned near the amino terminus of the fusion protein. The fusion protein has a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit. In order to determine the role that the conserved leucines have for the oligomeric structure and biological activity of the NDV fusion protein, the heptadic leucines at positions 481,488, and 495 were changed individually and in combination to an alanine residue. Whereas single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein although cell surface expression of the mutants and sedimentation in sucrose gradients was similar to that of the wild type. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain resulted in secretion of an oligomeric structure. These results indicate that the conserved leucines do not play a role in oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative substitutions of a serine-to-alanine (position 473), glutamic acid-to-lysine (position 482) or an asparagine-to-lysine (position 485), the fusogenic ability of the protein was not significantly disrupted. A phenylalanine residue is at the amino terminus of the F1 protein of all paramyxovirus fusion proteins with the exception of the avirulent strains which have a leucine residue in this position. To explore the role of this phenylalanine in the fusion activity of the protein, this residue was changed to leucine (F117L) or to glycine (F117G) by site-specific mutagenesis while maintaining the cleavage site sequence of virulent strains of NDV. Whereas both the wild-type and the F117G proteins were proteolytically cleaved and F1 was detected, the leucine subsitution abolished cleavage. When co-expressed with the HN protein, the fusion protein with either a phenylalanine and glycine residue at position 117, but not a leucine, was shown to stimulate membrane fusion. However, incubation in trypsin activated the fusion activity of the F117L protein. Thus the presence of a leucine at position 117 of the precursor sequence blocks cleavage, but not fusion acitivity, and indicated that the phenylalanine at the amino terminus of the F1 subunit is not conserved for the fusion activity of the protein.

Newcastle disease virus (NDV), an avian paramyxovirus, enters the host cell by fusion of viral and host cell membranes. The fusion of two membranes is mediated by the viral fusion (F) protein. The F protein, like other class I fusion proteins, is thought to undergo major conformational changes during the fusion process. The exact mechanism that leads to major refolding of F protein is not clear. Recently, it has been proposed that disulfide bond reduction in the fusion protein of some viruses may be involved in the conformational changes in fusion proteins. In some viruses, the reduction of disulfide bonds in the fusion protein is mediated by host cell disulfide isomerases belonging to the protein disulfide isomerase (PDI) family. In this study, the role of disulfide bond isomerization in the entry of NDV was analyzed. Using inhibitors of thiol-disulfide isomerases, we found that blocking the reduction of disulfide bonds in the fusion protein inhibited cell-cell fusion as well as virus entry into the host cell. Also, over-expression of isomerases belonging to the PDI family significantly enhanced cell-cell fusion. Taken together, these results suggest that free thiols play an important role in fusion mediated by NDV glycoproteins. Using a thiol specific, membrane impermeable biotin, MPB, we found that free thiols are produced in cell surface-expressed NDV F protein. The production of free thiols was inhibited by inhibitors of thiol-disulfide isomerases. Over-expression of isomerases belonging to the PDI family enhanced detection of free thiols in F protein. In F protein, present in virions or in virus-like particles, free thiols were detected only after the particles were attached to target cells. Taken together, these results suggest that free thiols are produced in F protein and the production of free thiols is mediated by host cell thiol-disulfide isomerases. Using conformation sensitive antibodies, we also studied the conformation of cell surface-expressed F protein in the presence ofthiol-disulfide isomerase inhibitors or in cells over-expressing thiol-disulfide isomerases. In the presence of thiol-disulfide isomerase inhibitors, the cell surface-expressed F protein was in a prefusion conformation while in cells over-expressing thiol-disulfide isomerases the F protein was in a post-fusion conformation. We also correlated the production of free thiols to the conformational changes in F protein. Using temperature-arrested intermediates or F protein with mutations in heptad repeat domains, which are defective in attaining intermediate conformations, we found that free thiols are produced before any of the proposed conformational changes in F protein. Also, the production of free thiols in F protein was found to be independent of its activation by hemagglutinin-neuraminidase (HN) protein. These results suggest that free thiols are probably required for the activation of F protein during membrane fusion.

Le métapneumovirus humain (hMPV) est un virus responsable d'infections aiguës des voies respiratoires telles que des bronchiolites, des bronchites ou des pneumonies, principalement chez les populations à risques que sont les jeunes enfants de moins de 5 ans, ainsi que les personnes âgées ou immunodéprimées. Découvert en 2001, ce virus et sa pathogénèse ne restent encore aujourd'hui que partiellement caractérisés. De ce fait et malgré les besoins, il n'y a aucun vaccin ou traitement thérapeutique spécifique et efficace contre le HMPV disponible sur le marché. Dans ce contexte, mon projet de thèse s'est articulé autour de deux axes principaux : (i) L'étude de la protéine de fusion F du virus hMPV, protéine majeure antigénique de surface et responsable de l'entrée du virus dans la cellule cible. Elle a pour particularité d'induire de manière autonome la fusion membranaire in vitro et d'être associée à des effets cytopathiques variable selon les souches virales. De par son rôle clé pour le virus hMPV, la protéine F a déjà fait l'objet de plusieurs études structurales et fonctionnelles mais les déterminants de cette activité fusogénique ne sont pas encore entièrement caractérisés. Nous nous sommes donc intéressés à l'identification de déterminants du phénotype viral hyperfusogénique, localisés dans les domaines heptad repeats de la protéine F du hMPV. (ii) L'atténuation de deux souches virales cliniques (CAN98-75 et C-85473) par délétion de gènes accessoires dans le but de développer des candidats vaccinaux adaptés aux enfants en bas âge. Différents virus ont été générés par génétique inverse et les délétions des gènes accessoires SH et G dans les deux fonds génétiques viraux ont été étudiées pour leur impact sur l'infectivité, la réplication et la pathogénèse virale in vitro et in vivo ainsi que leur contribution pour le développement de virus atténués candidats vaccinaux
Human metapneumovirus (hMPV) is a major pathogen responsible of acute respiratory tract infections, such as bronchiolitis or pneumonia, affecting especially infants, under five years old, elderly individuals and immunocompromised adults. Identified since 2001, this virus and its pathogenesis still remain largely unknown and no licensed vaccines or specific antivirals against hMPV are currently available. In this context, my research project was built over two main subjects: (i) The study of the fusion F glycoprotein which is the major antigenic protein of hMPV and is responsible of viral entry into host cell. By its crucial role for the virus, the F protein has already been characterized in several structural and/or functional studies. Thus, it has been described that the hMPV F protein induces membrane fusion autonomously, resulting in variable cytopathic effects in vitro, in a strain-dependent manner. However, as the determinants of the hMPV fusogenic activity are not well characterized yet, we focused on identification of some of these, located in heptad repeats domains of the protein. (ii) The evaluation of hMPV SH and G gene deletion for viral attenuation. Liveattenuated hMPV vaccine candidates for infants’ immunization has been constructed thank to this deletion approach at the beginning of hMPV vaccine development efforts. Despite encouraging results, these candidates have not been further characterized and the importance of the viral background has not been evaluated

