Journal articles: 'Pr55gag'

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Wyma, Donald J., Alexander Kotov, and Christopher Aiken. "Evidence for a Stable Interaction of gp41 with Pr55Gag in Immature Human Immunodeficiency Virus Type 1 Particles." Journal of Virology 74, no. 20 (October 15, 2000): 9381–87. http://dx.doi.org/10.1128/jvi.74.20.9381-9387.2000.

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ABSTRACT Assembly of infectious human immunodeficiency virus type 1 (HIV-1) virions requires incorporation of the viral envelope glycoproteins gp41 and gp120. Several lines of evidence have suggested that the cytoplasmic tail of the transmembrane glycoprotein, gp41, associates with Pr55Gag in infected cells to facilitate the incorporation of HIV-1 envelope proteins into budding virions. However, direct evidence for an interaction between gp41 and Pr55Gagin HIV-1 particles has not been reported. To determine whether gp41 is associated with Pr55Gag in HIV-1 particles, viral cores were isolated from immature HIV-1 virions by sedimentation through detergent. The cores contained a major fraction of the gp41 that was present on untreated virions. Association of gp41 with cores required the presence of the gp41 cytoplasmic tail. In HIV-1 particles containing a functional protease, a mutation that prevents cleavage of Pr55Gag at the matrix-capsid junction was sufficient for the detergent-resistant association of gp41 with the isolated cores. In addition to gp41, a major fraction of virion-associated gp120 was also detected on immature HIV-1 cores. Isolation of cores under conditions known to disrupt lipid rafts resulted in the removal of a raft-associated protein incorporated into virions but not the HIV-1 envelope proteins. These results provide biochemical evidence for a stable interaction between Pr55Gag and the cytoplasmic tail of gp41 in immature HIV-1 particles. Moreover, findings in this study suggest that the interaction of Pr55Gag with gp41 may regulate the function of the envelope proteins during HIV-1 maturation.

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Fritz, Joëlle V., Denis Dujardin, Julien Godet, Pascal Didier, Jan De Mey, Jean-Luc Darlix, Yves Mély, and Hugues de Rocquigny. "HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag." Journal of Virology 84, no. 3 (November 18, 2009): 1585–96. http://dx.doi.org/10.1128/jvi.01691-09.

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ABSTRACT During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55Gag. Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G2/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55Gag and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55Gag-TC. This TC motif is tethered to the C terminus of Pr55Gag and does not interfere with Pr55Gag trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55Gag-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55Gag-TC mutants confirm that the 41LXXLF domain of Gag-p6 is essential for Pr55Gag-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55Gag-Vpr complexes are still formed when Pr55Gag carries mutations impairing its multimerization. These findings suggest that Pr55Gag-Vpr recognition and complex formation occur early during Pr55Gag assembly.

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Chatel-Chaix, Laurent, Levon Abrahamyan, Céline Fréchina, Andrew J. Mouland, and Luc DesGroseillers. "The Host Protein Staufen1 Participates in Human Immunodeficiency Virus Type 1 Assembly in Live Cells by Influencing pr55Gag Multimerization." Journal of Virology 81, no. 12 (April 11, 2007): 6216–30. http://dx.doi.org/10.1128/jvi.00284-07.

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ABSTRACT Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. The activities of several host cell proteins and machineries are also critical to the completion of virus assembly and the release of infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55Gag. In this report, we tested whether Stau1 modulates pr55Gag assembly using a new and specific pr55Gag oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both the overexpression and knockdown of Stau1 increase the pr55Gag-pr55Gag BRET levels, suggesting a role for Stau1 in regulating pr55Gag oligomerization during assembly. This effect of Stau1 on pr55Gag oligomerization was observed only in membranes, a cellular compartment in which pr55Gag assembly primarily occurs. Consistently, expression of Stau1 harboring a vSrc myristylation signal led to a 6.5-fold enrichment of Stau1 in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55Gag-pr55Gag BRET, demonstrating that Stau1 acts on assembly when targeted to membranes. A role for Stau1 in the formation of particles is further supported by the detection of membrane-associated detergent-resistant pr55Gag complexes and the increase of virus-like particle release when Stau1 expression levels are modulated. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55Gag-pr55Gag interactions, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.

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Rue, Sarah M., Jason W. Roos, Patrick M. Tarwater, Janice E. Clements, and Sheila A. Barber. "Phosphorylation and Proteolytic Cleavage of Gag Proteins in Budded Simian Immunodeficiency Virus." Journal of Virology 79, no. 4 (February 15, 2005): 2484–92. http://dx.doi.org/10.1128/jvi.79.4.2484-2492.2005.

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ABSTRACT The lentiviral Gag polyprotein (Pr55Gag) is cleaved by the viral protease during the late stages of the virus life cycle. Proteolytic cleavage of Pr55Gag is necessary for virion maturation, a structural rearrangement required for infectivity that occurs in budded virions. In this study, we investigate the relationship between phosphorylation of capsid (CA) domains in Pr55Gag and its cleavage intermediates and their cleavage by the viral protease in simian immunodeficiency virus (SIV). First, we demonstrate that phosphorylated forms of Pr55Gag, several CA-containing cleavage intermediates of Pr55Gag, and the free CA protein are detectable in SIV virions but not in virus-producing cells, indicating that phosphorylation of these CA-containing Gag proteins may require an environment that is unique to the virion. Second, we show that the CA domain of Pr55Gag can be phosphorylated in budded virus and that this phosphorylation does not require the presence of an active viral protease. Further, we provide evidence that CA domains (i.e., incompletely cleaved CA) are phosphorylated to a greater extent than free (completely cleaved) CA and that CA-containing Gag proteins can be cleaved by the viral protease in SIV virions. Finally, we demonstrate that Pr55Gag and several of its intermediates, but not free CA, are actively phosphorylated in budded virus. Taken together, these data indicate that, in SIV virions, phosphorylation of CA domains in Pr55Gag and several of its cleavage intermediates likely precedes the cleavage of these domains by the viral protease.

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Nicholson, Michael G., Sheila A. Barber, and Janice E. Clements. "The SIVmac239 Pr55Gag isoform, SIV p43, suppresses proteolytic cleavage of Pr55Gag." Virology 360, no. 1 (March 2007): 84–91. http://dx.doi.org/10.1016/j.virol.2006.10.003.

