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1.
To elucidate the role of the C-terminal portion of Gag in the incorporation of human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virus particles, a series of HIV-1 Gag-Pol mutants with deletions in the C-terminalgag sequence was constructed and viral incorporation of the Gag-Pol deletion mutants was analyzed using cotransfecting 293T cells with a Pr55
gag
expression plasmid. The biological function of the incorporated HIV-1pol gene product was tested using an infectivity assay of the released virus particles which were pseudotyped with the murine leukemia virus Env. Analysis indicated that Gag-Pol deletion mutants, with a removal of the matrix (MA) and/or nucleocapsid (NC) or of the N-terminal two thirds of thegag coding sequence, could be incorporated efficiently into virus particles and produce significant amounts of infectious virions when assayed in a single-cycle infection assay. In contrast, mutations involving a deletion of the major homology region and the adjacent C-terminal capsid sequence significantly affected Gag-Pol incorporation. However, incorporation into virus particles of a Gag-Pol deletion mutant retaining both the major homology region and the adjacent C-terminal capsid intact was still severely impaired. This suggests that the capsid major homology region and the adjacent C-terminal capsid sequence in Gag-Pol are necessary but not sufficient for the incorporation of HIV-1 Pr160
gag-pol
into virus particles. 相似文献
2.
Differential membrane binding of the human immunodeficiency virus type 1 matrix protein. 总被引:19,自引:14,他引:5
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The human immunodeficiency virus type 1 matrix protein (p17MA) plays a central role at both the early and late stages of the virus life cycle. During viral assembly, the p17MA domain of Pr55gag promotes membrane association, which is essential for the formation of viral particles. When viral infection occurs, the mature p17MA dissociates from the plasma membrane and participates in the nuclear targeting process. Thus, p17MA contains a reversible membrane binding signal to govern its differential subcellular localization and biological functions. We previously identified a membrane binding signal within the amino-terminal 31 amino acids of the matrix domain of human immunodeficiency virus type 1 Gag, consisting of myristate and a highly basic region (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). Here we show that exposure of this membrane binding signal is regulated in different Gag protein contexts. Within full-length Pr55gag, the membrane targeting signal is exposed and can direct Pr55gag as well as heterologous proteins to the plasma membrane. However, in the context of p17MA alone, this signal is hidden and unable to confer plasma membrane binding. To investigate the molecular mechanism for regulation of membrane binding, a series of deletions within p17MA was generated by sequentially removing alpha-helical regions defined by the nuclear magnetic resonance structure. Removal of the last alpha helix (amino acids 97 to 109) of p17MA was associated with enhancement of binding to biological membranes in vitro and in vivo. Liposome binding experiments indicated that the C-terminal region of p17MA exerts a negative effect on the N-terminal MA membrane targeting domain by sequestering the myristate signal. We propose that mature p17MA adopts a conformation different from that of the p17MA domain within Pr55gag and present evidence to support this hypothesis. It is likely that such a conformational change results in an N-terminal myristyl switch which governs differential membrane binding. 相似文献
3.
4.
Antonella Bugatti Francesca Caccuri Federica Filippini Cosetta Ravelli Arnaldo Caruso 《The Journal of biological chemistry》2021,297(2)
HIV-1 matrix protein p17 variants (vp17s) derived from non-Hodgkin''s lymphoma (NHL) tissues of HIV-1–seropositive (HIV+) patients promote B-cell growth by activating the Akt signaling pathway. It is fundamental to understand the role played by vp17s in producing a microenvironment that fosters lymphoma development and progression. Therefore, we asked whether vp17s could be secreted from infected cells in their biologically active form. In this study, we show that two B-cell growth-promoting vp17s, NHL-a101 and NHL-a102, characterized by amino acid insertions at position 117 to 118 (Ala–Ala) or 125 to 126 (Gly–Asn), respectively, are secreted from HIV-1–infected Jurkat T cells during the active phase of viral replication. Secretion of biologically active vp17s also occurred in HeLa cells nucleofected with a plasmid expressing the entire Gag gene, following proteolytic cleavage of the Gag precursor polyprotein (Pr55Gag) by cellular aspartyl proteases. Binding of Pr55Gag to phosphatidylinositol-(4,5)-bisphosphate was indispensable for allowing the unconventional secretion of both wildtype p17 and vp17s. Indeed, here we demonstrate that inhibition of Pr55Gag binding to phosphatidylinositol-(4,5)-bisphosphate by using neomycin, or its enzymatic depletion achieved by overexpression of 5ptaseIV, significantly impair the secretion of p17s. We also demonstrated that heparan sulfate proteoglycans were involved in tethering p17s at the cell surface. This finding opens up an interesting way for investigating whether tethered p17s on the surface of HIV-1 reservoirs may represent a likely target for immune-mediated killing. 相似文献
5.