Newcastle disease virus (NDV) belongs to the Paramyxoviridae, a family of enveloped RNA viruses that includes many important human and animal pathogens. Although many aspects of the paramyxovirus life cycle are known in detail, our understanding of the mechanisms regulating paramyxovirus assembly and release are poorly understood. For many enveloped RNA viruses, it has recently become apparent that both viral and host cellular determinants coordinate the proper and efficient assembly of infectious progeny virions. Utilizing NDV as a model system to explore viral and cellular determinants of paramyxovirus assembly, we have shown that host cell membrane lipid raft domains serve as platforms of NDV assembly and release. This conclusion was supported by several key experimental results, including the exclusive incorporation of host cell membrane raftassociated molecules into virions, the association of structural components of the NDV particle with membrane lipid raft domains in infected cells and the strong correlation between the kinetics of viral protein dissociation from membrane lipid raft domains and incorporation into virions. Moreover, perturbation of infected cell membrane raft domains during virus assembly resulted in the disordered assembly of abnormal virions with reduced infectivity. These results further established membrane raft domains as sites of virus assembly and showed the integrity of these domains to be critical for the proper assembly of infectious virions. Although specific viral protein-protein interactions are thought to occur during paramyxovirus assembly, our understanding of how these interactions are coordinated is incomplete. While exploring the mechanisms underlying the disordered assembly of non-infectious virions in membrane raft-perturbed cells, we determined that the integrity of membrane raft domains was critical in the formation and virion incorporation of a complex consisting of the NDV attachment (HN) and fusion (F) proteins. The reduced virus-to-cell membrane fusion capacity of particles released from membrane raft-perturbed cells was attributed to an absence of the HN – F glycoprotein-containing complex within the virion envelope. This result also correlated with a reduction of these glycoprotein complexes in membrane lipid raft fractions of membrane raft-perturbed cells. Specifically, it was determined that the formation of newly synthesized HN and F polypeptides into the glycoprotein complex destined for virion incorporation was dependent on membrane lipid raft integrity. Finally, a novel virion complex between the ribonucleoprotein (RNP) structure and the HN attachment protein was identified and characterized. Unlike the glycoprotein complex, the detection of the RNP – HN protein-containing complex was not affected by membrane raft perturbation during virus assembly in the cell. The biological importance of this novel complex for the proper assembly of an infectious progeny virion is currently under investigation. The results presented in this thesis outline the role of host cell membrane lipid raft domains in the assembly and release processes of a model paramyxovirus. Furthermore, the present work extends our understanding of how these particular host cell domains mechanistically facilitate the ordered assembly and release of an enveloped RNA virus.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008.
Typescript. Includes bibliographical references (leaves 161-183). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Background Globally, the HIV/AIDS epidemic has cost over 35 million lives and approximately a further 37 million people are currently infected with HIV. In South Africa alone, more than 7 million people are HIV positive. Since the initiation of combination antiretroviral therapy (cART), viral replication can be supressed below the limit of detection by conventional testing. There is, however, no approved therapy for the cure of HIV. This is because HIV establishes viral reservoirs in memory CD4+ T-cells, where replication is low or arrested, allowing prolonged survival. Since there is little or no replication, a therapeutic strategy which targets the viral production and replication becomes ineffective and upon cessation of antiretroviral therapy a dramatic viral relapse occurs. The eradication of HIV, therefore, requires the targeted killing of the reservoir cells, or latency reversal followed by the prevention of further infection using cART. Targeting of cell-surface antigens for therapeutic purposes is the basis of immunotherapy. FDA-approved monoclonal antibodies such as Trastuzumab have been used to treat breast cancer via the human epidermal growth factor 2 (HER2) receptor. Immunotoxins (ITs) composed of an antibody fragment fused to apoptosis-inducing protein toxins targeting cellsurface antigens have been used for therapy of refractory leukaemia. The anti-CD22 recombinant IT Moxetumomab pasudotox based on Pseudomonas aeruginosa exotoxin A (ETA) has been FDA approved to treat hairy cell leukaemia. Moxetumomab pasudotox targets the antigen CD22 found on the surface of tumour cells. The HIV neutralizing VHH-nanobody J3, isolated from an immunised Llama has demonstrated anti-HIV properties against more than 95 % of HIV strains in vitro. As part of an ongoing project to develop a J3-ETA IT, this work sought to produce a J3-SNAP fusion protein by osmotic stress expression in the presence of compatible solutes in the periplasmic space of E. coli. SNAP-tag is a self-labelling protein that covalently binds benzylguanine (BG)-modified substrates in a 1:1 stoichiometric ratio. When recombinantly fused to any protein of interest, SNAP-tag allows the stable labelling of the protein of interest of in vitro and in vivo imaging. The periplasmic space of bacteria has been reported as a dedicated compartment to express functional proteins of interest. Furthermore, osmotic stress expression in the presence of compatible solutes has been reported to result in up to a thousand-fold increase in protein yield for difficult to express proteins. This study ultimately aimed to understand whether a functional J3-SNAP or J3-ETA can be expressed under osmotic stress in the presence of compatible solutes, in the periplasmic space of E. coli. 11 Experimental work In this study, a SNAP-tag-based fusion protein and an ETA-based IT were designed using J3, an anti-HIV-1 Env VHH-nanobody isolated from an immunised llama. Using the SnapGene® software (v.5.0.8, GSL Biotech LLC, USA), in silico design and cloning of an ETA-based IT J3-ETA and SNAP-tag-based fusion protein J3-SNAP was performed. Molecular cloning of designed open reading frames (ORFs) was performed into appropriate bacterial expression plasmid vectors. Plasmid vectors confirmed to contain the required ORFs by Sanger sequencing were transformed into E. coli BL21-DE3. Histidine-tagged J3-SNAP was expressed by osmotic stress in the presence of compatible solutes. J3-SNAP was purified by IMAC and assessed by SDS-PAGE and Western blot analysis. To ascertain the binding of J3- SNAP to cells expressing HIV-1 Env in vitro, recombinant Env protein was transiently transfected into HEK293T-cells to generate an Env expressing cell line. Cell-surface binding of SNAP-Surface® Alexa Fluor® 488 -conjugated J3-SNAP on Env expressing HEK293Tcells was assessed by confocal microscopy analysis. Results Successful expression of J3-SNAP in E. coli BL21-DE3 was confirmed by SDS-PAGE and Western blot analysis. The J3-SNAP fusion protein was subsequently purified by IMAC. Purified J3-SNAP was conjugated to the benzyl guanine-modified fluorophore SNAPSurface® Alexa Fluor® 488 and full-length conjugated protein was confirmed by combinations of SDS-PAGE and Western blot analysis. Cell-surface binding of J3-SNAP to HIV-1 Env-expressing HEK293T-cells was demonstrated in vitro by confocal microscopy analysis. These results prompted the generation of the IT, J3-ETA, by replacing SNAP-tag with ETA. Conclusion Successful binding studies suggest using J3 to target HIV-1 Env. Accessing patient probes would allow for the confirmation of these results for future human applications. Future in vitro studies would need to confirm the selective elimination of Env expressing T-cells by J3-ETA and thereafter confirmed on Env-positive patient probes.

