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1
Podgorski, Jennifer, Joshua Calabrese, Lauren Alexandrescu, Deborah Jacobs-Sera, Welkin Pope, Graham Hatfull, and Simon White. "Structures of Three Actinobacteriophage Capsids: Roles of Symmetry and Accessory Proteins." Viruses 12, no. 3 (March 8, 2020): 294. http://dx.doi.org/10.3390/v12030294.
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Here, we describe the structure of three actinobacteriophage capsids that infect Mycobacterium smegmatis. The capsid structures were resolved to approximately six angstroms, which allowed confirmation that each bacteriophage uses the HK97-fold to form their capsid. One bacteriophage, Rosebush, may have a novel variation of the HK97-fold. Four novel accessory proteins that form the capsid head along with the major capsid protein were identified. Two of the accessory proteins were minor capsid proteins and showed some homology, based on bioinformatic analysis, to the TW1 bacteriophage. The remaining two accessory proteins are decoration proteins that are located on the outside of the capsid and do not resemble any previously described bacteriophage decoration protein. SDS-PAGE and mass spectrometry was used to identify the accessory proteins and bioinformatic analysis of the accessory proteins suggest they are used in many actinobacteriophage capsids.
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Guan, Zhanwen, Ling Zhong, Chunyan Li, Wenbi Wu, Meijin Yuan, and Kai Yang. "The Autographa californica Multiple Nucleopolyhedrovirusac54Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly." Journal of Virology 90, no. 8 (February 10, 2016): 4115–26. http://dx.doi.org/10.1128/jvi.02885-15.
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ABSTRACTBaculovirus DNAs are synthesized and inserted into preformed capsids to form nucleocapsids at a site in the infected cell nucleus, termed the virogenic stroma. Nucleocapsid assembly ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) requires the major capsid protein VP39 and nine minor capsid proteins, including VP1054. However, how VP1054 participates in nucleocapsid assembly remains elusive. In this study, the VP1054-encoding gene (ac54) was deleted to generate theac54-knockout AcMNPV (vAc54KO). In vAc54KO-transfected cells, nucleocapsid assembly was disrupted, leading to the formation of abnormally elongated capsid structures. Interestingly, unlike cells transfected with AcMNPV mutants lacking other minor capsid proteins, in which capsid structures were distributed within the virogenic stroma,ac54ablation resulted in a distinctive location of capsid structures and VP39 at the periphery of the nucleus. The altered distribution pattern of capsid structures was also observed in cells transfected with AcMNPV lacking BV/ODV-C42 or in cytochalasind-treated AcMNPV-infected cells. BV/ODV-C42, along with PP78/83, has been shown to promote nuclear filamentous actin (F-actin) formation, which is another requisite for nucleocapsid assembly. Immunofluorescence using phalloidin indicated that the formation and distribution of nuclear F-actin were not affected byac54deletion. However, immunoelectron microscopy revealed that BV/ODV-C42, PP78/83, and 38K failed to integrate into capsid structures in the absence of VP1054, and immunoprecipitation further demonstrated that in transient expression assays, VP1054 interacted with BV/ODV-C42 and VP80 but not VP39. Our findings suggest that VP1054 plays an important role in the transport of capsid proteins to the nucleocapsid assembly site prior to the process of nucleocapsid assembly.IMPORTANCEBaculoviruses are large DNA viruses whose replication occurs within the host nucleus. The localization of capsids into the capsid assembly site requires virus-induced nuclear F-actin; the inhibition of nuclear F-actin formation results in the retention of capsid structures at the periphery of the nucleus. In this paper, we note that the minor capsid protein VP1054 is essential for the localization of capsid structures, the major capsid protein VP39, and the minor capsid protein 38K into the capsid assembly site. Moreover, VP1054 is crucial for correct targeting of the nuclear F-actin factors BV/ODV-C42 and PP78/83 for capsid maturation. However, the formation and distribution of nuclear F-actin are not affected by the lack of VP1054. We further reveal that VP1054 interacts with BV/ODV-C42 and a capsid transport-related protein, VP80. Taken together, our findings suggest that VP1054 plays a unique role in the pathway(s) for transport of capsid proteins.
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Burkert, Oliver, Susanne Kreßner, Ludwig Sinn, Sven Giese, Claudia Simon, and Hauke Lilie. "Biophysical characterization of polyomavirus minor capsid proteins." Biological Chemistry 395, no. 7-8 (July 1, 2014): 871–80. http://dx.doi.org/10.1515/hsz-2014-0114.
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Abstract The murine polyomavirus encodes three structural proteins, VP1, VP2 and VP3, which together form the viral capsid. The outer shell of this capsid is composed of the major capsid protein VP1, the inner shell consists of VP2 and its N-terminally truncated form VP3. These two minor capsid proteins interact with their identical C-terminal part in the central cavity of VP1 pentamers, building the capsid assembly unit. While the VP1 structure and functions are well studied, VP2 and VP3 are poorly understood. In order to get a detailed insight into the structure and function of the minor capsid proteins, they were produced recombinantly in Escherichia coli as inclusion bodies and refolded in vitro. The success of refolding was strictly dependent on the presence of detergent in the refolding buffer. VP2 and VP3 are monomeric and their structures exhibit a high α-helical content. The function of both proteins could be monitored by complex formation with VP1. Furthermore, we could demonstrate a hemolytic activity of VP2/VP3 in vitro, which fits well into a postulated membrane interaction of VP2 during viral infection.
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Vellinga, Jort, Stephanie Van der Heijdt, and Rob C. Hoeben. "The adenovirus capsid: major progress in minor proteins." Journal of General Virology 86, no. 6 (June 1, 2005): 1581–88. http://dx.doi.org/10.1099/vir.0.80877-0.
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Human adenoviruses have been the subject of intensive investigation since their discovery in the early 1950s: they have served as model pathogens, as probes for studying cellular processes and, more recently, as efficient gene-delivery vehicles for experimental gene therapy. As a result, a detailed insight into many aspects of adenovirus biology is now available. The capsid proteins and in particular the hexon, penton-base and fibre proteins (the so-called major capsid proteins) have been studied extensively and their structure and function in the virus capsid are now well-defined. On the other hand, the minor proteins in the viral capsid, i.e. proteins IIIa, VI, VIII and IX, have received much less attention. Only the last few years have witnessed a sharp increase in the number of studies on their structure and function. Here, a review of the minor capsid proteins is provided, with a focus on new insights into their position and role in the capsid and the opportunities that they provide for improving human adenovirus-derived gene-delivery vectors.
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Copeland, Anna Maria, William W. Newcomb, and Jay C. Brown. "Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment." Journal of Virology 83, no. 4 (December 10, 2008): 1660–68. http://dx.doi.org/10.1128/jvi.01139-08.
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ABSTRACT Replication of herpes simplex virus type 1 (HSV-1) involves a step in which a parental capsid docks onto a host nuclear pore complex (NPC). The viral genome then translocates through the nuclear pore into the nucleoplasm, where it is transcribed and replicated to propagate infection. We investigated the roles of viral and cellular proteins in the process of capsid-nucleus attachment. Vero cells were preloaded with antibodies specific for proteins of interest and infected with HSV-1 containing a green fluorescent protein-labeled capsid, and capsids bound to the nuclear surface were quantified by fluorescence microscopy. Results showed that nuclear capsid attachment was attenuated by antibodies specific for the viral tegument protein VP1/2 (UL36 gene) but not by similar antibodies specific for UL37 (a tegument protein), the major capsid protein (VP5), or VP23 (a minor capsid protein). Similar studies with antibodies specific for nucleoporins demonstrated attenuation by antibodies specific for Nup358 but not Nup214. The role of nucleoporins was further investigated with the use of small interfering RNA (siRNA). Capsid attachment to the nucleus was attenuated in cells treated with siRNA specific for either Nup214 or Nup358 but not TPR. The results are interpreted to suggest that VP1/2 is involved in specific attachment to the NPC and/or in migration of capsids to the nuclear surface. Capsids are suggested to attach to the NPC by way of the complex of Nup358 and Nup214, with high-resolution immunofluorescence studies favoring binding to Nup358.
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Sheaffer, Amy K., William W. Newcomb, Jay C. Brown, Min Gao, Sandra K. Weller, and Daniel J. Tenney. "Evidence for Controlled Incorporation of Herpes Simplex Virus Type 1 UL26 Protease into Capsids." Journal of Virology 74, no. 15 (August 1, 2000): 6838–48. http://dx.doi.org/10.1128/jvi.74.15.6838-6848.2000.