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Chatel-Chaix, Laurent, Jean-Francois Clément, Catherine Martel, Véronique Bériault, Anne Gatignol, Luc DesGroseillers, and Andrew J. Mouland. "Identification of Staufen in the Human Immunodeficiency Virus Type 1 Gag Ribonucleoprotein Complex and a Role in Generating Infectious Viral Particles." Molecular and Cellular Biology 24, no. 7 (April 1, 2004): 2637–48. http://dx.doi.org/10.1128/mcb.24.7.2637-2648.2004.

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ABSTRACT Staufen is a host protein that is selectively incorporated into human immunodeficiency virus type 1 (HIV-1) particles in a poorly defined process that involves the selection of HIV-1 genomic RNA for encapsidation and the activity of its third double-stranded RNA-binding domain (dsRBD3). To better understand this, we characterized its interactions with pr55Gag, the principal mediator of HIV-1 genomic RNA encapsidation. Chimeric proviruses harboring wild-type or mutant forms of Staufen were expressed in 293T cells. Cell fractionation analyses demonstrated that Staufen cosedimented with pr55Gag within detergent-resistant, trypsin-sensitive complexes that excluded mature capsid and matrix proteins. Coimmunoprecipitation and bioluminescence resonance energy transfer assays demonstrated a specific and direct interaction between Staufen and the nucleocapsid domain of pr55Gag in vitro and in live cells. This interaction is shown here to be mediated by Staufen's dsRBD3, with a contribution from its C-terminal domain. Immunoprecipitation and reverse transcription-PCR analyses showed that the 9-kb genomic RNA was found within Staufen-containing immune complexes. Spliced HIV-1 RNAs were not detected in these Staufen complexes, indicating a preferential association of Staufen with the 9-kb species. These results substantiate that Staufen and pr55Gag interact directly during HIV-1 expression. Knockdown of Staufen expression by small interfering RNAs in HIV-1-expressing cells demonstrated that this cellular protein was important for the generation of infectious virus. These data show that Staufen, pr55Gag, and genomic RNA are part of the same intracellular complex and support a role for Staufen in pr55Gag function in viral assembly, genomic RNA encapsidation, and the generation of infectious viral particles.

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Ding, Lingmei, Aaron Derdowski, Jaang-Jiun Wang, and Paul Spearman. "Independent Segregation of Human Immunodeficiency Virus Type 1 Gag Protein Complexes and Lipid Rafts." Journal of Virology 77, no. 3 (February 1, 2003): 1916–26. http://dx.doi.org/10.1128/jvi.77.3.1916-1926.2003.

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ABSTRACT Formation of human immunodeficiency virus type 1 (HIV-1) particles takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein. A functional assembly domain (the M domain) within the N-terminal portion of Pr55Gag mediates the interaction of Gag with cellular membranes. However, the determinants that provide specificity for assembly on the plasma membrane, as opposed to intracellular membranes, have not been identified. Recently, it was reported that Pr55Gag interacts with lipid raft microdomains of the plasma membrane. We sought to identify the domains within Pr55Gag that contribute to lipid raft association of Gag. Here we demonstrate that the I domain is required for interaction with detergent-resistant membrane fractions (DRMs). Mutation of key I-domain residues or loss of myristylation abrogated the association of Gag with DRMs. Thus, the I domain and the M domain combine to mediate Gag-lipid raft interactions as defined by these biochemical criteria. However, Gag protein complexes defined by flotation studies were much denser than classical lipid rafts, failed to incorporate classical lipid raft marker proteins, and were not disrupted by cholesterol extraction. Large sheets of Gag protein were identified in DRM fractions upon examination by electron microscopy. These results indicate that HIV-1 Pr55Gag forms detergent-resistant complexes at the cellular periphery that are distinct from lipid raft microdomains.

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Gerber, Pehuén Pereyra, Mercedes Cabrini, Carolina Jancic, Luciana Paoletti, Claudia Banchio, Catalina von Bilderling, Lorena Sigaut, et al. "Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate." Journal of Cell Biology 209, no. 3 (May 4, 2015): 435–52. http://dx.doi.org/10.1083/jcb.201409082.

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During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4+ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55Gag membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.

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Bardy, Martine, Bernard Gay, Stéphanie Pébernard, Nathalie Chazal, Marianne Courcoul, Robert Vigne, Etienne Decroly, and Pierre Boulanger. "Interaction of human immunodeficiency virus type 1 Vif with Gag and Gag–Pol precursors: co-encapsidation and interference with viral protease-mediated Gag processing." Journal of General Virology 82, no. 11 (November 1, 2001): 2719–33. http://dx.doi.org/10.1099/0022-1317-82-11-2719.

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Interactions of human immunodeficiency virus type 1 (HIV-1) Vif protein with various forms of Gag and Gag–Pol precursors expressed in insect cells were investigated in vivo and in vitro by co-encapsidation, co-precipitation and viral protease (PR)-mediated Gag processing assays. Addressing of Gag to the plasma membrane, its budding as extracellular virus-like particles (VLP) and the presence of the p6 domain were apparently not required for Vif encapsidation, as non-N-myristoylated Δp6-Gag and Vif proteins were co-encapsidated into intracellular VLP. Encapsidation of Vif occurred at significantly higher copy numbers in extracellular VLP formed from N-myristoylated, budding-competent Gag–Pol precursors harbouring an inactive PR domain or in chimaeric VLP composed of Gag and Gag–Pol precursors compared with the Vif content of Pr55Gag VLP. Vif encapsidation efficiency did not seem to correlate directly with VLP morphology, since these chimaeric VLP were comparable in size and shape to Pr55Gag VLP. Vif apparently inhibited PR-mediated Pr55Gag processing in vitro, with preferential protection of cleavage sites at the MA–CA and CA–NC junctions. Vif was resistant to PR action in vitro under conditions that allowed full Gag processing, and no direct interaction between Vif and PR was detected in vivo or in vitro. This suggested that inhibition by Vif of PR-mediated Gag processing resulted from interaction of Vif with the Gag substrate and not with the enzyme. Likewise, the higher efficiency of Vif encapsidation by Gag–Pol precursor compared with Pr55Gag was probably not mediated by direct binding of Vif to the Gag–Pol-embedded PR domain, but more likely resulted from a particular conformation of the Gag structural domains of the Gag–Pol precursor.

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Bussienne, Charlotte, Roland Marquet, Jean-Christophe Paillart, and Serena Bernacchi. "Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role." International Journal of Molecular Sciences 22, no. 6 (March 11, 2021): 2871. http://dx.doi.org/10.3390/ijms22062871.