To enter and infect cells, viruses must overcome the barrier presented by the cell membrane. Enveloped viruses, which possess their own lipid bilayer, fuse their viral membrane with the cell membrane. Non-enveloped viruses, whose outer surface is composed of proteins, penetrate through the hydrophobic interior of the cell membrane. Viruses accomplish the processes by coupling conformational changes in viral "entry proteins" to membrane disruption. This dissertation investigates the membrane disruption mechanisms of rotavirus, a non-enveloped virus, and vesicular stomatitis virus (VSV), an enveloped virus. Rotavirus uses proteins of its outer capsid to penetrate the membrane and deliver a transcriptionally-active core particle into the cell cytoplasm. \(VP5^*\), an outer capsid protein, undergoes a foldback rearrangement that translocates three clustered hydrophobic loops by \(\sim 180^{\circ}\). This rearrangement resembles the foldback rearrangements of enveloped virus fusion proteins. In the first half of my dissertation, I show that the hydrophobicity of the \(VP5^*\) apex is required for membrane disruption during rotavirus cell entry by mutating hydrophobic residues within the loop to hydrophilic residues. One particular mutation diminishes liposome interaction by the protein, blocks membrane penetration by virus particles in cells, and reduces particle infectivity by 10,000-fold. VSV uses its fusion protein, G, to fuse at low pH. Unlike other viral fusion proteins, pH-induced conformational changes in G are reversible. In the second half of my dissertation, I measure the fusion kinetics of individual VSV particles using a single-particle fusion assay previously developed for influenza virus. I find that hemifusion by VSV consists of at least two steps, an initial step that is pH-dependent and reversible, and a second step that is pH-independent. At low pHs, the second step becomes the sole rate-limiting step. I also show that at pH 6.6, the VSV particle enters a stable intermediate state that binds tightly to membranes but does not precede to fusion. This dissertation uses a variety of experimental approaches to arrive at a more detailed understanding of how viruses use their entry proteins to either penetrate or fuse with the cell membrane.

RESUMO: O Cell Fusing Agent Vírus (CFAV), considerado como o primeiro “flavivírus específicos de insectos” (ISF), parece estar exclusivamente adaptado aos seus hospedeiros, não replicando em células de vertebrados. Apesar de ter sido identificado há mais de três décadas (1975), a verdade é que muito pouco se conhece sobre a sua biologia. Dado o seu parentesco filogenético com alguns outros flavivírus encontrados naturalmente em mosquitos de diferentes géneros colhidos em diferentes regiões do globo, este vírus poderá ser usado como modelo para o estudo de ISF. No entanto, necessitam do desenvolvimento de ferramentas básicas, tais como clones moleculares ou baterias de soros contendo anticorpos que reconheçam uma ou mais proteínas codificadas pelo genoma viral, produzidas, por exemplo, a partir de antigénios virais produzidos de forma recombinante. Com este trabalho pretendeu-se a optimização de protocolos que permitiram a expressão e purificação parcial de quatro proteínas [duas proteínas estruturais (C e E) e duas não estruturais (NS3hel e NS5B)] do CFAV em E. coli, todas elas produzidas como proteínas de fusão com “caudas” (tags) de hexahistidina nos seus extremos carboxilo. Para a expansão do CFAV foram utilizadas células Aedes albopictus (C6/36). Após a realização da extracção do RNA viral e a obtenção de cDNA, procedeu-se amplificação, por RT-PCR, das regiões codificantes das proteínas C, E, NS3hel e NS5B, utilizando primers específicos. Os quatro fragmentos de DNA foram independentemente inseridos no vector pJTE1.2/blunt usando E. coli NovaBlue como hospedeira de clonagem e, posteriormente, inseridos em vectores de expressão pET-28b e pET-29b usando E. coli BL21(DE3)pLysS e Rosetta(DE3)pLysS como hospedeiras de expressão. Após da indução, expressão e purificação das proteínas recombinantes C, E, NS3hel e NS5B, foi confirmada a autenticidade destas proteínas produzidas através do método Western Blot com um anticorpo anti-histidina. --------- ABSTRACT: The Cell Fusing Agent virus (CFAV) considered as the first "insect- specific flavivirus" (ISF) and seems to be uniquely adapted to their hosts, not replicating in vertebrate cells. Although it has been known for more than three decades (1975), the truth is very little is known about its biology. Given its close phylogenetic relationship with other flavivirus naturally circulating in various genera of mosquitoes collected from different regions of the globe, this virus could be used as a model for the study of ISF. However, such studies require the development of experimental basic tools, such as molecular clones or serum batteries containing antibodies that recognize one or more proteins encoded by the viral genome, produced, for example, from viral antigens recombinant produced. In this work, we carried out the optimization of protocols that allowed the expression and partial purification of four proteins [two structural proteins (C and E) and two nonstructural proteins (NS3hel and NS5B)] CFAV in E. coli as fusion protein for c-terminal hexahistidine tags. For the expansion of the CFAV we used Aedes albopictus (C6/36) cells. After completion of the viral RNA extraction and cDNA obtained, amplification of the coding regions of the C, E, NS5B and NS3hel proteins was carried out by RT-PCR using specific primers. The four DNA fragments were independently inserted into the vector pJTE1.2/blunt using E. coli NovaBlue as cloning host and then inserted into expression vectors pET-28b and pET-29b using E. coli BL21(DE3)pLysS and Rosetta(DE3)pLysS as expression host. After induction, expression and purification of recombinant C, E, NS3hel and NS5B proteins Western Blot analyses with an anti-histidine antibody confirmed the authenticity of these proteins produced.

This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.

The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.

mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.

Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.