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ABSTRACT Herpes simplex virus type 1 (HSV-1) capsids are initially assembled with an internal protein scaffold. The scaffold proteins, encoded by overlapping in-frame UL26 and UL26.5 transcripts, are essential for formation and efficient maturation of capsids. UL26 encodes an N-terminal protease domain, and its C-terminal oligomerization and capsid protein-binding domains are identical to those of UL26.5. The UL26 protease cleaves itself, releasing minor scaffold proteins VP24 and VP21, and the more abundant UL26.5 protein, releasing the major scaffold protein VP22a. Unlike VP21 and VP22a, which are removed from capsids upon DNA packaging, we demonstrate that VP24 (containing the protease domain) is quantitatively retained. To investigate factors controlling UL26 capsid incorporation and retention, we used a mutant virus that fails to express UL26.5 (ΔICP35 virus). Purified ΔICP35 B capsids showed altered sucrose gradient sedimentation and lacked the dense scaffold core seen in micrographs of wild-type B capsids but contained capsid shell proteins in wild-type amounts. Despite C-terminal sequence identity between UL26 and UL26.5, ΔICP35 capsids lacking UL26.5 products did not contain compensatory high levels of UL26 proteins. Therefore, HSV capsids can be maintained and/or assembled on a minimal scaffold containing only wild-type levels of UL26 proteins. In contrast to UL26.5, increased expression of UL26 did not compensate for the ΔICP35growth defect. While indirect, these findings are consistent with the view that UL26 products are restricted from occupying abundant UL26.5 binding sites within the capsid and that this restriction is not controlled by the level of UL26 protein expression. Additionally, ΔICP35 capsids contained an altered complement of DNA cleavage and packaging proteins, suggesting a previously unrecognized role for the scaffold in this process.
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Wang, Nan, Dongming Zhao, Jialing Wang, Yangling Zhang, Ming Wang, Yan Gao, Fang Li, et al. "Architecture of African swine fever virus and implications for viral assembly." Science 366, no. 6465 (October 17, 2019): 640–44. http://dx.doi.org/10.1126/science.aaz1439.
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African swine fever virus (ASFV) is a giant and complex DNA virus that causes a highly contagious and often lethal swine disease for which no vaccine is available. Using an optimized image reconstruction strategy, we solved the ASFV capsid structure up to 4.1 angstroms, which is built from 17,280 proteins, including one major (p72) and four minor (M1249L, p17, p49, and H240R) capsid proteins organized into pentasymmetrons and trisymmetrons. The atomic structure of the p72 protein informs putative conformational epitopes, distinguishing ASFV from other nucleocytoplasmic large DNA viruses. The minor capsid proteins form a complicated network below the outer capsid shell, stabilizing the capsid by holding adjacent capsomers together. Acting as core organizers, 100-nanometer-long M1249L proteins run along each edge of the trisymmetrons that bridge two neighboring pentasymmetrons and form extensive intermolecular networks with other capsid proteins, driving the formation of the capsid framework. These structural details unveil the basis of capsid stability and assembly, opening up new avenues for African swine fever vaccine development.
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Gasparovic, M. L., G. V. Gee, and W. J. Atwood. "JC Virus Minor Capsid Proteins Vp2 and Vp3 Are Essential for Virus Propagation." Journal of Virology 80, no. 21 (November 1, 2006): 10858–61. http://dx.doi.org/10.1128/jvi.01298-06.
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ABSTRACT Virus-encoded capsid proteins play a major role in the life cycles of all viruses. The JC virus capsid is composed of 72 pentamers of the major capsid protein Vp1, with one of the minor coat proteins Vp2 or Vp3 in the center of each pentamer. Vp3 is identical to two-thirds of Vp2, and these proteins share a DNA binding domain, a nuclear localization signal, and a Vp1-interacting domain. We demonstrate here that both the minor proteins and the myristylation site on Vp2 are essential for the viral life cycle, including the proper packaging of its genome.
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Wills, Elizabeth, Luella Scholtes, and Joel D. Baines. "Herpes Simplex Virus 1 DNA Packaging Proteins Encoded by UL6, UL15, UL17, UL28, and UL33 Are Located on the External Surface of the Viral Capsid." Journal of Virology 80, no. 21 (August 18, 2006): 10894–99. http://dx.doi.org/10.1128/jvi.01364-06.
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ABSTRACT Studies to localize the herpes simplex virus 1 portal protein encoded by UL6, the putative terminase components encoded by UL15, UL 28, and UL33, the minor capsid proteins encoded by UL17, and the major scaffold protein ICP35 were conducted. ICP35 in B capsids was more resistant to trypsin digestion of intact capsids than pUL6, pUL15, pUL17, pUL28, or pUL33. ICP35 required sectioning of otherwise intact embedded capsids for immunoreactivity, whereas embedding and/or sectioning decreased the immunoreactivities of pUL6, pUL17, pUL28, and pUL33. Epitopes of pUL15 were recognized roughly equally well in both sectioned and unsectioned capsids. These data indicate that pUL6, pUL17, pUL28, pUL33, and at least some portion of pUL15 are located at the external surface of the capsid.
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Pawlowski, Alice, Anni M. Moilanen, Ilona A. Rissanen, Juha A. E. Määttä, Vesa P. Hytönen, Janne A. Ihalainen, and Jaana K. H. Bamford. "The Minor Capsid Protein VP11 of Thermophilic Bacteriophage P23-77 Facilitates Virus Assembly by Using Lipid-Protein Interactions." Journal of Virology 89, no. 15 (May 13, 2015): 7593–603. http://dx.doi.org/10.1128/jvi.00262-15.
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ABSTRACTThermus thermophilusbacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner-membrane-containing double-stranded DNA (dsDNA) viruses infecting thermophilic bacteria and halophilic archaea. The viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of the minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with a predominantly α-helical secondary structure and high thermal stability. The high proportion of basic amino acids in the protein enables electrostatic interaction with negatively charged molecules, including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function of VP11 is elucidated by demonstrating the interactions of VP11 withThermus-derived LUVs and with the major capsid proteins by means of the dynamic-light-scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers the binding of the major capsid protein VP17, thus facilitating a controlled incorporation of the two different major protein species into the assembling capsid.IMPORTANCEThe study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein charge is important in order to form electrostatic interactions with the lipid surface, thereby facilitating the incorporation of other capsid proteins on the membrane surface. This implies an alternative function for basic proteins present in the virions of other lipid-containing thermophilic viruses, whose proposed role in genome packaging is based on their capability to bind DNA. The unique minor capsid protein of bacteriophage P23-77 resembles in its characteristics the scaffolding proteins of tailed phages, though it constitutes a substantial part of the mature virion.
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Wang, Wen-Hung, Chung-Wen Kuo, Li-Kwan Chang, Chen-Chia Hung, Tzu-Hsuan Chang, and Shih-Tung Liu. "Assembly of Epstein-Barr Virus Capsid in Promyelocytic Leukemia Nuclear Bodies." Journal of Virology 89, no. 17 (June 17, 2015): 8922–31. http://dx.doi.org/10.1128/jvi.01114-15.
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ABSTRACTThe Epstein-Barr virus (EBV) capsid contains a major capsid protein, VCA; two minor capsid proteins, BDLF1 and BORF1; and a small capsid protein, BFRF3. During the lytic cycle, these capsid proteins are synthesized and imported into the host nucleus for capsid assembly. This study finds that EBV capsid proteins colocalize with promyelocytic leukemia (PML) nuclear bodies (NBs) in P3HR1 cells during the viral lytic cycle, appearing as nuclear speckles under a confocal laser scanning microscope. In a glutathioneS-transferase pulldown study, we show that BORF1 interacts with PML-NBsin vitro. BORF1 also colocalizes with PML-NBs in EBV-negative Akata cells after transfection and is responsible for bringing VCA and the VCA-BFRF3 complex from the cytoplasm to PML-NBs in the nucleus. Furthermore, BDLF1 is dispersed throughout the cell when expressed alone but colocalizes with PML-NBs when BORF1 is also present in the cell. In addition, this study finds that knockdown of PML expression by short hairpin RNA does not influence the intracellular levels of capsid proteins but reduces the number of viral particles produced by P3HR1 cells. Together, these results demonstrate that BORF1 plays a critical role in bringing capsid proteins to PML-NBs, which may likely be the assembly sites of EBV capsids. The mechanisms elucidated in this study are critical to understanding the process of EBV capsid assembly.IMPORTANCECapsid assembly is an important event during the Epstein-Barr virus (EBV) lytic cycle, as this process is required for the production of virions. In this study, confocal microscopy revealed that the EBV capsid protein BORF1 interacts with promyelocytic leukemia (PML) nuclear bodies (NBs) in the host nucleus and is responsible for transporting the other EBV capsid proteins, including VCA, BDLF1, and BFRF3, to these subnuclear locations prior to initiation of capsid assembly. This study also found that knockdown of PML expression by short hairpin RNA significantly reduces EBV capsid assembly capabilities. This enhanced understanding of capsid assembly offers potential for the development of novel antiviral strategies and therapies that can prevent the propagation and spread of EBV.
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Salmon, Brandy, Dorothy Nalwanga, Ying Fan, and Joel D. Baines. "Proteolytic Cleavage of the Amino Terminus of the UL15 Gene Product of Herpes Simplex Virus Type 1 Is Coupled with Maturation of Viral DNA into Unit-Length Genomes." Journal of Virology 73, no. 10 (October 1, 1999): 8338–48. http://dx.doi.org/10.1128/jvi.73.10.8338-8348.1999.