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Protein post-translational modifications (PTMs) play key roles in eukaryotes since they finely regulate numerous mechanisms used to diversify the protein functions and to modulate their signaling networks. Besides, these chemical modifications also take part in the viral hijacking of the host, and also contribute to the cellular response to viral infections. All domains of the human immunodeficiency virus type 1 (HIV-1) Gag precursor of 55-kDa (Pr55Gag), which is the central actor for viral RNA specific recruitment and genome packaging, are post-translationally modified. In this review, we summarize the current knowledge about HIV-1 Pr55Gag PTMs such as myristoylation, phosphorylation, ubiquitination, sumoylation, methylation, and ISGylation in order to figure out how these modifications affect the precursor functions and viral replication. Indeed, in HIV-1, PTMs regulate the precursor trafficking between cell compartments and its anchoring at the plasma membrane, where viral assembly occurs. Interestingly, PTMs also allow Pr55Gag to hijack the cell machinery to achieve viral budding as they drive recognition between viral proteins or cellular components such as the ESCRT machinery. Finally, we will describe and compare PTMs of several other retroviral Gag proteins to give a global overview of their role in the retroviral life cycle.

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Sandefur, Stephanie, Vasundhara Varthakavi, and Paul Spearman. "The I Domain Is Required for Efficient Plasma Membrane Binding of Human Immunodeficiency Virus Type 1 Pr55Gag." Journal of Virology 72, no. 4 (April 1, 1998): 2723–32. http://dx.doi.org/10.1128/jvi.72.4.2723-2732.1998.

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ABSTRACT The interaction of the human immunodeficiency virus type 1 (HIV-1) Pr55Gag molecule with the plasma membrane of an infected cell is an essential step of the viral life cycle. Myristic acid and positively charged residues within the N-terminal portion of MA constitute the membrane-binding domain of Pr55Gag. A separate assembly domain, termed the interaction (I) domain, is located nearer the C-terminal end of the molecule. The I domain is required for production of dense retroviral particles, but has not previously been described to influence the efficiency of membrane binding or the subcellular distribution of Gag. This study used a series of Gag-green fluorescent protein fusion constructs to define a region outside of MA which determines efficient plasma membrane interaction. This function was mapped to the nucleocapsid (NC) region of Gag. The minimal region in a series of C-terminally truncated Gag proteins conferring plasma membrane fluorescence was identified as the N-terminal 14 amino acids of NC. This same region was sufficient to create a density shift in released retrovirus-like particles from 1.13 to 1.17 g/ml. The functional assembly domain previously termed the I domain is thus required for the efficient plasma membrane binding of Gag, in addition to its role in determining the density of released particles. We propose a model in which the I domain facilitates the interaction of the N-terminal membrane-binding domain of Pr55Gag with the plasma membrane.

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Urano, Emiko, Toru Aoki, Yuko Futahashi, Tsutomu Murakami, Yuko Morikawa, Naoki Yamamoto, and Jun Komano. "Substitution of the myristoylation signal of human immunodeficiency virus type 1 Pr55Gag with the phospholipase C-δ1 pleckstrin homology domain results in infectious pseudovirion production." Journal of General Virology 89, no. 12 (December 1, 2008): 3144–49. http://dx.doi.org/10.1099/vir.0.2008/004820-0.

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The matrix domain (MA) of human immunodeficiency virus type 1 Pr55Gag is covalently modified with a myristoyl group that mediates efficient viral production. However, the role of myristoylation, particularly in the viral entry process, remains uninvestigated. This study replaced the myristoylation signal of MA with a well-studied phosphatidylinositol 4,5-biphosphate-binding plasma membrane (PM) targeting motif, the phospholipase C-δ1 pleckstrin homology (PH) domain. PH–Gag–Pol PM targeting and viral production efficiencies were improved compared with Gag–Pol, consistent with the estimated increases in Gag–PM affinity. Both virions were recovered in similar sucrose density-gradient fractions and had similar mature virion morphologies. Importantly, PH–Gag–Pol and Gag–Pol pseudovirions had almost identical infectivity, suggesting a dispensable role for myristoylation in the virus life cycle. PH–Gag–Pol might be useful in separating the myristoylation-dependent processes from the myristoylation-independent processes. This the first report demonstrating infectious pseudovirion production without myristoylated Pr55Gag.

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Ono, Akira, Dimiter Demirov, and Eric O. Freed. "Relationship between Human Immunodeficiency Virus Type 1 Gag Multimerization and Membrane Binding." Journal of Virology 74, no. 11 (June 1, 2000): 5142–50. http://dx.doi.org/10.1128/jvi.74.11.5142-5150.2000.

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ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Gag precursor, Pr55Gag, is necessary and sufficient for the assembly and release of viruslike particles. Binding of Gag to membrane and Gag multimerization are both essential steps in virus assembly, yet the domains responsible for these events have not been fully defined. In addition, the relationship between membrane binding and Gag-Gag interaction remains to be elucidated. To investigate these issues, we analyzed, in vivo, the membrane-binding and assembly properties of a series of C-terminally truncated Gag mutants. Pr55Gag was truncated at the C terminus of matrix (MAstop), between the N- and C-terminal domains of capsid (CA146stop), at the C terminus of capsid (p41stop), at the C terminus of p2 (p43stop), and after the N-terminal 35 amino acids of nucleocapsid (NC35stop). The ability of these truncated Gag molecules to assemble and release viruslike particles and their capacity to copackage into particles when coexpressed with full-length Gag were determined. We demonstrate that the amount of truncated Gag incorporated into particles is incrementally increased by extension from CA146 to NC35, suggesting that multiple sites in this region are involved in Gag multimerization. Using membrane flotation centrifugation, we observe that MA shows significantly reduced membrane binding relative to full-length Gag but that CA146 displays steady-state membrane-binding properties comparable to those of Pr55Gag. The finding that the CA146 mutant, which contains only matrix and the N-terminal domain of capsid, exhibits levels of steady-state membrane binding equivalent to those of full-length Gag indicates that strong Gag-Gag interaction domains are not required for the efficient binding of HIV-1 Gag to membrane.

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Gupta, Kalpana, David Ott, Thomas J. Hope, Robert F. Siliciano, and Jef D. Boeke. "A Human Nuclear Shuttling Protein That Interacts with Human Immunodeficiency Virus Type 1 Matrix Is Packaged into Virions." Journal of Virology 74, no. 24 (December 15, 2000): 11811–24. http://dx.doi.org/10.1128/jvi.74.24.11811-11824.2000.