The final step of paramyxovirus infection requires the assembly of viral structural components at the plasma membrane of infected cells followed by budding of virions. While the matrix (M) protein of some paramyxoviruses has been suggested to play a central role in the assembly and release of virus particles, the specific viral and host protein requirements are still unclear. Using Newcastle disease virus (NDV) as a prototype paramyxovirus, we explored the role of each of the NDV structural proteins in virion assembly and release. For these studies, we established a virus-like particle (VLP) system for NDV. The key viral proteins required for particle formation and the specific viral protein-protein interactions required for assembly and release of particles were explored in chapter 2. First we found that co-expression of all four proteins resulted in the release of VLPs with densities and efficiencies of release (1.18 to 1.16 g/cm3and 83.8%±1.1, respectively) similar to that of authentic virions. Expression of M protein alone, but not NP, F-K115Q or HN proteins individually, resulted in efficient VLP release. No combination of proteins in the absence of M protein resulted in particle release. Expression of any combination of proteins that included M protein yielded VLPs, although with different densities and efficiencies of release. To address the roles of NP, F and HN proteins in VLP assembly, the interactions of proteins in VLPs formed with different combinations of viral proteins were characterized by co-immunoprecipitation. The co-localization of M protein with cell surface F and HN proteins in cells expressing all combinations of viral proteins was characterized. Taken together, the results show that M protein is necessary and sufficient for NDV budding. Furthermore, they suggest that M protein – HN protein and M protein - NP interactions are responsible for incorporation of HN protein and NP proteins into VLPs and that F protein is incorporated indirectly due to interactions with NP and HN protein. Since the vacuolar protein sorting (VPS) system is involved in the release of several enveloped RNA viruses, chapter 3 describes studies which explored the role of the VPS system on NDV particle release. First, we characterized the effects of three dominant negative mutant proteins of the VPS pathway on particle release. Expression of dominant negative mutants of CHMP3, Vps4 and AIP1 proteins inhibited M protein particle release as well as release of complete VLPs. Mutation of a YANL sequence in the NDV M protein to AANA inhibited particle release while replacement of this sequence with either of the classical late domain motifs, PTAP or YPDL, completely restored particle release. The host protein AIP1, which binds YXXL late domain sequences, is incorporated into M protein particles. These results suggest that an intact VPS pathway is necessary for NDV VLP release and that the YANL sequence is an NDV M protein L domain. The sequence and structure of the Newcastle disease virus (NDV) fusion (F) protein are consistent with its classification as a type 1 glycoprotein. We have previously reported, however, that F protein can be detected in at least two topological forms with respect to membranes in both a cell-free protein synthesizing system containing membranes as well as infected COS-7 cells (J. Virol. 2004 77:1951). One form is the classical type 1 glycoprotein while the other is a polytopic form in which approximately 200 amino acids of the amino terminal end as well as the cytoplasmic domain (CT) are translocated across membranes. Furthermore, we detected CT sequences on surfaces of F protein expressing cells and antibodies specific for these sequences inhibited red blood cell fusion to HN and F protein expressing cells suggesting a role for surface expressed CT sequences in cell-cell fusion. In chapter 4, we extended these findings and found that the alternate form of the F protein can also be detected in infected and transfected avian cells, the natural host cells of NDV. Furthermore, the alternate form of F protein was also found in virions released from both infected COS-7 cells and avian cells by Western analysis. Mass spectrometry confirmed its presence in virions released from avian cells. Two different polyclonal antibodies raised against sequences of the CT domain of the F protein slowed plaque formation in both avian and COS-7 cells. Antibody specific for the CT domain also inhibited single cycle infections as detected by immunofluorescence of viral proteins in infected cells. The potential roles of this alternate form of the NDV F protein in infection are discussed. Virus-like particles (VLPs) generated from different viruses have been shown to have potential as good vaccines. Chapter 5 explored the potential of NDV VLPs as a vaccine for NDV or as a vaccine vector for human pathogens. Significant quantities of NDV VLPs can be produced from tissue culture cells. These VLPs are as pure as virions prepared in eggs. In addition, some rules for incorporation of viral proteins into VLPs were also explored. We found that the cytoplasmic domain of the fusion (F) protein is necessary for its incorporation into VLPs. We found that an HN protein with an HA tag at its carboxyl terminus was incorporated into VLPs. We also found that the HN and F proteins of NDV, strain B1, can be incorporated into VLPs with M and NP of strain AV. The demonstration of specific domains required for protein incorporation into particles is important in using NDV VLPs as a vaccine vector for important human pathogens. In conclusion, this dissertation presents results that show that the M protein plays a central role in NDV assembly and release, a finding that is consistent with findings with other paramyxoviruses. More importantly, this work extends the current knowledge of paramyxovirus assembly and release by providing the first direct evidence of interactions between paramyxovirus proteins. These interactions between viral proteins provide a rational basis for incorporation of viral proteins into particles. This work also provides a clearer understanding of the role of the host vacuolar protein sorting machinery in NDV budding. A clear understanding of virus assembly and budding process contributes to the design of strategies for therapeutic intervention and in the development of safer, more economical and effective vaccines.

Orientadores: Adriano Rodrigues Azzoni, Anete Pereira de Souza
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Apesar de seguros e simples de produzir, o uso de vetores não virais como o DNA plasmidial (DNAp) em estudos de terapia gênica e vacinação por DNA tem sido limitado pela baixa eficiência quando comparados aos vetores virais. Essa limitação provém principalmente da reduzida capacidade de superar as barreiras físicas, enzimáticas e difusionais encontradas durante o tráfego intracelular para o interior do núcleo das células alvo. Dentro deste contexto, o presente trabalho demonstra a utilização de cadeias leves modificadas de Dineína (Lc8 e Rp3) como vetores não-virais de entrega gênica. A escolha de cadeias leves de Dineína justifica-se pela possibilidade de utilizar o transporte retrógrado celular mediado por complexos motores de Dineína para facilitar o tráfego de material genético exógeno através do citoplasma em direção à periferia nuclear. Através da adição de pequenos domínios peptídicos, ricos em aminoácidos polares positivos (arginina e lisina), ao N-terminal de cadeias leves de Dineína foi possível conferir a estas proteínas a habilidade de interagir com material genético condensando-o em partículas. Ensaios de transfecção demonstraram que tais partículas apresentam elevada eficiência de entrega do material genético exógeno ao núcleo de células HeLa, eficiência esta superior àquela apresentada pelo peptídeo protamina, amplamente estudado como vetor não-viral de entrega gênica. A formação de complexos ternários utilizando-se DNA plasmidial, cadeias leves de Dineína modificadas e lipídios catiônicos apresentou eficiência de entrega superior àquelas apresentadas na ausência do lipídio. Adicionalmente, complexos de entrega formados apenas com DNA plasmidial e cadeias leves de Dineína modificadas apresentaram baixo efeito citotóxico em células HeLa, característica esta de grande relevância uma vez que a toxicidade dos vetores de entrega gênica atua como importante fator limitante em sua aplicação clínica. O mecanismo envolvido no processo de entrega gênica mediado por cadeias leves de Dineína modificadas também foi estudado, podendo ser observado que (1) a entrada dos complexos de entrega na célula é altamente dependente do processo de endocitose, (2) a eficiência de entrega observada depende da rede de microtúbulos e (3) parte significativa dos complexos de entrega é degradada na via de endossoma/lisossomo celular. Os vetores não-virais de entrega gênica descritos no presente estudo associam elevada eficiência de transfecção, baixa toxicidade celular e relativo baixo custo de produção, uma vez que as cadeias leves de Dineína recombinantes são produzidas em sistema heterólogo utilizando-se Escherichia coli. Ressalta-se ainda a possibilidade de adição de novos domínios peptídicos às cadeias leves de Dineína modificadas, agregando novas funções/capacidades que poderiam resultar em maior eficiência de entrega gênica através da otimização dos processos de internalização celular ou escape endossomal. A abordagem de se utilizar a via de transporte retrógrado celular para o desenvolvimento de vetores não-virais para entrega gênica é pouco explorada pela comunidade científica e o presente estudo apresenta-se entre os poucos da área, esperando assim contribuir para o desenvolvimento de vetores não-virais mais eficientes e seguros
Abstract: The use of non viral vectors such as plasmidial DNA (pDNA) in gene therapy and DNA vaccination protocols has been limited due to its low transfection efficiency when compared to viral vectors. This limitation occurs mainly due to the physical, enzymatic and diffusion barriers faced during the transport of the genetic material to the nucleus of target eukaryotic cells. Regarding this subject, the present work demonstrates the feasibility of using modified Dynein light chains (Lc8 and Rp3) as non viral vectors for gene delivery. The use of Dynein light chains relies on the possibility to exploit the Dynein based cellular retrograde transport in order to improve the exogenous genetic material transport across the citosol towards the nuclear periphery. By adding small peptide domains, based in positively charged aminoacids (arginine and lysine) to the N-terminal of Dynein light chains, the resulting recombinant proteins were able to interact and condense genetic material into delivery particles. Transfection assays demonstrated that these particles are highly efficient to delivery plasmidial DNA to nucleus of HeLa cells when compared to the transfection efficiency presented by protamine, a well characterized non viral vector peptide. Ternary complexes formed by modified Dynein light chains, pDNA and a cationic lipid showed even higher transfection efficiency. Additionally, the light chain based non viral delivery vectors presented low citotoxic effect to HeLa cells, a valuable feature as toxicity is regarded as one of the main concerns on delivery vectors development. The mechanism by which the modified Dynein light chain based vectors mediates gene delivery was also investigated and we could observe that (1) the internalization process deeply relies on endocytosis, (2) it depends on the microtubule network and (3) a significant fraction of the delivery complexes are trapped and degraded in the endocytic pathway. The non viral vectors developed in the present study combine high transfection efficiency, low toxicity and relative low production cost, as all modified proteins were produced in Escherichia coli prokaryotic host. Its noteworthy that additional peptide domains can be further associated to the delivery vectors described providing it with new abilities such as higher internalization or endosomal escape capacity. The approach to use the cellular retrograde transport in order to develop non viral vectors is poorly exploited by the scientific community and the present study stands among few in the field hopefully contributing to the development of more efficient and safer non viral vectors for gene delivery
Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular