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ABSTRACT The UL15 gene of herpes simplex virus type 1 (HSV-1), like UL6, UL17, UL28, UL32, and UL33, is required for cleavage of concatameric DNA into genomic lengths and for packaging of cleaved genomes into preformed capsids. A previous study indicated that the UL15 gene encodes minor capsid proteins. In the present study, we have shown that the amino-terminal 509 amino acids of the UL15-encoded protein are sufficient to confer capsid association inasmuch as a carboxyl-terminally truncated form of the UL15-encoded protein with an M r of approximately 55,000 readily associated with capsids. This and previous studies have shown that, whereas three UL15-encoded proteins with apparent M rs of 83,000, 80,000, and 79,000 associated with wild-type B capsids, only the full-length 83,000-M r protein associated with B capsids purified from cells infected with viruses lacking functional UL6, UL17, UL28, UL32, and UL33 genes (B. Salmon and J. D. Baines, J. Virol. 72:3045–3050, 1998). Thus, all viral mutants that fail to cleave viral DNA into genomic-length molecules also fail to produce capsid-associated UL15 80,000- and 79,000-M r proteins. In contrast, the 80,000- and 79,000-M r proteins were readily detected in capsids purified from cells infected with a UL25 null virus that cleaves, but does not package, DNA. The conclusion that the amino terminus of the 83,000-M r protein is truncated to produce the 80,000- and/or 79,000-M r protein was supported by the following observations. (i) Whereas the C termini of the 83,000-, 80,000-, and 79,000-M r proteins are identical, immunoreactivity dependent on the first 35 amino acids of the UL15 83,000-M r protein was absent from the 80,000- and 79,000-M r proteins. (ii) The 79,000- and 80,000-M r proteins were detected in capsids from cells infected with HSV-1(UL15M36V), an engineered virus encoding valine rather than methionine at codon 36. Thus, initiation at codon 36 is unlikely to account for production of the 80,000- and/or 79,000-M r protein. Taken together, these data strongly suggest that capsid-associated UL15-encoded protein is proteolytically cleaved near the N terminus and indicate that this modification is tightly linked to maturation of genomic DNA.
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Dubielzig, Ralf, Jason A. King, Stefan Weger, Andrea Kern, and Jürgen A. Kleinschmidt. "Adeno-Associated Virus Type 2 Protein Interactions: Formation of Pre-Encapsidation Complexes." Journal of Virology 73, no. 11 (November 1, 1999): 8989–98. http://dx.doi.org/10.1128/jvi.73.11.8989-8998.1999.
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ABSTRACT The nonstructural adeno-associated virus type 2 Rep proteins are known to control viral replication and thus provide the single-stranded DNA genomes required for packaging into preformed capsids. In addition, complexes between Rep proteins and capsids have previously been observed in the course of productive infections. Such complexes have been interpreted as genome-linked Rep molecules associated with the capsid upon successful DNA encapsidation. Here we demonstrate via coimmunoprecipitation, cosedimentation, and yeast two-hybrid analyses that the Rep-VP association also occurs in the absence of packageable genomes, suggesting that such complexes could be involved in the preparation of empty capsids for subsequent encapsidation steps. The Rep domain responsible for the observed Rep-VP interactions is situated within amino acids 322 to 482. In the presence of all Rep proteins, Rep52 and, to a lesser extent, Rep78 are most abundantly recovered with capsids, whereas Rep68 and Rep40 vary in association depending on their expression levels. Rep78 and Rep52 are bound to capsids to roughly the same extent as the minor capsid protein VP2. Complexes of Rep78 and Rep52 with capsids differ in their respective detergent stabilities, indicating that they result from different types of interactions. Rep-VP interaction studies suggest that Rep proteins become stably associated with the capsid during the assembly process. Rep-capsid complexes can reach even higher complexity through additional Rep-Rep interactions, which are particularly detergent labile. Coimmunoprecipitation and yeast two-hybrid data demonstrate the interaction of Rep78 with Rep68, of Rep68 with Rep52, and weak interactions of Rep40 with Rep52 and Rep78. We propose that the large complexes arising from these interactions represent intermediates in the DNA packaging pathway.
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San Martín, Carmen, Juha T. Huiskonen, Jaana K. H. Bamford, Sarah J. Butcher, Stephen D. Fuller, Dennis H. Bamford, and Roger M. Burnett. "Minor proteins, mobile arms and membrane–capsid interactions in the bacteriophage PRD1 capsid." Nature Structural Biology 9, no. 10 (September 9, 2002): 756–63. http://dx.doi.org/10.1038/nsb837.
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Agranovsky, Alexey. "Enhancing Capsid Proteins Capacity in Plant Virus-Vector Interactions and Virus Transmission." Cells 10, no. 1 (January 7, 2021): 90. http://dx.doi.org/10.3390/cells10010090.
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Vector transmission of plant viruses is basically of two types that depend on the virus helper component proteins or the capsid proteins. A number of plant viruses belonging to disparate groups have developed unusual capsid proteins providing for interactions with the vector. Thus, cauliflower mosaic virus, a plant pararetrovirus, employs a virion associated p3 protein, the major capsid protein, and a helper component for the semi-persistent transmission by aphids. Benyviruses encode a capsid protein readthrough domain (CP-RTD) located at one end of the rod-like helical particle, which serves for the virus transmission by soil fungal zoospores. Likewise, the CP-RTD, being a minor component of the luteovirus icosahedral virions, provides for persistent, circulative aphid transmission. Closteroviruses encode several CPs and virion-associated proteins that form the filamentous helical particles and mediate transmission by aphid, whitefly, or mealybug vectors. The variable strategies of transmission and evolutionary ‘inventions’ of the unusual capsid proteins of plant RNA viruses are discussed.
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Agranovsky, Alexey. "Enhancing Capsid Proteins Capacity in Plant Virus-Vector Interactions and Virus Transmission." Cells 10, no. 1 (January 7, 2021): 90. http://dx.doi.org/10.3390/cells10010090.
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Vector transmission of plant viruses is basically of two types that depend on the virus helper component proteins or the capsid proteins. A number of plant viruses belonging to disparate groups have developed unusual capsid proteins providing for interactions with the vector. Thus, cauliflower mosaic virus, a plant pararetrovirus, employs a virion associated p3 protein, the major capsid protein, and a helper component for the semi-persistent transmission by aphids. Benyviruses encode a capsid protein readthrough domain (CP-RTD) located at one end of the rod-like helical particle, which serves for the virus transmission by soil fungal zoospores. Likewise, the CP-RTD, being a minor component of the luteovirus icosahedral virions, provides for persistent, circulative aphid transmission. Closteroviruses encode several CPs and virion-associated proteins that form the filamentous helical particles and mediate transmission by aphid, whitefly, or mealybug vectors. The variable strategies of transmission and evolutionary ‘inventions’ of the unusual capsid proteins of plant RNA viruses are discussed.
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Yamauchi, Yohei, Kaoru Wada, Fumi Goshima, Hiroki Takakuwa, Tohru Daikoku, Masao Yamada, and Yukihiro Nishiyama. "The UL14 protein of herpes simplex virus type 2 translocates the minor capsid protein VP26 and the DNA cleavage and packaging UL33 protein into the nucleus of coexpressing cells." Journal of General Virology 82, no. 2 (February 1, 2001): 321–30. http://dx.doi.org/10.1099/0022-1317-82-2-321.
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The herpes simplex virus type 2 (HSV-2) gene UL14 encodes a 32 kDa protein which is a minor component of the virion tegument and is expressed late in infection. The UL14 protein shows varied localization patterns in HSV-2-infected and singly expressing cells, suggesting the possibility that it is multifunctional. We have investigated the influence of the UL14 protein on the intracellular localization of capsid proteins and DNA cleavage and packaging proteins in coexpressing cells. VP26 is the minor capsid protein; it binds to hexons of the outer capsid shell and is predominantly cytoplasmic upon sole expression. We have found that VP26 coexpressed with the UL14 protein showed mutual and predominant relocation into the nucleus. At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32 and UL33) that are required for DNA cleavage and packaging. We have found that the UL33 protein, which was also cytoplasmic by sole expression, was relocated to the nucleus upon expression with the UL14 protein, which again seemed to be a result of mutual influence. Coexpression experiments also suggested the possibility of a mutual influence between the UL14 and UL17 proteins, and the UL17 protein and VP26. Our results suggest that the UL14 protein can influence the intracellular localization patterns of a number of proteins belonging to the capsid or the DNA encapsidation machinery.
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Haupt, Sophie, Tanya Stroganova, Eugene Ryabov, Sang Hyon Kim, Gill Fraser, George Duncan, Mike A. Mayo, Hugh Barker, and Michael Taliansky. "Nucleolar localization of potato leafroll virus capsid proteins." Journal of General Virology 86, no. 10 (October 1, 2005): 2891–96. http://dx.doi.org/10.1099/vir.0.81101-0.
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Potato leafroll virus (PLRV) encodes two capsid proteins, major protein (CP) and minor protein (P5), an extended version of the CP produced by occasional translational ‘readthrough’ of the CP gene. Immunogold electron microscopy showed that PLRV CP is located in the cytoplasm and also localized in the nucleus, preferentially targeting the nucleolus. The nucleolar localization of PLRV CP was also confirmed when it was expressed as a fusion with green fluorescent protein (GFP) via an Agrobacterium vector. Mutational analysis identified a particular sequence within PLRV CP involved in nucleolar targeting [the nucleolar localization signal (NoLS)]. Minor protein P5 also contains the same NoLS, and was targeted to the nucleolus when it was expressed as a fusion with GFP from Agrobacterium. However, P5–GFP lost its nucleolar localization in the presence of replicating PLRV.