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ABSTRACT Active nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) is essential for the productive infection of nondividing cells. Nuclear import of the PIC is mediated by the HIV-1 matrix protein, which also plays several critical roles during viral entry and possibly during virion production facilitating the export of Pr55Gag and genomic RNA. Using a yeast two-hybrid screen, we identified a novel human virion-associated matrix-interacting protein (VAN) that is highly conserved in vertebrates and expressed in most human tissues. Its expression is upregulated upon activation of CD4+ T cells. VAN is efficiently incorporated into HIV-1 virions and, like matrix, shuttles between the nucleus and cytoplasm. Furthermore, overexpression of VAN significantly inhibits HIV-1 replication in tissue culture. We propose that VAN regulates matrix nuclear localization and, by extension, both nuclear import of the PIC and export of Pr55Gag and viral genomic RNA during virion production. Our data suggest that this regulatory mechanism reflects a more global process for regulation of nucleocytoplasmic transport.

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Porter, Kristen A., Lauren N. Kelley, Annette George, Jonathan A. Harton, and Karen M. Duus. "Class II Transactivator (CIITA) Enhances Cytoplasmic Processing of HIV-1 Pr55Gag." PLoS ONE 5, no. 6 (June 24, 2010): e11304. http://dx.doi.org/10.1371/journal.pone.0011304.

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Tanwar, Hanumant S., Keith K. Khoo, Megan Garvey, Lynne Waddington, Andrew Leis, Marcel Hijnen, Tony Velkov, Geoff J. Dumsday, William J. McKinstry, and Johnson Mak. "The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV." PLOS Pathogens 13, no. 2 (February 21, 2017): e1006221. http://dx.doi.org/10.1371/journal.ppat.1006221.

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Gomez, Candace Y., and Thomas J. Hope. "Mobility of Human Immunodeficiency Virus Type 1 Pr55Gag in Living Cells." Journal of Virology 80, no. 17 (September 1, 2006): 8796–806. http://dx.doi.org/10.1128/jvi.02159-05.

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ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.

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Turpin, JA, CA Schaeffer, SJ Terpening, L. Graham, M. Bu, and WG Rice. "Reverse Transcription of Human Immunodeficiency Virus Type 1 is Blocked by Retroviral Zinc Finger Inhibitors." Antiviral Chemistry and Chemotherapy 8, no. 1 (February 1997): 60–69. http://dx.doi.org/10.1177/095632029700800107.

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The Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys zinc fingers of retroviral nucleocapsid (NC) proteins are prime antiviral targets due to conservation of the Cys and His chelating residues and the absolute requirement of these fingers in both early and late phases of retroviral replication. Certain 2,2′-dithiobisbenzamides (DIBAs) chemically modify the Cys residues of the fingers, thereby inhibiting in vitro replication of human immunodeficiency virus type 1 (HIV-1). We examined the consequences of DIBA interaction with cell-free virions and their subsequent ability to initiate new rounds of infection. The DIBAs entered intact virions and chemically modified the p7NC proteins, resulting in extensive disulphide cross-linkage among zinc fingers of adjacent p7NC molecules. Likewise, treatment of Pr55gag-laden pseudovirions, used as a model of virion particles, with DIBAs resulted in Pr55gag cross-linkage. In contrast, monomeric p7NC protein did not form cross-linkages after DIBA treatment, indicating that the retroviral zinc finger proteins must exist in close proximity for cross-linkage to occur. Cross-linkage of p7NC in virions correlated with loss of infectivity and decreased proviral DNA synthesis during acute infection, even though DIBAs did not inhibit virus attachment to host cells or reverse transcriptase enzymatic activity. Thus, DIBA-type molecules impair the ability of HIV-1 virions to initiate reverse transcription through their action on the retroviral zinc finger, thereby blocking further rounds of replication.

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Wagner, Ralf, Ludwig Deml, Holger Fließbach, Gerhard Wanner, and Hans Wolf. "Assembly and Extracellular Release of Chimeric HIV-1 Pr55gag Retrovirus-like Particles." Virology 200, no. 1 (April 1994): 162–75. http://dx.doi.org/10.1006/viro.1994.1175.

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Bouyac, M., M. Courcoul, G. Bertoia, Y. Baudat, D. Gabuzda, D. Blanc, N. Chazal, et al. "Human immunodeficiency virus type 1 Vif protein binds to the Pr55Gag precursor." Journal of virology 71, no. 12 (1997): 9358–65. http://dx.doi.org/10.1128/jvi.71.12.9358-9365.1997.

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Derdowski, Aaron, Lingmei Ding, and Paul Spearman. "A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55Gag I Domain Mediates Gag-Gag Interactions." Journal of Virology 78, no. 3 (February 1, 2004): 1230–42. http://dx.doi.org/10.1128/jvi.78.3.1230-1242.2004.

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ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein (Gag). One of the essential steps in the assembly process is the multimerization of Gag. We have developed a novel fluorescence resonance energy transfer (FRET) assay for the detection of protein-protein interactions between Gag molecules. We demonstrate that Gag multimerization takes place primarily on cellular membranes, with the majority of these interactions occurring on the plasma membrane. However, distinct sites of Gag-Gag interaction are also present at punctate intracellular locations. The I domain is a functional assembly domain within the nucleocapsid region of Gag that affects particle density, the subcellular localization of Gag, and the formation of detergent-resistant Gag protein complexes. Results from this study provide evidence that the I domain mediates Gag-Gag interactions. Using Gag-fluorescent protein fusion constructs that were previously shown to define the minimal I domain within HIV-1 Pr55Gag, we show by FRET techniques that protein-protein interactions are greatly diminished when Gag proteins lacking the I domain are expressed. Gag-Tsg101 interactions are also seen in living cells and result in a shift of Tsg101 to the plasma membrane. The results within this study provide direct evidence that the I domain mediates protein-protein interactions between Gag molecules. Furthermore, this study establishes FRET as a powerful tool for the detection of protein-protein interactions involved in retrovirus assembly.

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Grobelny, D., Q. Chen, D. Tyssen, G. Tachedjian, K. Sebire, L. Buchanan, and C. Birch. "Antiviral Activity of DG-35-VIII, a Potent Inhibitor of the Protease of Human Immunodeficiency Virus." Antiviral Chemistry and Chemotherapy 8, no. 2 (April 1997): 99–106. http://dx.doi.org/10.1177/095632029700800203.

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The protease of human immunodeficiency virus (HIV) is an important target for antiretroviral drug therapy. The synthesis and in vitro antiviral activity of a novel protease inhibitor, DG-35-VIII, which contains an hydroxyethylhydrazide core unit, is described. DG-35-VIII had potent activity against HIV-1 and related viruses (HIV-2 and simian immunodeficiency virus) in an acutely infected T lymphocyte line (MT-2) and was also active in cells chronically infected with HIV-1, where it inhibited processing of the Pr55gag and Pr160 gag-pal precursor proteins.