A common phenotype observed in most cancers is chromosomal instability. This includes both structural and numerical chromosomal aberrations, which can promote carcinogenesis. The fusion gene CBFB/MYH11 is created by the structural chromosomal inversion(16)(p13.1q22), resulting in the fusion protein CBFβ-SMMHC, which blocks differentiation in hematopoietic progenitor cells. This mutation alone, however, is not sufficient for transformation, and at least one additional cooperating mutation is necessary. The role of wildtype Cbfb in modulating the oncogenic function of the fusion protein Cbfβ-SMMHC in mice was examined. Transgenic mice expressing the fusion protein, but lacking a wild-type copy of Cbfb, were created to model the effects of these combined mutations. It was found that wild-type Cbfb is necessary for maintaining normal hematopoietic differentiation. Consequently, complete loss of wild-type Cbfb accelerates leukemogenesis in Cbfb/MYH11 mice compared to mice expressing both the fusion and wild-type proteins. While there is no evidence in human patient samples that loss of wild-type Cbfb expression cooperates with the fusion protein to cause transformation, it is apparent from these experiments that wild-type Cbfβ does play a role in maintaining genomic integrity in the presence of Cbfβ-SMMHC. Experiments have also shown that loss of Cbfb leads to accumulation of hematopoietic progenitor cells, which may acquire additional cooperating mutations. Not unlike CBFB/MYH11, the human papillomavirus (HPV) E6 and E7 proteins are not sufficient for cellular transformation. Instead, high risk HPV E7 causes numerical chromosomal aberrations, which can lead to accumulation of additional cooperating mutations. Expression of HPV-16 E7 and subsequent downregulation of the retinoblastoma protein (Rb) has been shown to induce polyploidy in human keratinocytes. Polyploidy predisposes cells to aneuploidy and can eventually lead to transformation in HPV positive cells. There are several possible mechanisms through which E7 may lead to polyploidization, including abrogation of the spindle assembly checkpoint, cleavage failure, abrogation of the postmitotic checkpoint, and re-replication. Rb-defective mouse and human cells were found to undergo normal mitosis and complete cytokinesis. Furthermore, DNA re-replication was not found to be a major mechanism to polyploidization in HPV-E7 cells upon microtubule disruption. Interestingly, upon prolonged mitotic arrest, cells were found to adapt to the spindle assembly checkpoint and halt in a G1-like state with 4C DNA content. This post-mitotic checkpoint is abrogated by E7-induced Rb-downregulation leading to S-phase induction and polyploidy. This dissertation explores two examples of the multi-step pathway in human cancers. While certain genes or genetic mutations are often characteristic of specific cancers, those mutations are often not sufficient for transformation. The genetic or chromosomal abnormalities that they produce often stimulate the additional mutations necessary for oncogenesis. The studies with Cbfb/MYH11 and HPV E7 further exemplify the significance of numerical and structural chromosomal aberrations in multi-step carcinogenesis.

Les syncytines sont des gènes d'enveloppes rétrovirales (env) capturés qui sont essentiels pour l'établissement du placenta chez les mammifères. Il a été proposé que la diversité des syncytines capturées explique pourquoi le placenta est l'organe le plus variable chez les mammifères. Ici nous avons employé deux approches pour étudier le lien entre la capture d'env et l'émergence et la diversité des structures placentaires. D'abord, nous avons étudié la placentation des Hyaenidae, les seuls carnivores à présenter un placenta très invasif hémochorial, comme l'humain. Comme tous les carnivores, les hyènes expriment la syncytin-Car1 précédemment décrite, mais nous avons identifié une nouvelle env, capturée uniquement chez ces dernières, que nous avons nommée Hyena-Env2. Ce nouveau gène est présent au même locus chez toutes les hyènes, ayant été capturé pendant la radiation de la famille. Il est non-fusiogène mais a néanmoins été conservé pendant plus de 10 millions d'années et est exprimé à l'interface materno-fœtale du placenta, ce qui en fait un gène candidat pour expliquer le passage à la placentation hémochoriale qui a eu lieu chez les Hyaenidae. Ensuite, nous avons cherché des gènes syncytine dans le genre non-mammifère Mabuya, des lézards vivipares présentant un type rare de placenta très complexe et proche de celui des mammifères. Nous avons identifié une env qui a été capturée et conservée dans ce genre depuis sa radiation, il y a 25 millions d'années. Ce gène, que nous avons appelé syncytin-Mab1, est capable d'induire la fusion cellule-cellule et est exprimé dans une couche de cellules fusionnées à l'interface materno-fœtale du placenta, deux propriétés canoniques de syncytine. Nous avons aussi identifié le récepteur de syncytin-Mab1, MPZL1, et avons montré que leur interaction induit son activation et sa phosphorylation. L'activation de MPZL1 a été liée à la migration et à l'invasion cellulaire, indiquant que cette interaction env-récepteur pourrait jouer un rôle dans l'invasion placentaire du tissu maternel observée chez les Mabuya. Pour conclure, la caractérisation de ces deux nouvelles env indique que les gènes de type syncytine ont pu jouer un rôle à la fois dans l'émergence du placenta de Mabuya et dans la structure atypique du placenta des hyènes, supportant la notion que la capture d'env est une force évolutive majeure
Syncytins are captured retroviral envelope genes (env) that are essential for the establishment of placental structures in mammals. The syncytins present in different mammalian families are highly diverse, resulting from distinct capture events, and it has been suggested that this might play a role in making the placenta the most diverse structure in mammals. Here we used two different approaches to investigate the links between env capture and emergence and diversity of placental structures. First, we investigated placentation in Hyaenidae, the only carnivorans that present a highly invasive hemochorial placenta, as is also found in humans. Hyenas express the previously identified syncytin-Car1 gene, as do all carnivorans, but we identified a new hyena-specific captured env that we named Hyena-Env2. This new gene is present at the same locus in all hyenas, having been captured during the radiation of this family. It is non-fusiogenic but still conserved over at least 10 million years of evolution and expressed at the materno-fetal interface in the hyena placenta, making it a candidate gene for explaining the endotheliochorial to hemochorial placental transition that occurred in Hyeanidae. Second, we searched for syncytin-like genes in the non-mammalian Mabuya lizards, which are viviparous and present a rare type of highly complex placenta that is very reminiscent of mammalian placentas. We identified an env gene that was captured and conserved in this genus since its radiation 25 million years ago. This gene, that we named syncytin-Mab1, is able to mediate cell-cell fusion in vitro and is expressed in a fused cell layer at the materno-fetal interface of the placenta in vivo, characteristic features of canonical mammalian syncytin genes. We also identified the cellular gene MPZL1 as the cognate receptor of syncytin-Mab1 and showed that their interaction induces activation and phosphorylation of the former. MPZL1 activation has been linked with cell migration and invasion, indicating that this env-receptor interaction could play a role in the placental invasion of maternal tissues observed in Mabuya. In conclusion, the characterization of these two novel env genes indicates that syncytin-like env might have played a role both in the emergence of the Mabuya placenta and the atypical placental structure of hyenas, reinforcing the notion that env capture is a major driving force in evolution