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Florin, Luise, Cornelia Sapp, Rolf E. Streeck, and Martin Sapp. "Assembly and Translocation of Papillomavirus Capsid Proteins." Journal of Virology 76, no. 19 (October 1, 2002): 10009–14. http://dx.doi.org/10.1128/jvi.76.19.10009-10014.2002.
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ABSTRACT The major and minor capsid proteins of polyomavirus are preassembled in the cytoplasm and translocated to the nucleus only as a VP1-VP2/VP3 complex. In this study, we describe independent nuclear translocation of the L1 major protein and the L2 minor capsid protein of human papillomavirus type 33 by several approaches. First, we observed that expression and nuclear translocation of L2 in natural lesions precede expression of L1. Second, using a cell culture system for coexpression, we found that accumulation of L2 in nuclear domain 10 (ND10) subnuclear structures precedes L1 by several hours. In contrast, complexes of L2 and mutants of L1 forced to assemble in the cytoplasm are translocated directly to ND10, like L2 expressed alone. Interestingly, accumulation of wild-type L1 is observed only after L2-induced release of the ND10-associated protein Sp100. Third, nuclear translocation of L2 but not of L1 was blocked by the proteasome inhibitor MG132. Our data suggest that L1 and L2 interaction occurs after L2-induced reorganization of ND10 subnuclear domains.
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Ayalew, Lisanework E., Amit Gaba, Pankaj Kumar, and Suresh K. Tikoo. "Conserved regions of bovine adenovirus-3 pVIII contain functional domains involved in nuclear localization and packaging in mature infectious virions." Journal of General Virology 95, no. 8 (August 1, 2014): 1743–54. http://dx.doi.org/10.1099/vir.0.065763-0.
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Adenoviruses are non-enveloped DNA viruses that replicate in the nucleus of infected cells. One of the core proteins, named pVIII, is a minor capsid protein connecting the core with the inner surface of the capsid. Here, we report the characterization of minor capsid protein pVIII encoded by the L6 region of bovine adenovirus (BAdV)-3. Anti-pVIII serum detected a 24 kDa protein at 12–48 h post-infection and an additional 8 kDa protein at 24–48 h post-infection. While the 24 kDa protein was detected in empty capsids, only the C-terminal-cleaved 8 kDa protein was detected in the mature virion, suggesting that amino acids147–216 of the conserved C-terminus of BAdV-3 pVIII are incorporated in mature virions. Detection of hexon protein associated with both precursor (24 kDa) and cleaved (8 kDa) forms of pVIII suggest that the C-terminus of pVIII interacts with the hexon. The pVIII protein predominantly localizes to the nucleus of BAdV-3-infected cells utilizing the classical importin α/β dependent nuclear import pathway. Analysis of mutant pVIII demonstrated that amino acids 52–72 of the conserved N-terminus bind to importin α-3 with high affinity and are required for the nuclear localization.
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Albright, Brandon S., Athena Kosinski, Renata Szczepaniak, Elizabeth A. Cook, Nigel D. Stow, James F. Conway, and Sandra K. Weller. "The Putative Herpes Simplex Virus 1 Chaperone Protein UL32 Modulates Disulfide Bond Formation during Infection." Journal of Virology 89, no. 1 (October 15, 2014): 443–53. http://dx.doi.org/10.1128/jvi.01913-14.
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ABSTRACTDuring DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.IMPORTANCEAlthough functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.
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Buck, Christopher B., Naiqian Cheng, Cynthia D. Thompson, Douglas R. Lowy, Alasdair C. Steven, John T. Schiller, and Benes L. Trus. "Arrangement of L2 within the Papillomavirus Capsid." Journal of Virology 82, no. 11 (March 26, 2008): 5190–97. http://dx.doi.org/10.1128/jvi.02726-07.
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ABSTRACT Papillomaviruses are a family of nonenveloped DNA tumor viruses. Some sexually transmitted human papillomavirus (HPV) types, including HPV type 16 (HPV16), cause cancer of the uterine cervix. Papillomaviruses encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein, L2, is not required for capsid formation, it is thought to participate in encapsidation of the viral genome and plays a number of essential roles in the viral infectious entry pathway. The abundance of L2 and its arrangement within the virion remain unclear. To address these questions, we developed methods for serial propagation of infectious HPV16 capsids (pseudoviruses) in cultured human cell lines. Biochemical analysis of capsid preparations produced using various methods showed that up to 72 molecules of L2 can be incorporated per capsid. Cryoelectron microscopy and image reconstruction analysis of purified capsids revealed an icosahedrally ordered L2-specific density beneath the axial lumen of each L1 capsomer. The relatively close proximity of these L2 density buttons to one another raised the possibility of homotypic L2 interactions within assembled virions. The concept that the N and C termini of neighboring L2 molecules can be closely apposed within the capsid was supported using bimolecular fluorescence complementation or “split GFP” technology. This structural information should facilitate investigation of L2 function during the assembly and entry phases of the papillomavirus life cycle.
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El Mehdaoui, Slimane, Antoine Touzé, Sylvie Laurent, Pierre-Yves Sizaret, Denis Rasschaert, and Pierre Coursaget. "Gene Transfer Using Recombinant Rabbit Hemorrhagic Disease Virus Capsids with Genetically Modified DNA Encapsidation Capacity by Addition of Packaging Sequences from the L1 or L2 Protein of Human Papillomavirus Type 16." Journal of Virology 74, no. 22 (November 15, 2000): 10332–40. http://dx.doi.org/10.1128/jvi.74.22.10332-10340.2000.
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ABSTRACT The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.
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Johnson, Karyn N., and L. Andrew Ball. "Virions of Pariacoto virus contain a minor protein translated from the second AUG codon of the capsid protein open reading frame." Journal of General Virology 84, no. 10 (October 1, 2003): 2847–52. http://dx.doi.org/10.1099/vir.0.19419-0.
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Virions of the alphanodavirus Pariacoto virus (PaV) have T=3 icosahedral symmetry and are assembled from multiple copies of a precursor protein that is cleaved into two mature capsid proteins after assembly. The crystal structure of PaV shows that the N-terminal ∼30 amino acid residues of the subunits surrounding the 5-fold axes interact extensively with icosahedrally ordered regions of the encapsidated positive-sense genomic RNAs. We found that wild-type PaV particles also contain a minor capsid protein that is truncated by 24 residues at its N terminus. Reverse genetic experiments showed that translation of this protein initiated at the second AUG of the capsid protein open reading frame. When either the longer or shorter version of the capsid protein was expressed independently of the other, it assembled into virus particles and underwent maturational cleavage. Virions that lacked the shorter capsid protein retained infectivity for cultured insect cells and Galleria mellonella larvae.
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Pasdeloup, David, Danielle Blondel, Anabela L. Isidro, and Frazer J. Rixon. "Herpesvirus Capsid Association with the Nuclear Pore Complex and Viral DNA Release Involve the Nucleoporin CAN/Nup214 and the Capsid Protein pUL25." Journal of Virology 83, no. 13 (April 22, 2009): 6610–23. http://dx.doi.org/10.1128/jvi.02655-08.
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ABSTRACT After penetrating the host cell, the herpesvirus capsid is transported to the nucleus along the microtubule network and docks to the nuclear pore complex before releasing the viral DNA into the nucleus. The viral and cellular interactions involved in the docking process are poorly characterized. However, the minor capsid protein pUL25 has recently been reported to be involved in viral DNA uncoating. Here we show that herpes simplex virus type 1 (HSV-1) capsids interact with the nucleoporin CAN/Nup214 in infected cells and that RNA silencing of CAN/Nup214 delays the onset of viral DNA replication in the nucleus. We also show that pUL25 interacts with CAN/Nup214 and another nucleoporin, hCG1, and binds to the pUL36 and pUL6 proteins, two other components of the herpesvirus particle that are known to be important for the initiation of infection and viral DNA release. These results identify CAN/Nup214 as being a nuclear receptor for the herpesvirus capsid and pUL25 as being an interface between incoming capsids and the nuclear pore complex and as being a triggering element for viral DNA release into the nucleus.
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Chen, Liu, Guangqing Liu, Zheng Ni, Bin Yu, Tao Yun, Yi Song, Jionggang Hua, Shuangmao Li, and Jianping Chen. "Minor structural protein VP2 in rabbit hemorrhagic disease virus downregulates the expression of the viral capsid protein VP60." Journal of General Virology 90, no. 12 (December 1, 2009): 2952–55. http://dx.doi.org/10.1099/vir.0.015081-0.
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Rabbit hemorrhagic disease virus (RHDV) has two structural proteins: the major capsid protein VP60 and the minor capsid protein VP2. VP2 is speculated to play an important role in the virus life cycle. To investigate the effect of VP2 on VP60 expression, three types of experiment (baculovirus–insect cell system, mammalian–luciferase assay system and in vitro coupled transcription/translation system) were used to express VP60 alone or co-expressed with VP2. Both forms of VP60 were able to form virus-like particles in insect cells. Western blot analysis and dual-luciferase assays demonstrated that the presence of VP2 results in downregulation of the expression of VP60 in vivo. Real-time RT-PCR of mRNA levels showed that downregulation of VP60 occurs at the transcriptional level. The ability of the viral minor structural protein VP2 to regulate capsid protein levels may contribute to effective virus infection.