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Ono, Akira, and Eric O. Freed. "Binding of Human Immunodeficiency Virus Type 1 Gag to Membrane: Role of the Matrix Amino Terminus." Journal of Virology 73, no. 5 (May 1, 1999): 4136–44. http://dx.doi.org/10.1128/jvi.73.5.4136-4144.1999.

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ABSTRACT Binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55Gag, to membrane is an indispensable step in virus assembly. Previously, we reported that a matrix (MA) residue 6 substitution (6VR) imposed a virus assembly defect similar to that observed with myristylation-defective mutants, suggesting that the 6VR change impaired membrane binding. Intriguingly, the 6VR mutation had no effect on Gag myristylation. The defective phenotype imposed by 6VR was reversed by changes at other positions in MA, including residue 97. In this study, we use several biochemical methods to demonstrate that the residue 6 mutation, as well as additional substitutions in MA amino acids 7 and 8, reduce membrane binding without affecting N-terminal myristylation. This effect is observed in the context of Pr55Gag, a truncated Gag containing only MA and CA, and in MA itself. The membrane binding defect imposed by the 6VR mutation is reversed by second-site changes in MA residues 20 and 97, both of which, when present alone, increase membrane binding to levels greater than those for the wild type. Both reduced and enhanced membrane binding imposed by the MA substitutions depend upon the presence of the N-terminal myristate. The results support the myristyl switch model recently proposed for the regulation of Gag membrane binding, according to which membrane binding is determined by the degree of exposure or sequestration of the N-terminal myristate moiety. Alternatively, insertion of the myristate into the lipid bilayer might be a prerequisite event for the function of other distinct MA-encoded membrane binding domains.

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Sandefur, Stephanie, Rita M. Smith, Vasundhara Varthakavi, and Paul Spearman. "Mapping and Characterization of the N-Terminal I Domain of Human Immunodeficiency Virus Type 1 Pr55Gag." Journal of Virology 74, no. 16 (August 15, 2000): 7238–49. http://dx.doi.org/10.1128/jvi.74.16.7238-7249.2000.

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ABSTRACT Human immunodeficiency virus (HIV) type 1 particles assemble at the plasma membrane of cells in a manner similar to that of the type C oncoretroviruses. The Pr55Gag molecule directs the assembly process and is sufficient for particle assembly in the absence of all other viral gene products. The I domain is an assembly domain that has been previously localized to the nucleocapsid (NC) region of Gag. In this study we utilized a series of Gag-green fluorescent protein (GFP) fusion proteins to precisely identify sequences that constitute the N-terminal I domain of Pr55Gag. The minimal sequence required for the I domain was localized to the extreme N terminus of NC. Two basic residues (arginine 380 and arginine 384) within the initial seven residues of NC were found to be critical for the function of the N-terminal I domain. The presence of positive charge alone in these two positions, however, was not sufficient to mediate the formation of dense Gag particles. The I domain was required for the formation of detergent-resistant complexes of Gag protein, and confocal microscopy demonstrated that the I domain was also required for the formation of punctate foci of Gag proteins at the plasma membrane. Electron microscopic analysis of cells expressing Gag-GFP fusion constructs with an intact I domain revealed numerous retrovirus-like particles (RVLPs) budding from the plasma membrane, while I domain-deficient constructs failed to generate visible RVLPs. These results provide evidence that Gag-Gag interactions mediated by the I domain play a central role in the assembly of HIV particles.

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Nicholson, Michael G., Sarah M. Rue, Janice E. Clements, and Sheila A. Barber. "An internal ribosome entry site promotes translation of a novel SIV Pr55Gag isoform." Virology 349, no. 2 (June 2006): 325–34. http://dx.doi.org/10.1016/j.virol.2006.01.034.

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Bernacchi, Serena, Ekram W. Abd El-Wahab, Noé Dubois, Marcel Hijnen, Redmond P. Smyth, Johnson Mak, Roland Marquet, and Jean-Christophe Paillart. "HIV-1 Pr55Gag binds genomic and spliced RNAs with different affinity and stoichiometry." RNA Biology 14, no. 1 (December 18, 2016): 90–103. http://dx.doi.org/10.1080/15476286.2016.1256533.

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Cruz, Pedro E., Cristina C. Peixoto, José L. Moreira, and Manuel J. T. Carrondo. "Production and quality analysis of Pr55gag particles produced in baculovirus-infected insect cells." Journal of Chemical Technology & Biotechnology 72, no. 2 (June 1998): 149–58. http://dx.doi.org/10.1002/(sici)1097-4660(199806)72:2<149::aid-jctb886>3.0.co;2-c.

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Scotti, Nunzia, Fiammetta Alagna, Enrico Ferraiolo, Gelsomina Formisano, Lorenza Sannino, Luigi Buonaguro, Angelo De Stradis, et al. "High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts." Planta 229, no. 5 (February 21, 2009): 1109–22. http://dx.doi.org/10.1007/s00425-009-0898-2.

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Cruz, Pedro E., Pedro C. Martins, Paula M. Alves, Cristina C. Peixoto, Helena Santos, Jos� L. Moreira, and Manuel J. T. Carrondo. "Proteolytic activity in infected and noninfected insect cells: Degradation of HIV-1 Pr55gag particles." Biotechnology and Bioengineering 65, no. 2 (October 20, 1999): 133–43. http://dx.doi.org/10.1002/(sici)1097-0290(19991020)65:2<133::aid-bit2>3.0.co;2-x.

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30

von Poblotzki, Andreas, Ralf Wagner, Matthias Niedrig, Gerhard Wanner, Hans Wolf, and Susanne Modrow. "Identification of a Region in the Pr55gag-Polyprotein Essential for HIV-1 Particle Formation." Virology 193, no. 2 (April 1993): 981–85. http://dx.doi.org/10.1006/viro.1993.1210.

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Chege, Gerald K., Enid G. Shephard, Ann Meyers, Joanne van Harmelen, Carolyn Williamson, Alisson Lynch, Clive M. Gray, Edward P. Rybicki, and Anna-Lise Williamson. "HIV-1 subtype C Pr55gag virus-like particle vaccine efficiently boosts baboons primed with a matched DNA vaccine." Journal of General Virology 89, no. 9 (September 1, 2008): 2214–27. http://dx.doi.org/10.1099/vir.0.83501-0.