A formulação HIVBr18, previamente desenvolvida e testada, é uma vacina de DNA que codifica 18 epítopos CD4, promíscuos e conservados do HIV-1, e que após imunização de camundongos transgênicos para diversas moléculas de HLA de classe II humanas, observou-se proliferação de linfócitos T CD4+ e CD8+ e produção de IFN-? direcionadas a múltiplos epítopos codificados pela vacina. Abordamos aqui estratégias baseadas na fusão ou combinação dos epítopos codificados pela vacina HIVBr18 à glicoproteína D (gD) do HSV-1, e também na coadministração de plasmídeos que codificam citocinas (IL-2, -12, -15 e GM-CSF) visando aumentar a imunogenicidade de HIVBr18. A sequencia de DNA que codifica os 18 peptídeos da vacina HIVBr18 foi amplificada por PCR e clonada em um plasmídeo que abrigava a sequencia da gD do HSV-1. dando origem ao plasmídeo pVAX-gDh-HIVBr18. Animais imunizados com gDh-HIVBr18 apresentaram resposta imunológica similar ao grupo que recebeu somente HIVBr18, não sendo diferente também daqueles que receberam plasmídeos gDh-HIVBr18 que sofreram alterações nas sequências para melhorar o padrão de distribuição hidrofóbica e permitir a migração da proteína de fusão para o meio extracelular. Construímos e testamos um plasmídeo bicistrônico que expressa gDh e HIVBr18 isoladamente, mas também não observamos aumento na resposta imune induzida. A coadministração com o plasmídeo HIVBr18 e plasmídeos que codificam as citocinas IL-12, IL-15 e GM-CSF, proporciona um aumento na magnitude da resposta imunológica induzida contra o pool de peptídeos codificados pela vacina, entretanto sem alteração da amplitude da resposta. Além disso, o plasmídeo de GM-CSF induziu maior número de células T CD4+ polifuncionais. Demonstramos também que a coadministração do plasmídeo que codifica GM-CSF, induz uma resposta imune celular de maior magnitude mesmo em uma condição de dose reduzida. Entretanto, observamos que esta citocina não é um bom adjuvante quando utilizamos como vetor de imunização um adenovírus que expressa os 18 epítopos
The formulation HIVBr18, previously developed and tested, is based on a DNA vaccine encoding 18 conserved and promiscuous HIV-1 CD4 epitopes and after immunization of transgenic mice for many human HLA class II molecules using this DNA vaccine, could be observed proliferation of CD4+ and CD8+ T cells and IFN-y production directed to multiple epitopes encoded by the vaccine. We intend to explore here, strategies based on fusion or combination of epitopes encoded by HIVBr18 vaccine with glycoprotein D (gD) of HSV- 1 and also the coadministration of cytokine-encoding plasmids (pIL-2, -12, -15 and pGM -CSF) aiming to enhance immunogenicity of HIVBr18. The DNA sequence of epitopes encoded by HIVBr18 vaccine was amplified by PCR and cloned into a plasmid that contained the sequence of gD, giving rise to plasmid pVAX-gDh-HIVBr18. After mice immunization, animals immunized with this construct showed similar immune response to the group that received HIVBr18, and also the group of animals that received gDh-HIVBr18 plasmid that had been modified by exchange in peptides order to assure to the molecule a better hydrophobic distribution and allow translocation to the extracellular face of cell membrane. We constructed and injected mice with a bicistronic plasmid expressing gDh and HIVBr18, simultaneously and isolated, but no increase in the magnitude of the immune response was observed. HIVBr18 coadministration with cytokine-encoding plasmids pIL-12, pIL-15 and pGM-CSF, provides an increase in the magnitude of immune response induced against the peptides encoded by the vaccine, and similar breadth. In addition, co-immunization with pGM-CSF induced greater number of polyfunctional CD4 + T cells. We also demonstrate that, even in a low dose approach coadministration of pGM-CSF induced a higher immune response than HIVBr18 alone in the same dose. However, we observed that this cytokine is not a good adjuvant when used in combination with an adenovirus that expresses the 18 HIV-1 epitopes.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

Chemokines are small immune system proteins mediating leukocyte migration and activation, and are important in many aspects of health and diseases. Some chemokines also have the ability to block HIV-1 infection by binding to the HIV-1 co-receptors CCR5 (CC chemokine receptor 5) and CXCR4 (CXC chemokine receptor 4). The first part of this work is to determine the mechanism of action of a human herpesvirus-8 encoded viral chemokine analog vMIP-II (viral macrophage inflammatory protein-II) by characterizing its interactions with endothelial surface glycosaminoglycans (GAGs) and cell surface receptors. Nuclear magnetic resonance (NMR), mutagenesis and molecular-docking were conducted and results show that vMIP-II tightly binds glycosaminoglycans using residues distributed along one face of the protein, such as R18, R46 and R48, and that there is a shift in the GAG binding site between the monomer and dimer form of vMIP-II where the N-terminus is involved in GAG binding for the dimer. This study, for the first time, provides a model that explains the mechanism of how quaternary structure affects chemokine-GAG binding. Mutagenesis and competition binding assays were conducted to study the receptor-binding mechanism of vMIP-II. Preliminary results suggest that vMIP-II uses the same positively charged binding surface comprising R18, K45, R46 and R48 to interact with the negatively charged N-termini of CCR5 and CXCR4. NMR studies on how vMIP-II interacts with N-terminal peptides of CCR5 and CXCR4 is on-going. The second part of this work was to rationally design HIV-1 entry inhibitors based on our knowledge of the mechanisms of chemokine-receptor binding and HIV-1 cell entry. We successfully designed two chimeric HIV entry inhibitors composed of CCR5-targeting RANTES variants (5P12-RANTES and 5P14-RANTES) linked to a gp41 targeting C-peptide, C37. In in vitro assays, chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed the highest anti-viral potency yet published with IC50 values as low as 0.001 nM against certain virus strains. On human peripheral blood mononuclear cells, the chimeric inhibitors also exhibited very strong inhibition against R5-tropic and X4-tropic viruses, with IC50 values as low as 0.015 nM and 0.44 nM, respectively. A clear delivery mechanism was observed and characterized. These fully recombinant inhibitors can be easily produced at low cost and are excellent candidates for HIV microbicides.