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Nakanishi, Akira, Akiko Nakamura, Robert Liddington, and Harumi Kasamatsu. "Identification of Amino Acid Residues within Simian Virus 40 Capsid Proteins Vp1, Vp2, and Vp3 That Are Required for Their Interaction and for Viral Infection." Journal of Virology 80, no. 18 (September 15, 2006): 8891–98. http://dx.doi.org/10.1128/jvi.00781-06.
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ABSTRACT Interaction of simian virus 40 (SV40) major capsid protein Vp1 with the minor capsid proteins Vp2 and Vp3 is an integral aspect of the SV40 architecture. Two Vp3 sequence elements mediate Vp1 pentamer binding in vitro, Vp3 residues 155 to 190, or D1, and Vp3 residues 222 to 234, or D2. Of the two, D1 but not D2 was necessary and sufficient to direct the interaction with Vp1 in vivo. Rational mutagenesis of Vp3 residues (Phe157, Ile158, Pro164, Gly165, Gly166, Leu177, and Leu181) or Vp1 residues (Val243 and Leu245), based on a structural model of the SV40 Vp1 pentamer complexed with Vp3 D1, was carried out to disrupt the interaction between Vp1 and Vp3 and to study the consequences of these mutations for viral viability. Altering these residues to bulky, charged residues blocked the interaction in vitro. When these alterations were introduced into the viral genome, they reduced viral viability. Mutants with alterations in Vp1 Val243, Leu245, or both to glutamate were nearly nonviable, whereas those with Vp3 alterations reduced, but did not eliminate, viability. Our results defined the residues of Vp1 and the minor capsid proteins that are essential for both the interaction of the capsid proteins and viral viability in permissive cells.
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Reddy, Vijay, and Glen Nemerow. "Structure and function of cement proteins in human adenovirus." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1603. http://dx.doi.org/10.1107/s205327331408396x.
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Human adenoviruses (HAdVs) are large (~150nm in diameter, 150MDa) nonenveloped double-stranded DNA (dsDNA) viruses that cause respiratory, ocular, and enteric diseases. The capsid shell of adenovirus (Ad) comprises multiple copies of three major capsid proteins (MCP: hexon, penton base and fiber) and four minor/cement proteins (IIIa, VI, VIII and IX) that are organized with pseudo T=25 icosahedral symmetry. In addition, six other proteins (V, VII, μ, IVa2, terminal protein and protease) are encapsidated along with the 36Kb dsDNA genome inside the capsid. The crystal structures of all three MCPs are known and so is their organization in the capsid from prior X-ray crystallography and cryoEM analyses. However structures and locations of various cement proteins are of considerable debate. We have determined and refined the structure of an entire human adenovirus employing X-ray crystallpgraphic methods at 3.8Å resolution. Adenovirus cement proteins play crucial roles in virion assembly, disassembly, cell entry and infection. Based on the refined crystal structure of adenovirus, we have determined the structure of the cement protein VI, a key membrane-lytic molecule and its associations with proteins V and VIII, which together glue peripentonal hexons beneath vertex region and connect them to rest of the capsid. Following virion maturation, the cleaved N-terminal pro-peptide of VI is observed deep in the peripentonal hexon cavity, detached from the membrane-lytic domain. Furthermore, we have significantly revised the recent cryoEM models for proteins IIIa and IX and both are located on the capsid exterior. Together, the cement proteins exclusively stabilize the hexon shell, thus rendering penton vertices the weakest links of the adenovirus capsid. Adenovirus cement protein structures reveal the molecular basis of the maturation cleavage of VI that is needed for endosome rupture and delivery of the virion into cytoplasm.
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Nelson, Christian D. S., Luisa J. Ströh, Gretchen V. Gee, Bethany A. O'Hara, Thilo Stehle, and Walter J. Atwood. "Modulation of a Pore in the Capsid of JC Polyomavirus Reduces Infectivity and Prevents Exposure of the Minor Capsid Proteins." Journal of Virology 89, no. 7 (January 21, 2015): 3910–21. http://dx.doi.org/10.1128/jvi.00089-15.
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ABSTRACTJC polyomavirus (JCPyV) infection of immunocompromised individuals results in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). The viral capsid of JCPyV is composed primarily of the major capsid protein virus protein 1 (VP1), and pentameric arrangement of VP1 monomers results in the formation of a pore at the 5-fold axis of symmetry. While the presence of this pore is conserved among polyomaviruses, its functional role in infection or assembly is unknown. Here, we investigate the role of the 5-fold pore in assembly and infection of JCPyV by generating a panel of mutant viruses containing amino acid substitutions of the residues lining this pore. Multicycle growth assays demonstrated that the fitness of all mutants was reduced compared to that of the wild-type virus. Bacterial expression of VP1 pentamers containing substitutions to residues lining the 5-fold pore did not affect pentamer assembly or prevent association with the VP2 minor capsid protein. The X-ray crystal structures of selected pore mutants contained subtle changes to the 5-fold pore, and no other changes to VP1 were observed. Pore mutant pseudoviruses were not deficient in assembly, packaging of the minor capsid proteins, or binding to cells or in transport to the host cell endoplasmic reticulum. Instead, these mutant viruses were unable to expose VP2 upon arrival to the endoplasmic reticulum, a step that is critical for infection. This study demonstrated that the 5-fold pore is an important structural feature of JCPyV and that minor modifications to this structure have significant impacts on infectious entry.IMPORTANCEJCPyV is an important human pathogen that causes a severe neurological disease in immunocompromised individuals. While the high-resolution X-ray structure of the major capsid protein of JCPyV has been solved, the importance of a major structural feature of the capsid, the 5-fold pore, remains poorly understood. This pore is conserved across polyomaviruses and suggests either that these viruses have limited structural plasticity in this region or that this pore is important in infection or assembly. Using a structure-guided mutational approach, we showed that modulation of this pore severely inhibits JCPyV infection. These mutants do not appear deficient in assembly or early steps in infectious entry and are instead reduced in their ability to expose a minor capsid protein in the host cell endoplasmic reticulum. Our work demonstrates that the 5-fold pore is an important structural feature for JCPyV.
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Ogasawara, Masahiro, Tatsuo Suzutani, Itsuro Yoshida, and Masanobu Azuma. "Role of the UL25 Gene Product in Packaging DNA into the Herpes Simplex Virus Capsid: Location of UL25 Product in the Capsid and Demonstration that It Binds DNA." Journal of Virology 75, no. 3 (February 1, 2001): 1427–36. http://dx.doi.org/10.1128/jvi.75.3.1427-1436.2001.
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ABSTRACT Recent studies have suggested that the herpes simplex type 1 (HSV-1) UL25 gene product, a minor capsid protein, is required for encapsidation but not cleavage of replicated viral DNA. This study set out to investigate the potential interactions of UL25 protein with other virus proteins and determine what properties it has for playing a role in DNA encapsidation. The UL25 protein is found in 42 ± 17 copies per B capsid and is present in both pentons and hexons. We introduced green fluorescent protein (GFP) as a fluorescent tag into the N terminus of UL25 protein to identify its location in HSV-1-infected cells and demonstrated the relocation of UL25 protein from the cytoplasm into the nucleus at the late stage of HSV-1 infection. To clarify the cause of this relocation, we analyzed the interactions of UL25 protein with other virus proteins. The UL25 protein associates with VP5 and VP19C of virus capsids, especially of the penton structures, and the association with VP19C causes its relocation into the nucleus. Gel mobility shift analysis shows that UL25 protein has the potential to bind DNA. Moreover, the amino-terminal one-third of the UL25 protein is particularly important in DNA binding and forms a homo-oligomer. In conclusion, the UL25 gene product forms a tight connection with the capsid being linked with VP5 and VP19C, and it may play a role in anchoring the genomic DNA.
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Bissett, Sara L., Anna Godi, Maxime J. J. Fleury, Antoine Touze, Clementina Cocuzza, and Simon Beddows. "Naturally Occurring Capsid Protein Variants of Human Papillomavirus Genotype 31 Represent a Single L1 Serotype." Journal of Virology 89, no. 15 (May 20, 2015): 7748–57. http://dx.doi.org/10.1128/jvi.00842-15.
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ABSTRACTWe investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of oncogenic human papillomavirus (HPV) genotype 31 (HPV31) to determine the impact on capsid antigenicity. L1L2 pseudoviruses (PsVs) representing the three HPV31 variant lineages, variant lineages A, B, and C, exhibited comparable particle-to-infectivity ratios and morphologies. Lineage-specific L1L2 PsVs demonstrated subtle differences in susceptibility to neutralization by antibodies elicited following vaccination or preclinical L1 virus-like particle (VLP) immunization or by monoclonal antibodies; however, these differences were generally of a low magnitude. These data indicate that the diagnostic lineage-specific single nucleotide polymorphisms within the HPV31 capsid genes have a limited effect on L1 antibody-mediated neutralization and that the three HPV31 variant lineages belong to a single L1 serotype. These data contribute to our understanding of HPV L1 variant antigenicity.IMPORTANCEThe virus coat (capsid) of the human papillomavirus contains major (L1) and minor (L2) capsid proteins. These proteins facilitate host cell attachment and viral infectivity and are the targets for antibodies which interfere with these events. In this study, we investigated the impact of naturally occurring variation within these proteins upon susceptibility to viral neutralization by antibodies induced by L1 VLP immunization. We demonstrate that HPV31 L1 and L2 variants exhibit similar susceptibility to antibody-mediated neutralization and that for the purposes of L1 VLP-based vaccines, these variant lineages represent a single serotype.