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A DNA vaccine expressing human immunodeficiency virus type 1 (HIV-1) southern African subtype C Gag (pTHGag) and a recombinant baculovirus Pr55gag virus-like particle prepared using a subtype C Pr55gag protein (Gag VLP) was tested in a prime–boost inoculation regimen in Chacma baboons. The response of five baboons to Gag peptides in a gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay after three pTHGag immunizations ranged from 100 to 515 spot-forming units (s.f.u.) per 106 peripheral blood mononuclear cells (PBMCs), whilst the response of two baboons to the Gag VLP vaccine ranged from 415 to 465 s.f.u. per 106 PBMCs. An increase in the Gag-specific response to a range of 775–3583 s.f.u. per 106 PBMCs was achieved by boosting with Gag VLPs the five baboons that were primed with pTHGag. No improvement in Gag responses was achieved in this prime–boost inoculation regimen by increasing the number of pTHGag inoculations to six. IFN-γ responses were mapped to several peptides, some of which have been reported to be targeted by PBMCs from HIV-1 subtype C-infected individuals. Gag VLPs, given as a single-modality regimen, induced a predominantly CD8+ T-cell IFN-γ response and interleukin-2 was a major cytokine within a mix of predominantly Th1 cytokines produced by a DNA–VLP prime–boost modality. The prime–boost inoculation regimen induced high serum p24 antibody titres in all baboons, which were several fold above that induced by the individual vaccines. Overall, this study demonstrated that these DNA prime/VLP boost vaccine regimens are highly immunogenic in baboons, inducing high-magnitude and broad multifunctional responses, providing support for the development of these products for clinical trials.

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Dietrich, Lars, Lorna S. Ehrlich, Tracy J. LaGrassa, Dana Ebbets-Reed, and Carol Carter. "Structural Consequences of Cyclophilin A Binding on Maturational Refolding in Human Immunodeficiency Virus Type 1 Capsid Protein." Journal of Virology 75, no. 10 (May 15, 2001): 4721–33. http://dx.doi.org/10.1128/jvi.75.10.4721-4733.2001.

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ABSTRACT While several cellular proteins are incorporated in the human immunodeficiency virus type 1 virion, cyclophilin (CyP) A is the only one whose absence has been demonstrated to impair infectivity. Incorporation of the cytosolic protein results from interaction with a highly exposed Pro-rich loop in the N-terminal region of the capsid (CA) domain of the precursor polyprotein, Pr55Gag. Even when prevented from interacting with CyP A, Pr55Gag still forms particles that proceed to mature into morphologically wild-type virions, suggesting that CyP A influences a postassembly event. The nature of this CyP A influence has yet to be elucidated. Here, we show that while CyP A binds both Gag and mature CA proteins, the two binding interactions are actually different. Tryptophan 121 (W121) in CyP A distinguished the two proteins: a phenylalanine substitution (W121F) impaired binding of mature CA protein but not of Gag. This indicates the occurrence of a maturation-dependent switch in the conformation of the Pro-rich loop. A structural consequence of Gag binding to CyP A was to block this maturational refolding, resulting in a 24-kDa CA protein retaining the immature Pro-rich loop conformation. Using trypsin as a structure probe, we demonstrate that the conformation of the C-terminal region in mature CA is also a product of maturational refolding. Binding to wild-type CyP A altered this conformation, as indicated by a reduction in the accessibility of Cys residue(s) in the region to chemical modification. Hence, the end result of binding to CyP A, whether the Pro-rich loop is in the context of Gag or mature CA protein, is a structurally modified mature CA protein. The postassembly role of CyP A may be mediated through these modified mature CA proteins.

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Deml, Ludwig, Cornelia Speth, Manfred P. Dierich, Hans Wolf, and Ralf Wagner. "Recombinant HIV-1 Pr55gag virus-like particles: potent stimulators of innate and acquired immune responses." Molecular Immunology 42, no. 2 (February 2005): 259–77. http://dx.doi.org/10.1016/j.molimm.2004.06.028.

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McKinstry, William J., Marcel Hijnen, Hanumant S. Tanwar, Lindsay G. Sparrow, Sureshbabu Nagarajan, Son T. Pham, and Johnson Mak. "Expression and purification of soluble recombinant full length HIV-1 Pr55Gag protein in Escherichia coli." Protein Expression and Purification 100 (August 2014): 10–18. http://dx.doi.org/10.1016/j.pep.2014.04.013.

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LeBlanc, Jason J., Omar Perez, and Thomas J. Hope. "Probing the Structural States of Human Immunodeficiency Virus Type 1 Pr55gag by Using Monoclonal Antibodies." Journal of Virology 82, no. 5 (December 19, 2007): 2570–74. http://dx.doi.org/10.1128/jvi.01717-07.

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ABSTRACT Gag-FP (fluorescent protein) fusion constructs are commonly used to study human immunodeficiency virus type 1 assembly, yielding diffuse signals throughout the cytoplasm along with punctate signals routinely described as virus-like particles (VLPs) representing assembled but unprocessed Gag. However, these particles cannot be accurately described as VLPs, since fluorescence microscopy cannot provide structural resolution. We demonstrate here that the inability of a monoclonal p24 antibody to bind its cognate epitope when unprocessed Gag is assembled distinguishes VLPs from unassembled, monomeric Gag. Furthermore, we show that assembled and unassembled Gag punctate signals travel along microtubules. These monoclonal antibody studies provide a new tool for examining retroviral assembly.

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Aoki, T., S. Shimizu, E. Urano, Y. Futahashi, M. Hamatake, H. Tamamura, K. Terashima, T. Murakami, N. Yamamoto, and J. Komano. "Improvement of lentiviral vector-mediated gene transduction by genetic engineering of the structural protein Pr55Gag." Gene Therapy 17, no. 9 (April 22, 2010): 1124–33. http://dx.doi.org/10.1038/gt.2010.61.

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Bojak, Alexandra, Jens Wild, Hans Wolf, and Ralf Wagner. "Efficiency of a myogenic DNA vaccine is strictly dependent upon cellular localization of HIV-1 Pr55gag." Vaccine 20, no. 15 (May 2002): 1980–84. http://dx.doi.org/10.1016/s0264-410x(02)00082-8.

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Archambault, D., Z. M. Wang, J. C. Lacal, A. Gazit, A. Yaniv, J. E. Dahlberg, and S. R. Tronick. "Development of an enzyme-linked immunosorbent assay for equine infectious anemia virus detection using recombinant Pr55gag." Journal of Clinical Microbiology 27, no. 6 (1989): 1167–73. http://dx.doi.org/10.1128/jcm.27.6.1167-1173.1989.