Dissertação de mestrado em Bioinformatics
Fusion Peptides (FPs) play an important role in viral fusion. They are segments of fusion proteins that include conserved hydrophobic domains absolutely required for the fusogenic activity of glycoproteins from divergent virus families. FPs from different viruses are very different, which is intriguing and makes it difficult to find patterns that could characterize them. However, the development of therapeutics targeting fusion peptides requires a detailed knowledge about their properties. Most of the studies made in this field were more focused on Influenza, HIV and all retroviruses fusion peptides, but one cannot generalize that information for all viral families, since they are different even at the sequence level. Hence, machine learning can be a good tool to unveil hidden patterns that characterize these peptides. Creating a model capable of separating fusion peptides (positive cases) from non-fusion peptides (negative cases) using their amino acid (AA) sequence as the basis for generating features, requires the usage of well annotated and reviewed proteins. Currently, the information about these peptides is very dispersed and there are no complete databases available to access and use this data. In the scope of this dissertation, an extensive search on these fusion peptides was performed, which resulted in 468 sequences found for 207 out of 255 viruses. From that universe 111 sequences, with experimentally validated FPs were used in subsequent analysis. Multiple alignments and phylogenetic trees analysis suggested clusters per class and per family, which led to consensus sequences per virus family. For this work, eight different machine learning models were trained and tested, using a five-fold cross validation process, on different datasets to identify and classify fusion peptides. To prove the value of the developed models, three different datasets composed by well annotated sequences from UniProt and NCBI were used. Ensembles of the created models using one dataset showed good overall performance with scores of accuracy and recall above 90 %. These are promising results on predicting the most plausible regions where the FP is located within an entire fusion protein sequence, which can be very useful in future research.
Os péptidos de fusão têm um papel importante no mecanismo de fusão viral. Estes péptidos são segmentos de proteínas de fusão que incluem domínios hidrofóbicos conservados absolutamente indispensáveis à atividade fusogénica de glicoproteínas de diversas famílias de vírus. É intrigante que cada vírus tenha um péptido de fusão diferente, o que dificulta a identificação de padrões que os caracterizem, mesmo que o desenvolvimento de novos fármacos dependa do conhecimento detalhado sobre as propriedades dos mesmos. A maioria dos estudos feitos nesta área incidem principalmente sobre os vírus Influenza, VIH e os péptidos de fusão dos retrovírus, contudo não é possível inferir informação para outros vírus devido às diferenças ao nível sequencial. Tendo em conta todos estes factos, machine learning pode ser uma boa ferramenta para revelar padrões que estejam mais impercetíveis à primeira vista que caracterizem péptidos de fusão. Para criar modelos capazes de distinguir claramente um péptido de fusão de uma outra sequência, recorrendo aos aminoácidos presentes na sua sequência, é necessário usar informação que esteja bem anotada e revista. Atualmente, a informação relativa a estes péptidos encontra-se dispersa por várias bases de dados, não existindo assim nenhum local onde a informação esteja centralizada e completa. No âmbito desta dissertação fez-se uma pesquisa exaustiva sobre péptidos de fusão, resultando em 468 sequências para 207 vírus de um universo de 255 vírus, usando com confiança 111 dessas sequências para efeitos de machine learning. Neste trabalho foram treinados oito modelos de machine learning diferentes, e testados usando 5-fold cross-validation, em diferentes datasets de forma a identificar e classificar péptidos de fusão. Para comprovar a utilidade dos modelos, este foram usados em três datasets diferentes compostos por sequências retiradas da UniProt e do NCBI. O conjunto de modelos final obteve uma percentagem de exemplos corretamente classificados e recall a rondar os 90 %. Estes resultados são promissores na medida em que prevêem corretamente a região mais provável do péptido de fusão, dentro de uma sequência de uma proteína de fusão, resultados que podem ser deveras proveitosos para investigadores desta área científica.