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Condezo, Gabriela N., Roberto Marabini, Silvia Ayora, José M. Carazo, Raúl Alba, Miguel Chillón, and Carmen San Martín. "Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k." Journal of Virology 89, no. 18 (July 15, 2015): 9653–64. http://dx.doi.org/10.1128/jvi.01453-15.
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ABSTRACTAdenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating.IMPORTANCEAdenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the molecular details of capsid assembly. Here, we provide evidence supporting one of the two current models for capsid architecture. We also show for the first time the location of the packaging protein L1 52/55k in particles lacking the virus genome and how this location changes during maturation. Our results contribute to clarifying standing questions in adenovirus capsid architecture and provide new details on the role of L1 52/55k protein in assembly.
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Mannová, Petra, David Liebl, Nina Krauzewicz, Anna Fejtová, Jitka Štokrová, Zdena Palková, Beverly E. Griffin, and Jitka Forstová. "Analysis of mouse polyomavirus mutants with lesions in the minor capsid proteins." Journal of General Virology 83, no. 9 (September 1, 2002): 2309–19. http://dx.doi.org/10.1099/0022-1317-83-9-2309.
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Polyomavirus mutants E, Q and H, expressing non-myristylated VP2, were generated by replacing the N-terminal glycine residue with glutamic acid, glutamine or histidine, respectively. Viruses mutated in either VP2 or VP3 translation initiation codons were also prepared. All mutated genomes, when transfected into murine host cells, gave rise to viral particles. Infectivity of VP2− and VP3− viruses, as measured by the number of cells expressing viral antigens, was dramatically diminished, indicative of defects in the early stages of infection. In contrast, the absence of a myristyl moiety on VP2 did not substantially affect the early steps of virus infection. No differences in numbers of cells expressing early or late viral antigens were observed between wild-type (wt) and E or Q myr− viruses during the course of a life cycle. Furthermore, no delay in virus DNA replication was detected. However, when cells were left for longer in culture, the number of infected cells, measured by typical virus bursts, was much lower when mutant rather than wt genomes were used. In situ, cell fractionation studies revealed differences in the interaction of viral particles with host cell structures. The infectivity of mutants was affected not only by loss of the myristyl group on VP2, but also, and to a greater extent, by alterations of the N-terminal amino acid composition.
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O'Hara, Maureen, Frazer J. Rixon, Nigel D. Stow, Jill Murray, Mary Murphy, and Valerie G. Preston. "Mutational Analysis of the Herpes Simplex Virus Type 1 UL25 DNA Packaging Protein Reveals Regions That Are Important after the Viral DNA Has Been Packaged." Journal of Virology 84, no. 9 (February 24, 2010): 4252–63. http://dx.doi.org/10.1128/jvi.02442-09.
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ABSTRACT The herpes simplex virus type 1 (HSV-1) UL25 gene encodes a minor capsid protein, pUL25, that is essential for packaging the full-length viral genome. Six regions which contain disordered residues have been identified in the high-resolution structure of pUL25. To investigate the significance of these flexible regions, a panel of plasmids was generated encoding mutant proteins, with each member lacking the disordered residues in one of the six regions. In addition, UL25 constructs were produced, which specified proteins that contained missense mutations individually affecting two of the four regions on the surface of pUL25 predicted from evolutionary trace analysis to be important in protein-protein interactions. The impacts of these mutations on viral DNA packaging, virus assembly, and growth were examined. Of the nine mutant proteins analyzed, five failed to complement the growth of a UL25 deletion mutant in Vero cells. These noncomplementing proteins fell into three classes. Proteins in one class did not alter the DNA packaging phenotype of an HSV-1 UL25 deletion mutant, whereas proteins from the other two classes allowed the UL25 null mutant to package full-length viral DNA. Subsequent analysis of the latter classes of mutant proteins demonstrated that one class enabled the null virus to release enveloped virus particles from U2OS cells, whereas the other class prevented egress of mature HSV-1 capsids from the nucleus. These findings reveal a new role for pUL25 in virion assembly, consistent with its flexible structure and location on the capsid.
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Gordon-Shaag, Ariela, Yael Yosef, Mahmoud Abd El-Latif, and Ariella Oppenheim. "The Abundant Nuclear Enzyme PARP Participates in the Life Cycle of Simian Virus 40 and Is Stimulated by Minor Capsid Protein VP3." Journal of Virology 77, no. 7 (April 1, 2003): 4273–82. http://dx.doi.org/10.1128/jvi.77.7.4273-4282.2003.
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ABSTRACT The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) functions in DNA damage surveillance and repair and at the decision between apoptosis and necrosis. Here we show that PARP binds to simian virus 40 (SV40) capsid proteins VP1 and VP3. Furthermore, its enzymatic activity is stimulated by VP3 but not by VP1. Experiments with purified mutant proteins demonstrated that the PARP binding domain in VP3 is localized to the 35 carboxy-terminal amino acids, while a larger peptide of 49 amino acids was required for full stimulation of its activity. The addition of 3-aminobenzamide (3-AB), a known competitive inhibitor of PARP, demonstrated that PARP participates in the SV40 life cycle. The titer of SV40 propagated on CV-1 cells was reduced by 3-AB in a dose-dependent manner. Additional experiments showed that 3-AB did not affect viral DNA replication or capsid protein production. PARP did not modify the viral capsid proteins in in vitro poly(ADP-ribosylation) assays, implying that it does not affect SV40 infectivity. On the other hand, it greatly reduced the magnitude of the host cytopathic effects, a hallmark of SV40 infection. Additional experiments suggested that the stimulation of PARP activity by VP3 leads the infected cell to a necrotic pathway, characterized by the loss of membrane integrity, thus facilitating the release of mature SV40 virions from the cells. Our studies identified a novel function of the minor capsid protein VP3 in the recruitment of PARP for the SV40 lytic process.
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Vlasak, Marketa, Merja Roivainen, Manuela Reithmayer, Irene Goesler, Pia Laine, Luc Snyers, Tapani Hovi, and Dieter Blaas. "The Minor Receptor Group of Human Rhinovirus (HRV) Includes HRV23 and HRV25, but the Presence of a Lysine in the VP1 HI Loop Is Not Sufficient for Receptor Binding." Journal of Virology 79, no. 12 (June 15, 2005): 7389–95. http://dx.doi.org/10.1128/jvi.79.12.7389-7395.2005.
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ABSTRACT Like all 10 minor receptor group human rhinoviruses (HRVs), HRV23 and HRV25, previously classified as major group viruses, are neutralized by maltose binding protein (MBP)-V33333 (a soluble recombinant concatemer of five copies of repeat 3 of the very-low-density lipoprotein receptor fused to MBP), bind to low-density lipoprotein receptor in virus overlay blots, and replicate in intercellular adhesion molecule 1 (ICAM-1)-negative COS-7 cells. From phylogenetic analysis of capsid protein VP1-coding sequences, they are also known to cluster together with other minor group strains. Therefore, they belong to the minor group; there are now 12 minor group and 87 major group HRV serotypes. Sequence comparison of the VP1 capsid proteins of all HRVs revealed that the lysine in the HI loop, strictly conserved in the 12 minor group HRVs, is also present in 9 major group serotypes that are neutralized by soluble ICAM-1. Despite the presence of this lysine, they are not neutralized by MBP-V33333 and fail to replicate in COS-7 cells and in HeLa cells in the presence of an ICAM-1-blocking antibody. These nine serotypes are therefore “true” major group viruses.
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Tellinghuisen, Timothy L., and Richard J. Kuhn. "Nucleic Acid-Dependent Cross-Linking of the Nucleocapsid Protein of Sindbis Virus." Journal of Virology 74, no. 9 (May 1, 2000): 4302–9. http://dx.doi.org/10.1128/jvi.74.9.4302-4309.2000.
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ABSTRACT The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway.
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Fan, Wan H., Ashley P. E. Roberts, Marion McElwee, David Bhella, Frazer J. Rixon, and Rebecca Lauder. "The Large Tegument Protein pUL36 Is Essential for Formation of the Capsid Vertex-Specific Component at the Capsid-Tegument Interface of Herpes Simplex Virus 1." Journal of Virology 89, no. 3 (November 19, 2014): 1502–11. http://dx.doi.org/10.1128/jvi.02887-14.