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Alfadhli, Ayna, Andrew Mack, Christopher Ritchie, Isabel Cylinder, Logan Harper, Philip R. Tedbury, Eric O. Freed, and Eric Barklis. "Trimer Enhancement Mutation Effects on HIV-1 Matrix Protein Binding Activities." Journal of Virology 90, no. 12 (March 30, 2016): 5657–64. http://dx.doi.org/10.1128/jvi.00509-16.

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ABSTRACTThe HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.IMPORTANCEThe HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of Env CT domains with the MA domains of the viral precursor Gag proteins. Despite this knowledge, little is known about the mechanisms by which MA facilitates the virion incorporation of Env proteins. To help elucidate this process, we examined the binding activities of an MA mutant that stabilizes MA trimers. We found that the mutant proteins organized similarly to WT proteins on membranes, and that mutant and WT proteins revealed only slight differences in their binding to RNAs or lipids. However, the mutant proteins showed better binding to Env CTs than the WT proteins, and CT binding correlated with MA trimerization. Our results suggest that multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.

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Speth, Cornelia, Simon Bredl, Magdalena Hagleitner, Jens Wild, Manfred Dierich, Hans Wolf, Josef Schroeder, Ralf Wagner, and Ludwig Deml. "Human immunodeficiency virus type-1 (HIV-1) Pr55gag virus-like particles are potent activators of human monocytes." Virology 382, no. 1 (December 2008): 46–58. http://dx.doi.org/10.1016/j.virol.2008.08.043.

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Cen, Shan, Yue Huang, Ahmad Khorchid, Jean-Luc Darlix, Mark A. Wainberg, and Lawrence Kleiman. "The Role of Pr55gag in the Annealing of tRNA3Lys to Human Immunodeficiency Virus Type 1 Genomic RNA." Journal of Virology 73, no. 5 (May 1, 1999): 4485–88. http://dx.doi.org/10.1128/jvi.73.5.4485-4488.1999.

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ABSTRACT During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA3 Lys, is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA3 Lys in HIV-1 is independent of polyprotein processing and is facilitated in vitro by p7 nucleocapsid (NCp7). We have previously shown that mutations in clusters of basic amino acids flanking the first Cys-His box in NC sequence inhibit annealing of tRNA3 Lys in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55 gag or Pr160 gag-pol . In vivo placement of tRNA3 Lys is measured with total viral RNA as the source of primer tRNA-template in an in vitro reverse transcription assay. Cotransfection of COS cells with a plasmid coding for either mutant Pr55 gag or mutant Pr160 gag-pol , and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55 gag inhibit tRNA3 Lysplacement. The NC mutations in Pr55 gag reduce viral infectivity by 95% and are trans-dominant-negative, i.e., they inhibit genomic placement of tRNA3 Lys even in the presence of wild-type Pr55 gag . This dominant phenotype may indicate that the mutant Pr55 gag is disrupting an ordered Pr55 gag structure responsible for the annealing of tRNA3 Lys to genomic RNA.

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Harila, Kirsi, Ian Prior, Mathilda Sjöberg, Antti Salminen, Jorma Hinkula, and Maarit Suomalainen. "Vpu and Tsg101 Regulate Intracellular Targeting of the Human Immunodeficiency Virus Type 1 Core Protein Precursor Pr55gag." Journal of Virology 80, no. 8 (April 15, 2006): 3765–72. http://dx.doi.org/10.1128/jvi.80.8.3765-3772.2006.

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ABSTRACT Assembly of human immunodeficiency virus type 1 (HIV-1) is directed by the viral core protein Pr55 gag . Depending on the cell type, Pr55 gag accumulates either at the plasma membrane or on late endosomes/multivesicular bodies. Intracellular localization of Pr55 gag determines the site of virus assembly, but molecular mechanisms that define cell surface or endosomal targeting of Pr55 gag are poorly characterized. We have analyzed targeting of newly synthesized Pr55 gag in HeLa H1 cells by pulse-chase studies and subcellular fractionations. Our results indicated that Pr55 gag was inserted into the plasma membrane and, when coexpressed with the viral accessory protein Vpu, Pr55 gag remained at the plasma membrane and virions assembled at this site. In contrast, Pr55 gag expressed in the absence of Vpu was initially inserted into the plasma membrane, but subsequently endocytosed, and virus assembly was partially shifted to internal membranes. This endocytosis of Pr55 gag required the host protein Tsg101. These results identified a previously unknown role for Vpu and Tsg101 as regulators for the endocytic uptake of Pr55 gag and suggested that the site of HIV-1 assembly is determined by factors that regulate the endocytosis of Pr55 gag .

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VerPlank, L., F. Bouamr, T. J. LaGrassa, B. Agresta, A. Kikonyogo, J. Leis, and C. A. Carter. "Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55Gag." Proceedings of the National Academy of Sciences 98, no. 14 (June 26, 2001): 7724–29. http://dx.doi.org/10.1073/pnas.131059198.

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44

Jacobs, E., D. Gheysen, D. Thines, M. Francotte, and M. de Wilde. "The HIV-1 Gag precursor Pr55gag synthesized in yeast is myristoylated and targeted to the plasma membrane." Gene 79, no. 1 (June 1989): 71–81. http://dx.doi.org/10.1016/0378-1119(89)90093-0.

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Gheysen, Dirk, Eric Jacobs, Françoise de Foresta, Clotilde Thiriart, Myriam Francotte, Denise Thines, and Michel De Wilde. "Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells." Cell 59, no. 1 (October 1989): 103–12. http://dx.doi.org/10.1016/0092-8674(89)90873-8.

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46

Cheng, Xiaogang, Michael Belshan, and Lee Ratner. "Hsp40 Facilitates Nuclear Import of the Human Immunodeficiency Virus Type 2 Vpx-Mediated Preintegration Complex." Journal of Virology 82, no. 3 (November 21, 2007): 1229–37. http://dx.doi.org/10.1128/jvi.00540-07.

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ABSTRACT Human immunodeficiency virus type 2 (HIV-2) Vpx is required for nuclear translocation of the viral preintegration complex (PIC) in quiescent cells. In order to decipher the mechanism of action of Vpx, a cDNA library was screened with the yeast two-hybrid assay, resulting in the identification of heat shock protein 40, Hsp40/DnaJB6, as a Vpx-interactive protein. Interaction with Vpx was confirmed by glutathione S-transferase (GST) pull-down and coimmunoprecipitation assays. Overexpression of Hsp40/DnaJB6 enhanced Vpx nuclear import, whereas overexpression of a nuclear localization mutant of Hsp40/DnaJB6 (H31Q) or down-regulation of Hsp40/DnaJB6 by small interfering RNA (siRNA) reduced the nuclear import of Vpx. Hsp40/DnaJB6 competed with the Pr55Gag precursor protein for the binding of Vpx and incorporation into virus-like particles. Overexpression of Hsp40/DnaJB6 promoted viral PIC nuclear import, whereas siRNA down-regulation of Hsp40/DnaJB6 inhibited PIC nuclear import. These results demonstrate a role for Hsp40/DnaJB6 in the regulation of HIV-2 PIC nuclear transport.