Chu Ling Hon Matthew.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (leaves 201-223).
Abstracts in English and Chinese.
Declaration --- p.i
Thesis/Assessment Committee --- p.ii
Abstract --- p.iii
摘要 --- p.vi
Acknowledgements --- p.viii
General abbreviations --- p.xi
Abbreviations of chemicals --- p.xv
Table of Contents --- p.xvi
List of Figures --- p.xxiii
List of tables --- p.xxviii
Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Severe Acute Respiratory Syndrome (SARS) - Three Years in Review --- p.1
Chapter 1.1.1 --- Epidemiology --- p.1
Chapter 1.1.2 --- Clinical presentation --- p.3
Chapter 1.1.3 --- Diagnostic tests --- p.5
Chapter 1.2 --- Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) --- p.7
Chapter 1.2.1 --- SARS - Identification of the etiological agent --- p.7
Chapter 1.2.2 --- The coronaviruses --- p.9
Chapter 1.2.3 --- The genome organization of SARS-CoV --- p.11
Chapter 1.2.4 --- The life cycle of SARS-CoV --- p.13
Chapter 1.3 --- Spike Glycoprotein (S protein) of SARS-CoV --- p.15
Chapter 1.3.1 --- SARS-CoV S protein --- p.15
Chapter 1.3.2 --- S protein-driven infection --- p.17
Chapter 1.4 --- SARS-CoV S Protein Fusion Core --- p.22
Chapter 1.4.1 --- Heptad repeat and coiled coil --- p.22
Chapter 1.4.2 --- The six-helix coiled coil bundle structure --- p.25
Chapter 1.5 --- 3C-like Protease (3CLpro) of SARS-CoV --- p.28
Chapter 1.5.1 --- Extensive proteolytic processing of replicase polyproteins --- p.28
Chapter 1.5.2 --- SARS-CoV 3CLpro --- p.30
Chapter 1.5.3 --- Substrate Specificity of SARS-CoV 3CLpro --- p.31
Chapter 1.6 --- SARS Drug Development --- p.32
Chapter 1.6.1 --- Drug targets of SARS-CoV --- p.32
Chapter 1.6.2 --- Current anti-SARS drugs --- p.36
Chapter 1.7 --- Project Objectives --- p.39
Chapter 1.7.1 --- Characterization of SARS-CoV S protein fusion core --- p.39
Chapter 1.7.2 --- Characterization of SARS-CoV 3CLpr0 substrate specificity --- p.40
Chapter 2 --- Materials and Methods --- p.42
Chapter 2.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.42
Chapter 2.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.42
Chapter 2.1.2 --- Recombinant protein approach --- p.43
Chapter 2.1.2.1 --- Plasmids construction --- p.43
Chapter 2.1.2.2 --- Protein expression and purification --- p.52
Chapter 2.1.2.3 --- Amino acid analysis --- p.57
Chapter 2.1.2.4 --- GST-pulldown experiment --- p.58
Chapter 2.1.2.5 --- Laser light scattering --- p.61
Chapter 2.1.2.6 --- Size-exclusion chromatography --- p.62
Chapter 2.1.2.7 --- Circular dichroism spectroscopy --- p.62
Chapter 2.1.3 --- Synthetic peptide approach --- p.64
Chapter 2.1.3.1 --- Peptide synthesis --- p.64
Chapter 2.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.65
Chapter 2.1.3.3 --- Size-exclusion high-performance liquid chromato-graphy --- p.66
Chapter 2.1.3.4 --- Laser light scattering --- p.66
Chapter 2.1.3.5 --- Circular dichroism spectroscopy --- p.67
Chapter 2.2 --- Identification of SARS-CoV Entry Inhibitors --- p.70
Chapter 2.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.70
Chapter 2.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.74
Chapter 2.2.3 --- Molecular modeling --- p.75
Chapter 2.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.79
Chapter 2.3.1 --- Protein expression and purification --- p.79
Chapter 2.3.2 --- """Cartridge replacement"" solid-phase peptide synthesis" --- p.80
Chapter 2.3.3 --- Peptide cleavage assay and mass spectrometric analysis --- p.83
Chapter 3 --- Results --- p.84
Chapter 3.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.84
Chapter 3.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.84
Chapter 3.1.2 --- Recombinant protein approach --- p.87
Chapter 3.1.2.1 --- "Plasmids construction of pET-28a-His6-HRl, pGEX-6P-l-HR2 and pGEX-6P-l-2-Helix" --- p.87
Chapter 3.1.2.2 --- Protein expression and purification --- p.92
Chapter 3.1.2.3 --- GST-pulldown experiment --- p.101
Chapter 3.1.2.4 --- Laser light scattering --- p.103
Chapter 3.1.2.5 --- Size-exclusion chromatography --- p.105
Chapter 3.1.2.6 --- Circular dichroism spectroscopy --- p.107
Chapter 3.1.3 --- Synthetic peptide approach --- p.112
Chapter 3.1.3.1 --- Peptide synthesis --- p.112
Chapter 3.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.116
Chapter 3.1.3.3 --- Size-exclusion high-performance liquid chromatography --- p.117
Chapter 3.1.3.4 --- Laser light scattering --- p.122
Chapter 3.1.3.5 --- Circular dichroism spectroscopy --- p.124
Chapter 3.2 --- Identification of SARS-CoV Entry Inhibitors --- p.129
Chapter 3.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.129
Chapter 3.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.131
Chapter 3.2.3 --- Molecular modeling --- p.135
Chapter 3.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.141
Chapter 3.3.1 --- Protein expression and purification --- p.141
Chapter 3.3.2 --- Substrate specificity preference of SARS-CoV 3CLpr0 --- p.142
Chapter 3.3.3 --- "Primary and secondary screening using the ""cartridge replacement strategy""" --- p.142
Chapter 4 --- Discussion --- p.149
Chapter 4.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.149
Chapter 4.1.1 --- Design of recombinant proteins and synthetic peptides of HR regions --- p.149
Chapter 4.1.2 --- Recombinant protein approach --- p.151
Chapter 4.1.3 --- Synthetic peptide approach --- p.153
Chapter 4.1.4 --- Summary of the present and previous studies in the SARS-CoV S protein fusion core --- p.157
Chapter 4.2 --- Identification of SARS-CoV Entry Inhibitors --- p.167
Chapter 4.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.167
Chapter 4.2.2 --- Identification of peptide inhibitors --- p.168
Chapter 4.2.3 --- Identification of small molecule inhibitors --- p.172
Chapter 4.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.183
Chapter 4.3.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.184
Chapter 4.3.2 --- The development of the rapid and high-throughput screening strategy for protease substrate specificity --- p.188
Appendix --- p.191
Chapter I. --- Nucleotide Sequence of S protein of SARS-CoV --- p.191
Chapter II. --- Protein Sequence of S protein of SARS-CoV --- p.194
Chapter III. --- Protein Sequence of 3CLpro of SARS-CoV --- p.195
Chapter IV. --- Vector maps --- p.196
Chapter 1. --- Vector map and MCS of pET-28a --- p.196
Chapter 2. --- Vector map and MCS of pGEX-6P-l --- p.197
Chapter V. --- Electrophoresis markers --- p.198
Chapter 1. --- GeneRuler´ёØ 1 kb DNA Ladder --- p.198
Chapter 2. --- GeneRuler´ёØ 100bp DNA Ladder --- p.198
Chapter 3. --- High-range Rainbow Molecular Weight Markers --- p.199
Chapter 4. --- Low-range Rainbow Molecular Weight Markers --- p.199
Chapter VI. --- SDS-PAGE gel preparation protocol --- p.200
References --- p.201

Nipah virus (NiV) and Hendra virus (HeV) have been identified as the causes of outbreaks of fatal meningitis, encephalitis, and respiratory disease in Australia, Malaysia, Bangladesh, and India from 1994 until 2004. In order to accommodate the unique genomic characteristics of NiV and HeV, a new genus within the family Paramyxoviridae was created, named Henipavirus. NiV encodes two surface glycoproteins: the attachment glycoprotein (G) binds to the cellular receptor for the virus, while the fusion glycoprotein (F) mediates membrane fusion between the virus and cell membranes. Expression of F and G in the same cell results in cell-cell fusion in transfected cell monolayers, while expression of F and G on their own in cell monolayers does not result in fusion. Co-culture of singly-transfected F and G cells also does not result in fusion. Expression of NiV G in transgenic CRFK cells results in resistance to NiV- and HeV-induced cytopathic effect. Additionally, neither NiV nor HeV nucleic acid could be detected in CRFK-NiV G that had been exposed to NiV or HeV. NiV G expression also prevents NiV F+NiV G-mediated cell-cell fusion, but does not affect cell surface expression of either virus receptor, ephrin-B2 and ephrin-B3. Chimeric glycoproteins derived from NiV G and CDV H were constructed and characterized. None of the chimeric glycoproteins were able to fuse when coexpressed with either NiV F or CDV F. Only one of the chimeric glycoproteins (H145/G458) was detected on the cell surface by immunofluorescence assay (IFA). None of the chimeric glycoproteins altered cell surface expression levels of ephrin-B2 and ephrin-B3. Finally, recombinant NiV genomes (rNiV and rNiV eGFPG) were constructed, as well as chimeric CDV genomes with NiV ORF substitutions (rCDV eGFPH NiVFG and rCDV eGFPH NiVMFG). The only chimeric virus that was generated, rCDV eGFPH NiVFG, was assessed for its release from infected cells. rCDV eGFPH NiVFG was poorly released from infected cells without a freeze-thaw cycle, but was also found to induce the cellsurface down-regulation of the viral receptors ephrin-B2 and ephrin-B3.
October 2007