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ABSTRACTHerpesviruses have a characteristic particle structure comprising an icosahedral capsid, which contains the DNA genome and is, in turn, surrounded by a proteinaceous tegument layer and a lipid envelope. In herpes simplex virus, the interaction between the capsid and tegument is limited to the capsid vertices and involves two minor capsid proteins, pUL17 and pUL25, and the large inner tegument protein pUL36. pUL17 and pUL25 form a heterodimeric structure, the capsid vertex-specific component (CVSC), that lies on top of the peripentonal triplexes, while pUL36 has been reported to connect the CVSC to the penton. In this study, we used virus mutants with deletions in the genes for pUL36 and another inner tegument protein, pUL37, to analyze the contributions of these proteins to CVSC structure. Using electron cryomicroscopy and icosahedral reconstruction of mutants that express pUL17 and pUL25 but not pUL36, we showed that in contrast to accepted models, the CVSC is not formed from pUL17 and pUL25 on their own but requires a contribution from pUL36. In addition, the presence of full-length pUL36 results in weak density that extends the CVSC toward the penton, suggesting either that this extra density is formed directly by pUL36 or that pUL36 stabilizes other components of the vertex-tegument interface.IMPORTANCEHerpesviruses have complex particles that are formed as a result of a carefully controlled sequence of assembly steps. The nature of the interaction between two of the major particle compartments, the icosahedral capsid and the amorphous tegument, has been extensively studied, but the identity of the interacting proteins and their roles in forming the connections are still unclear. In this study, we used electron microscopy and three-dimensional reconstruction to analyze virus particles formed by mutants that do not express particular interacting proteins. We show that the largest viral protein, pUL36, which occupies the layer of tegument closest to the capsid, is essential for formation of structurally normal connections to the capsid. This demonstrates the importance of pUL36 in the initial stages of tegument addition and provides new insights into the process of virus particle assembly.
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Wild, Peter, Monika Engels, Claudia Senn, Kurt Tobler, Urs Ziegler, Elisabeth M. Schraner, Eva Loepfe, Mathias Ackermann, Martin Mueller, and Paul Walther. "Impairment of Nuclear Pores in Bovine Herpesvirus 1-Infected MDBK Cells." Journal of Virology 79, no. 2 (January 15, 2005): 1071–83. http://dx.doi.org/10.1128/jvi.79.2.1071-1083.2005.
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ABSTRACT Herpesvirus capsids originating in the nucleus overcome the nucleocytoplasmic barrier by budding at the inner nuclear membrane. The fate of the resulting virions is still under debate. The fact that capsids approach Golgi membranes from the cytoplasmic side led to the theory of fusion between the viral envelope and the outer nuclear membrane, resulting in the release of capsids into the cytoplasm. We recently discovered a continuum from the perinuclear space to the Golgi complex implying (i) intracisternal viral transportation from the perinuclear space directly into Golgi cisternae and (ii) the existence of an alternative pathway of capsids from the nucleus to the cytoplasm. Here, we analyzed the nuclear surface by high-resolution microscopy. Confocal microscopy of MDBK cells infected with recombinant bovine herpesvirus 1 expressing green fluorescent protein fused to VP26 (a minor capsid protein) revealed distortions of the nuclear surface in the course of viral multiplication. High-resolution scanning and transmission electron microscopy proved the distortions to be related to enlargement of nuclear pores through which nuclear content including capsids protrudes into the cytoplasm, suggesting that capsids use impaired nuclear pores as gateways to gain access to the cytoplasmic matrix. Close examination of Golgi membranes, rough endoplasmic reticulum, and outer nuclear membrane yielded capsid-membrane interaction of high identity to the budding process at the inner nuclear membrane. These observations signify the ability of capsids to induce budding at any cell membrane, provided the fusion machinery is present and/or budding is not suppressed by viral proteins.
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Embers, Monica E., Lynn R. Budgeon, Timothy D. Culp, Cynthia A. Reed, Martin D. Pickel, and Neil D. Christensen. "Differential antibody responses to a distinct region of human papillomavirus minor capsid proteins." Vaccine 22, no. 5-6 (January 2004): 670–80. http://dx.doi.org/10.1016/j.vaccine.2003.08.037.
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Booy, F. P., W. W. Newcomb, J. C. Brown, and A. C. Steven. "Herpesvirus Nucleocapsids Imaged in the Frozen-Hydrated State." Proceedings, annual meeting, Electron Microscopy Society of America 46 (1988): 164–65. http://dx.doi.org/10.1017/s0424820100102900.
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Herpesviruses are an extensive family of double-stranded DNA-containing animal viruses. The typical herpes-virion consists of a loosely-fitting envelope derived from the nuclear membrane of the host cell, within which a proteinaceous "tegument" layer encloses an icosahedral nucleocapsid. The capsid is a thick-walled shell of triangulation class T=16, whose major constituent is a protein of monomer mass ∽ 155kDa. Hexamers of this protein occupy the hexavalent sites of the icosahedral surface lattice. The nucleocapsid also contains several minor proteins and the linear genome of 80-150 MDaDespite investigations extending over more than 20 years, many basic questions concerning the HSV nucleocapsid structure remain unanswered; e.g. where are the minor proteins located? and what is the structure and chemical identity of the vertex capsomers? Moreover, although several models have been proposed for the packing of DNA inside the capsid, further, more detailed, evidence is needed to settle this issue. Conventional electron microscopic approaches having been constrained by difficulties in preserving and conveying the native structures of intact nucleocapsids, we have started to explore the potentialities of cryo-electron microscopy for this system.
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Preston, Valerie G., and Iris M. McDougall. "Regions of the Herpes Simplex Virus Scaffolding Protein That Are Important for Intermolecular Self-Interaction." Journal of Virology 76, no. 2 (January 15, 2002): 673–87. http://dx.doi.org/10.1128/jvi.76.2.673-687.2002.
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ABSTRACT The herpes simplex virus type 1 (HSV-1) scaffolding protein encoded by gene UL26.5 promotes the formation of the icosahedral capsid shell through its association with the major capsid protein VP5 and through intermolecular interactions with itself. Inside the capsid shell, the UL26.5 product together with the maturational protease, a minor protein, form a spherical structure which is broken down and released from the capsid during packaging of the viral genome. Selected residues from four internal regions of the HSV-1 scaffolding protein that have significant conservation of amino acids within the scaffolding proteins of alphaherpesviruses were mutated, and the properties of the proteins were examined. Only the HSV-1 scaffolding protein with mutations in the conserved N-terminal domain showed reduced interaction with the varicella-zoster virus homologue in a cell-based immunofluorescence assay, providing the first evidence that this domain in the HSV-1 protein is likely to be involved in intermolecular self-interaction. Scaffolding protein with mutations in this domain or in either of two other domains failed to assemble into scaffold-like particles but retained the ability to self-interact, although the aggregates were significant smaller than most of the aggregates formed by the wild-type protein. These results suggest that there are multiple domains involved in the intermolecular self-association of the HSV-1 scaffolding protein that can act independently of one another. This conclusion was supported by the observation that none of the mutant proteins with lesions in an individual domain, including a protein with mutations in a central region previously implicated in self-interaction (A. Pelletier, F. Dô, J. J. Brisebois, L. Lagacé, and M. G. Cordingley, J. Virol. 71:5197–5208, 1997), interfered with capsid assembly in a baculovirus expression system. A protein mutated in the central region and another conserved domain, both of which had been predicted to form coiled coils, was impaired for capsid formation but still retained the capacity to interact with VP5.
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Shishido-Hara, Yukiko, Shizuko Ichinose, Kayoko Higuchi, Yoshinobu Hara, and Kotaro Yasui. "Major and Minor Capsid Proteins of Human Polyomavirus JC Cooperatively Accumulate to Nuclear Domain 10 for Assembly into Virions." Journal of Virology 78, no. 18 (September 15, 2004): 9890–903. http://dx.doi.org/10.1128/jvi.78.18.9890-9903.2004.
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ABSTRACT The human polyomavirus JC (JCV) replicates in the nuclei of infected cells. Here we report that JCV virions are efficiently assembled at nuclear domain 10 (ND10), which is also known as promyelocytic leukemia (PML) nuclear bodies. The major capsid protein VP1, the minor capsid proteins VP2 and VP3, and a regulatory protein called agnoprotein were coexpressed from a polycistronic expression vector in COS-7 cells. We found that VP1 accumulated to distinct subnuclear domains in the presence of VP2/VP3 and agnoprotein, while VP1 expressed alone was distributed both in the cytoplasm and in the nucleus. Mutation analysis revealed that discrete intranuclear accumulation of VP1 requires the presence of either VP2 or VP3. However, VP2 or VP3 expressed in the absence of VP1 showed diffuse, not discrete, nuclear localization. The C-terminal sequence of VP2/VP3 contains two basic regions, GPNKKKRRK (cluster 1) and KRRSRSSRS (cluster 2). The deletion of cluster 2 abolished the accumulation of VP1 to distinct subnuclear domains. Deletion of the C-terminal 34 residues of VP2/VP3, including both cluster 1 and cluster 2, caused VP1 to localize both in the cytoplasm and in the nucleus. Using immunoelectron microscopy of cells that coexpressed VP1, VP2/VP3, and agnoprotein, we detected the assembly of virus-like particles in discrete locations along the inner nuclear periphery. Both in oligodendrocytes of the human brain and in transfected cells, discrete nuclear domains for VP1 accumulation were identified as ND10, which contains the PML protein. These results indicate that major and minor capsid proteins cooperatively accumulate in ND10, where they are efficiently assembled into virions.
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Bossis, Ioannis, Richard B. S. Roden, Ratish Gambhira, Rongcun Yang, Mitsuo Tagaya, Peter M. Howley, and Patricio I. Meneses. "Interaction of tSNARE Syntaxin 18 with the Papillomavirus Minor Capsid Protein Mediates Infection." Journal of Virology 79, no. 11 (June 1, 2005): 6723–31. http://dx.doi.org/10.1128/jvi.79.11.6723-6731.2005.