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Jalaguier, Pascal, Réjean Cantin, Halim Maaroufi, and Michel J. Tremblay. "Selective Acquisition of Host-Derived ICAM-1 by HIV-1 Is a Matrix-Dependent Process." Journal of Virology 89, no. 1 (October 15, 2014): 323–36. http://dx.doi.org/10.1128/jvi.02701-14.

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ABSTRACTHIV-1 acquires an impressive number of foreign components during its formation. Despite all previous efforts spent studying the nature and functionality of virus-anchored host molecules, the exact mechanism(s) through which such constituents are acquired by HIV-1 is still unknown. However, in the case of ICAM-1, one of the most extensively studied transmembrane proteins found associated with mature virions, the Pr55Gagprecursor polyprotein appears to be a potential interaction partner. We investigated and characterized at the molecular level the process of ICAM-1 incorporation using initially a Pr55Gag-based virus-like particle (VLP) model. Substitution of various domains of Pr55Gag, such as the nucleocapsid, SP2, or p6, had no effect on the acquisition of ICAM-1. We found that the structural matrix protein (MA) is mandatory for ICAM-1 incorporation within VLPs, and we confirmed this novel observation with the replication-competent HIV-1 molecular clone NL4.3. Additional studies suggest that the C-terminal two-thirds of MA, and especially 13 amino acids positioned inside the fifth α-helix, are important. Moreover, based on three-dimensional (3D) modeling of protein-protein interactions (i.e., protein-protein docking) and further validation by a virus capture assay, we found that a series of acidic residues in the MA domain interact with basic amino acids located in the ICAM-1 cytoplasmic tail. Our findings provide new insight into the molecular mechanism governing the acquisition of ICAM-1, a host molecule known to enhance HIV-1 infectivity in a significant manner. Altogether, these observations offer a new avenue for the development of antiviral therapeutics that are directed at a target of host origin.IMPORTANCEIntercellular adhesion molecule 1 (ICAM-1) is a cell surface host component known to be efficiently inserted within emerging HIV-1 particles. It has been demonstrated that host-derived ICAM-1 molecules act as a strong attachment factor and increase HIV-1 infectivity substantially. Despite previous efforts spent studying virus-associated host molecules, the precise mechanism(s) through which such constituents are inserted within emerging HIV-1 particles still remains obscure. Previous data suggest that the Pr55Gagprecursor polyprotein appears as a potential interaction partner with ICAM-1. In the present study, we demonstrate that the HIV-1 matrix domain plays a key role in the ICAM-1 incorporation process. Some observations were confirmed with whole-virus preparations amplified in primary human cells, thereby providing physiological significance to our data.

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Hermida-Matsumoto, Luz, and Marilyn D. Resh. "Human Immunodeficiency Virus Type 1 Protease Triggers a Myristoyl Switch That Modulates Membrane Binding of Pr55gag and p17MA." Journal of Virology 73, no. 3 (March 1, 1999): 1902–8. http://dx.doi.org/10.1128/jvi.73.3.1902-1908.1999.

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ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Pr55 gag gene product directs the assembly of virions at the inner surface of the cell plasma membrane. The specificity of plasma membrane binding by Pr55 gag is conferred by a combination of an N-terminal myristoyl moiety and a basic residue-rich domain. Although the myristate plus basic domain is also present in the p17MA proteolytic product formed upon Pr55 gag maturation, the ability of p17MA to bind to membranes is significantly reduced. It was previously reported that the reduced membrane binding of p17MA was due to sequestration of the myristate moiety by a myristoyl switch (W. Zhou and M. D. Resh, J. Virol. 70:8540–8548, 1996). Here we demonstrate directly that treatment of membrane-bound Pr55 gag in situ with HIV-1 protease generates p17MA, which is then released from the membrane. Pr55 gag was synthesized in reticulocyte lysates, bound to membranes, and incubated with purified HIV-1 protease. The p17MA product in the membrane-bound and soluble fractions was analyzed following proteolysis. Newly generated p17MA initially was membrane bound but then displayed a slow, time-dependent dissociation resulting in 65% solubilization. Residual p17MA could be extracted from the membranes with either high pH or high salt. Treatment of membranes from transfected COS-1 cells with protease revealed that Pr55 gag was present within sealed membrane vesicles and that the release of p17MA occurred only when detergent and salt were added. We present a model proposing that the HIV-1 protease is the “trigger” for a myristoyl switch mechanism that modulates the membrane associations of Pr55 gag and p17MA in virions and membranes.

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Maleeff, Beverly E., Timothy K. Hart, James A. Hoxie, and Peter J. Bugelski. "Structural studies of immunodeficiency viruses." Proceedings, annual meeting, Electron Microscopy Society of America 53 (August 13, 1995): 698–99. http://dx.doi.org/10.1017/s042482010013986x.

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Immunodeficiency viruses (IV) are retroviruses belonging to the subfamily Lentivirinae and include human (HIV) and simian (SIV) immunodeficiency viruses. In this study, we focus on the structure of proteins in SIV isolated from macaques (SIVmac). IV form by budding through the surface of an infected cell, where viral proteins and RNA are enveloped by the host cell bilayer membrane. Immature virions consist of this outer membrane studded with surface glycoprotein projections and a thick sub-envelope protein plaque composed of unprocessed Pr55gag protein. Viral maturation is marked by cleavage of this protein by a viral protease into the structural proteins p17, p24 and p15. Mature virus particles are icosahedral, approximately 120 nm in diameter, consisting of membrane surface glycoprotein projections, a thin submembrane p17 protein layer, a centrally located core capsid composed of p24 protein subunits surrounding the viral ribonucleic acid, and nucleocapsid proteins p7 and p6, derived from processed p15. One step in the development of anti-IV treatments is to understand the organization of the proteins in order to determine specific sites to target chemotherapeutic agents.

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Lynch, Alisson, Ann E. Meyers, Anna-Lise Williamson, and Edward P. Rybicki. "Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media." Virology Journal 9, no. 1 (2012): 210. http://dx.doi.org/10.1186/1743-422x-9-210.

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Journal articles on the topic 'Pr55gag'

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