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ABSTRACT The papillomavirus capsid mediates binding to the cell surface and passage of the virion to the perinuclear region during infection. To better understand how the virus traffics across the cell, we sought to identify cellular proteins that bind to the minor capsid protein L2. We have identified syntaxin 18 as a protein that interacts with bovine papillomavirus type 1 (BPV1) L2. Syntaxin 18 is a target membrane-associated soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (tSNARE) that resides in the endoplasmic reticulum (ER). The ectopic expression of FLAG-tagged syntaxin 18, which disrupts ER trafficking, blocked BPV1 pseudovirion infection. Furthermore, the expression of FLAG-syntaxin 18 prevented the passage of BPV1 pseudovirions to the perinuclear region that is consistent with the ER. Genetic studies identified a highly conserved L2 domain, DKILK, comprising residues 40 to 44 that mediated BPV1 trafficking through the ER during infection via an interaction with the tSNARE syntaxin 18. Mutations within the DKILK motif of L2 that did not significantly impact virion morphogenesis or binding at the cell surface prevented the L2 interaction with syntaxin 18 and disrupted BPV1 infection.
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Zheng, Honghong, Li Yu, Chunhong Wei, Dongwei Hu, Yunping Shen, Zhangliang Chen, and Yi Li. "Assembly of Double-Shelled, Virus-Like Particles in Transgenic Rice Plants Expressing Two Major Structural Proteins of Rice Dwarf Virus." Journal of Virology 74, no. 20 (October 15, 2000): 9808–10. http://dx.doi.org/10.1128/jvi.74.20.9808-9810.2000.
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ABSTRACT Rice dwarf virus (RDV) is a double-shelled particle that contains a major capsid protein (P8), a major core protein (P3), several minor core proteins, and viral genomic double-stranded RNA. Coexpression of P8 and P3 in transgenic rice plants resulted in formation of double-shelled, virus-like particles (VLPs) similar to the authentic RDV particles. The VLPs were not detected in transgenic rice plant cells expressing P8 alone. This in vivo result suggests that P8 interacted with P3 and that these two proteins provide the structural integrity required for the formation of VLPs in rice cells independently of other structural proteins, nonstructural proteins, or viral genomic double-stranded RNAs.
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Breiner, Bastian, Laura Preuss, Nora Roos, Marcel Conrady, Hauke Lilie, Thomas Iftner, and Claudia Simon. "Refolding and in vitro characterization of human papillomavirus 16 minor capsid protein L2." Biological Chemistry 400, no. 4 (April 24, 2019): 513–22. http://dx.doi.org/10.1515/hsz-2018-0311.
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Abstract The minor capsid protein L2 of papillomaviruses exhibits multiple functions during viral entry including membrane interaction. Information on the protein is scarce, because of its high tendency of aggregation. We determined suitable conditions to produce a functional human papillomavirus (HPV) 16 L2 protein and thereby provide the opportunity for extensive in vitro analysis with respect to structural and biochemical information on L2 proteins and mechanistic details in viral entry. We produced the L2 protein of high-risk HPV 16 in Escherichia coli as inclusion bodies and purified the protein under denaturing conditions. A successive buffer screen resulted in suitable conditions for the biophysical characterization of 16L2. Analytical ultracentrifugation of the refolded protein showed a homogenous monomeric species. Furthermore, refolded 16L2 shows secondary structure elements. The N-terminal region including the proposed transmembrane region of 16L2 shows alpha-helical characteristics. However, overall 16L2 appears largely unstructured. Refolded 16L2 is capable of binding to DNA indicating that the putative DNA-binding regions are accessible in refolded 16L2. Further the refolded protein interacts with liposomal membranes presumably via the proposed transmembrane region at neutral pH without structural changes. This indicates that 16L2 can initially interact with membranes via pre-existing structural features.
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Marabini, Roberto, Gabriela N. Condezo, Mart Krupovic, Rosa Menéndez-Conejero, Josué Gómez-Blanco, and Carmen San Martín. "Near-atomic structure of an atadenovirus reveals a conserved capsid-binding motif and intergenera variations in cementing proteins." Science Advances 7, no. 14 (March 2021): eabe6008. http://dx.doi.org/10.1126/sciadv.abe6008.
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Of five known adenovirus genera, high-resolution structures are available only for mammalian-infecting mastadenoviruses. We present the first high-resolution structure of an adenovirus with nonmammalian host: lizard atadenovirus LAdV-2. We find a large conformational difference in the internal vertex protein IIIa between mast- and atadenoviruses, induced by the presence of an extended polypeptide. This polypeptide, and α-helical clusters beneath the facet, likely correspond to genus-specific proteins LH2 and p32k. Another genus-specific protein, LH3, with a fold typical of bacteriophage tailspikes, contacts the capsid surface via a triskelion structure identical to that used by mastadenovirus protein IX, revealing a conserved capsid-binding motif and an ancient gene duplication event. Our data also suggest that mastadenovirus E1B-55 K was exapted from the atadenovirus-like LH3 protein. This work provides new information on the evolution of adenoviruses, emphasizing the importance of minor coat proteins for determining specific physicochemical properties of virions and most likely their tropism.
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Hewat, Elizabeth A., and Dieter Blaas. "Cryoelectron Microscopy Analysis of the Structural Changes Associated with Human Rhinovirus Type 14 Uncoating." Journal of Virology 78, no. 6 (March 15, 2004): 2935–42. http://dx.doi.org/10.1128/jvi.78.6.2935-2942.2004.
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ABSTRACT Release of the human rhinovirus (HRV) genome into the cytoplasm of the cell involves a concerted structural modification of the viral capsid. The intracellular adhesion molecule 1 (ICAM-1) cellular receptor of the major-group HRVs and the low-density lipoprotein (LDL) receptor of the minor-group HRVs have different nonoverlapping binding sites. While ICAM-1 binding catalyzes uncoating, LDL receptor binding does not. Uncoating of minor-group HRVs is initiated by the low pH of late endosomes. We have studied the conformational changes concomitant with uncoating in the major-group HRV14 and compared them with previous results for the minor-group HRV2. The structure of empty HRV14 was determined by cryoelectron microscopy, and the atomic structure of native HRV14 was used to examine the conformational changes of the capsid and its constituent viral proteins. For both HRV2 and HRV14, the transformation from full to empty capsid involves an overall 4% expansion and an iris type of movement of viral protein VP1 to open up a 10-Å-diameter channel on the fivefold axis to allow exit of the RNA genome. The β-cylinders formed by the N termini of the VP3 molecules inside the capsid on the fivefold axis all open up in HRV2, but we propose that only one opens up in HRV14. The release of VP4 is less efficient in HRV14 than in HRV2, and the N termini of VP1 may exit at different points. The N-terminal loop of VP2 is modified in both viruses, probably to detach the RNA, but it bends only inwards in HRV2.
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Faustino, Martins, Karguth, Artilheiro, Enguita, Ricardo, Santos, and Martins. "Structural and Functional Properties of the Capsid Protein of Dengue and Related Flavivirus." International Journal of Molecular Sciences 20, no. 16 (August 8, 2019): 3870. http://dx.doi.org/10.3390/ijms20163870.
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Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.
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Finnen, Renée L., Kimberly D. Erickson, Xiaojiang S. Chen, and Robert L. Garcea. "Interactions between Papillomavirus L1 and L2 Capsid Proteins." Journal of Virology 77, no. 8 (April 15, 2003): 4818–26. http://dx.doi.org/10.1128/jvi.77.8.4818-4826.2003.
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ABSTRACT The human papillomavirus (HPV) capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers on a T=7 icosahedral lattice, with substoichiometric amounts of the minor capsid protein, L2. In order to understand the arrangement of L2 within the HPV virion, we have defined and biochemically characterized a domain of L2 that interacts with L1 pentamers. We utilized an in vivo binding assay involving the coexpression of recombinant HPV type 11 (HPV11) L1 and HPV11 glutathione S-transferase (GST) L2 fusion proteins in Escherichia coli. In this system, L1 forms pentamers, GST=L2 associates with these pentamers, and L1+L2 complexes are subsequently isolated by using the GST tag on L2. The stoichiometry of L1:L2 in purified L1+L2 complexes was 5:1, indicating that a single molecule of L2 interacts with an L1 pentamer. Coexpression of HPV11 L1 with deletion mutants of HPV11 L2 defined an L1-binding domain contained within amino acids 396 to 439 near the carboxy terminus of L2. L2 proteins from eight different human and animal papillomavirus serotypes were tested for their ability to interact with HPV11 L1. This analysis targeted a hydrophobic region within the L1-binding domain of L2 as critical for L1 binding. Introduction of negative charges into this hydrophobic region by site-directed mutagenesis disrupted L1 binding. L1-L2 interactions were not significantly disrupted by treatment with high salt concentrations (2 M NaCl), weak detergents, and urea concentrations of up to 2 M, further indicating that L1 binding by this domain is mediated by strong hydrophobic interactions. L1+L2 protein complexes were able to form virus-like particles in vitro at pH 5.2 and also at pH 6.8, a pH that is nonpermissive for assembly of L1 protein alone. Thus, L1/L2 interactions are primarily hydrophobic, encompass a relatively short stretch of amino acids, and have significant effects upon in vitro assembly.