Analysis of the Assembly Function of the Human Immunodeficiency Virus Type 1 Gag Protein Nucleocapsid Domain
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Previous studies have shown that in addition to its function in specific RNA encapsidation, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is required for efficient virus particle assembly. However, the mechanism by which NC facilitates the assembly process is not clearly established. Formally, NC could act by constraining the Pr55gag polyprotein into an assembly-competent conformation or by masking residues which block the assembly process. Alternatively, the capacity of NC to bind RNA or make interprotein contacts might affect particle assembly. To examine its role in the assembly process, we replaced the NC domain in Pr55gag with polypeptide domains of known function, and the chimeric proteins were analyzed for their abilities to direct the release of virus-like particles. Our results indicate that NC does not mask inhibitory domains and does not act passively, by simply providing a stable folded monomeric structure. However, replacement of NC by polypeptides which form interprotein contacts permitted efficient virus particle assembly and release, even when RNA was not detected in the particles. These results suggest that formation of interprotein contacts by NC is essential to the normal HIV-1 assembly process.Human immunodeficiency virus type 1 (HIV-1) encodes three major genes, gag, pol, and env, which are commonly found in all mammalian retroviruses. It also encodes accessory genes whose protein products are important for regulation of its life cycle (6, 30, 35). However, of all the genes encoded by HIV-1, only the protein product of the gag gene has been found to be necessary and sufficient for the assembly of virus-like particles (11, 13, 17, 22, 32, 33). The HIV-1 Gag protein initially is expressed as a 55-kDa polyprotein precursor (Pr55gag), but during or shortly after particle release, Pr55gag ordinarily is cleaved by the viral protease (PR). The products of the protease action are the four major viral proteins matrix (MA), capsid (CA), nucleocapsid (NC), and p6, and the two spacer polypeptides p2 and p1, which represent sequences between CA and NC and between NC and p6, respectively (15, 19, 23, 30).The HIV-1 nucleocapsid proteins have two Cys-X2-Cys-X4-His-X4-Cys (Cys-His) motifs, reminiscent of the zinc finger motifs found in many DNA binding proteins, and NC has been shown to facilitate the specific encapsidation of HIV-1 genomic RNAs. In addition to its encapsidation function, NC influences virus particle assembly (7, 10, 17, 21, 40). In particular, Gag proteins lacking the NC domain fail to assemble virus particles efficiently. Nevertheless, some chimeric Gag proteins which carry foreign sequences in place of NC have been shown to assemble and release virus particles at wild-type (wt) levels (2, 37, 40). Thus, it appears that in some circumstances, the role that NC plays in virus particle assembly can be replaced. To date, it is not clear how NC affects particle assembly, although several possibilities might be envisioned. One possibility is that deletion of NC unmasks inhibitory sequences in p2 or the C terminus of CA. Alternatively, NC may simply provide a stable monomeric folded structure which locks CA or other Gag domains into an assembly-competent conformation. Another possibility is that NC facilitates assembly by forming essential protein-protein contacts between neighbor Prgag molecules, as suggested in cross-linking studies (21). Finally, the assembly role of NC may stem from its RNA binding capabilities, a hypothesis supported by studies of Campbell and Vogt (5), which have shown that RNA facilitates the in vitro assembly of retroviral Gag proteins into higher-order structures.To distinguish among possible mechanisms by which NC facilitates HIV-1 assembly, we replaced NC with polypeptides having known structural characteristics and examined particle assembly directed by these chimeric proteins. Using this approach, we have found that NC does not play a passive role in HIV-1 assembly as either a mask to assembly inhibitor domains or a nonspecific, stably folded structure. Rather, sequences known to form strong interprotein contacts were observed to enhance assembly, suggesting a similar role for the NC domain itself. With several assembly-competent chimeric proteins, we detected no particle-associated RNAs. These results suggest that while RNA may be essential to virus assembly in the context of the wt Pr55gag protein, it is dispensable for formation of virus-like particles from chimeric proteins. 相似文献
6.
Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism. 总被引:18,自引:13,他引:5
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The interaction of the human immunodeficiency virus (HIV) Gag protein with the plasma membrane of a cell is a critical event in the assembly of HIV particles. The matrix protein region (MA) of HIV type 1 (HIV-1) Pr55Gag has previously been demonstrated to confer membrane-binding properties on the precursor polyprotein. Both the myristic acid moiety and additional determinants within MA are essential for plasma membrane binding and subsequent particle formation. In this study, we demonstrated the myristylation-dependent membrane interaction of MA in an in vivo membrane-binding assay. When expressed within mammalian cells, MA was found both in association with cellular membranes and in a membrane-free form. In contrast, the intact precursor Pr55Gag molecule analyzed in an identical manner was found almost exclusively bound to membranes. Both membrane-bound and membrane-free forms of MA were myristylated and phosphorylated. Differential membrane binding was not due to the formation of multimers, as dimeric and trimeric forms of MA were also found in both membrane-bound and membrane-free fractions. To define the requirements for membrane binding of MA, we analyzed the membrane binding of a series of MA deletion mutants. Surprisingly, deletions within alpha-helical regions forming the globular head of MA led to a dramatic increase in overall membrane binding. The stability of the MA-membrane interaction was not affected by these deletions, and no deletion eliminated membrane binding of the molecule. These results establish that myristic acid is a primary determinant of the stability of the Gag protein-membrane interaction and provide support for the hypothesis that a significant proportion of HIV-1 MA molecules may adopt a conformation in which myristic acid is hidden and unavailable for membrane interaction. 相似文献
7.
8.
Nunzia Scotti Fiammetta Alagna Enrico Ferraiolo Gelsomina Formisano Lorenza Sannino Luigi Buonaguro Angelo De Stradis Alessandro Vitale Luigi Monti Stefania Grillo Franco M. Buonaguro Teodoro Cardi 《Planta》2009,229(5):1109-1122
Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency
virus Gag (Pr55gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at
very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments,
the native Pr55gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the
5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared
to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg
FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended
in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling
VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
IGV publication no. 330 相似文献
9.
Virion-Targeted Viral Inactivation of Human Immunodeficiency Virus Type 1 by Using Vpr Fusion Proteins 总被引:4,自引:0,他引:4
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Gary P. Kobinger Alessandra Borsetti Zilin Nie Johanne Mercier Nesrine Daniel Heinrich G. Gttlinger ric A. Cohen 《Journal of virology》1998,72(7):5441-5448
Inactivation of progeny virions with chimeric virion-associated proteins represents a novel therapeutic approach against human immunodeficiency virus (HIV) replication. The HIV type 1 (HIV-1) Vpr gene product, which is packaged into virions, is an attractive candidate for such a strategy. In this study, we developed Vpr-based fusion proteins that could be specifically targeted into mature HIV-1 virions to affect their structural organization and/or functional integrity. Two Vpr fusion proteins were constructed by fusing to the first 88 amino acids of HIV-1 Vpr the chloramphenicol acetyltransferase enzyme (VprCAT) or the last 18 C-terminal amino acids of the HIV-1 Vpu protein (VprIE). These Vpr fusion proteins were initially designed to quantify their efficiency of incorporation into HIV-1 virions when produced in cis from the provirus. Subsequently, CD4+ Jurkat T-cell lines constitutively expressing the VprCAT or the VprIE fusion protein were generated with retroviral vectors. Expression of the VprCAT or the VprIE fusion protein in CD4+ Jurkat T cells did not interfere with cellular viability or growth but conferred substantial resistance to HIV replication. The resistance to HIV replication was more pronounced in Jurkat-VprIE cells than in Jurkat-VprCAT cells. Moreover, the antiviral effect mediated by VprIE was dependent on an intact p6gag domain, indicating that the impairment of HIV-1 replication required the specific incorporation of Vpr fusion protein into virions. Gene expression, assembly, or release was not affected upon expression of these Vpr fusion proteins. Indeed, the VprIE and VprCAT fusion proteins were shown to affect the infectivity of progeny virus, since HIV virions containing the VprCAT or the VprIE fusion proteins were, respectively, 2 to 3 times and 10 to 30 times less infectious than the wild-type virus. Overall, this study demonstrated the successful transfer of resistance to HIV replication in tissue cultures by use of Vpr-based antiviral genes. 相似文献
10.
Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA 总被引:11,自引:6,他引:5
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The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65gag and Moloney murine leukemia virus Pr63gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200gag-pol coincided closely with the molar loss of Pr63gag. Enhancement of Pr200gag-pol and Pr70gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67gag, appeared, whereas Pr63gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67gag which appeared, 1 mol of Pr63gag was lost. 相似文献
11.
Composition, arrangement and cleavage of the mouse mammary tumor virus polyprotein precursor Pr77gag and p110gag. 总被引:14,自引:0,他引:14
The amino acid sequence relationship between the nonglycosylated structural proteins of murine mammary tumor virus and the polyproteins from infected cells immunoprecipitated with an anti-p27 serum were examined using two-dimensional tryptic peptide mapping procedures. The proteins were labeled with 14C-lysine and 14C-arginine so that all but one of the tryptic peptides released from a protein could be detected. Previous studies have shown that immunoprecipitation of mammary tumor cells with anti-p27 serum results in the isolation of seven proteins in the molecular weight range of 34,000–160,000 daltons; and that cell-free translation using viral genomic RNA yields three p27-related proteins of 160,000, 110,000 and 77,000 daltons, similar to the three high molecular weight proteins detected in vivo. The proteins of lower molecular weight were thought to be cleavage intermediates of Pr77gag. As judged from the peptide maps, Pr77gag contained the complete sequences of the four major internal proteins of the virion (p27, pp21, p14 and p10) and possibly a fifth highly basic protein (p8) also found in virions. The putative cleavage intermediates, as expected, lacked some tryptic peptides that could be assigned to one or more of the major virion proteins and thus allow a scheme for the cleavage events to be constructed. p110gag contained all the tryptic peptides found in Pr77gag, plus some additional peptides. A minor virion protein p30 was found to include the peptides of p14 as well as some of the additional peptides present in p110gag, suggesting a precursor-product relationship between the pr110gag and p30. The data obtained from these studies lead us to propose that there are three protein precursors which include, at least in part, the gag gene region of the virion—p160 (potentially a gag/pol precursor), p110gag and Pr77gag—and that the arrangement of the virion proteins within the gag gene (pr77gag) is p10-pp21-p27-p14. 相似文献
12.
Szu-Wei Huang Sheng-Fan Wang Yu-Ting Lin Chia-Hung Yen Chih-Hao Lee Wing-Wai Wong Hung-Chin Tsai Chia-Jui Yang Bor-Shen Hu Yu-Huei Lin Chin-Tien Wang Jaang-Jiun Wang Zixin Hu Daniel R. Kuritzkes Yen-Hsu Chen Yi-Ming Arthur Chen 《PloS one》2014,9(12)
The circulating recombinant form (CRF) 07_BC is the most prevalent HIV-1 strain among injection drug users (IDUs) in Taiwan. It contains a 7 amino-acid deletion in its p6gag. We conducted a cohort study to compare viral loads and CD4 cell count changes between patients infected with subtype B and CRF07_BC and to elucidate its mechanism. Twenty-one patients infected with CRF07_BC and 59 patients with subtype B were selected from a cohort of 667 HIV-1/AIDS patients whom have been followed up for 3 years. Generalized estimated equation was used to analyze their clinical data and the results showed that patients infected with CRF07_BC had significantly lower viral loads (about 58,000 copies per ml less) than patients with subtype B infection (p = 0.002). The replicative capacity of nine CRF07_BC and four subtype B isolates were compared and the results showed that the former had significantly lower replicative capacity than the latter although all of them were CCR5- tropic and non-syncytium inducing viruses. An HIV-1-NL4-3 mutant virus which contains a 7 amino-acid deletion in p6gag (designated as 7d virus) was generated and its live cycle was investigated. The results showed that 7d virus had significantly lower replication capacity, poorer protease-mediated processing and viral proteins production. Electron microscopic examination of cells infected with wild-type or 7d virus demonstrated that the 7d virus had poorer and slower viral maturation processes: more viruses attached to the cell membrane and higher proportion of immature virions outside the cells. The interaction between p6gag and Alix protein was less efficient in cells infected with 7d virus. In conclusion, patients infected with CRF07_BC had significantly lower viral loads than patients infected with subtype B and it may due to the deletion of 7 amino acids which overlaps with Alix protein-binding domain of the p6gag. 相似文献
13.
Extensive Diversification of Human Immunodeficiency Virus Type 1 Subtype B Strains during Dual Infection of a Chimpanzee That Progressed to AIDS 总被引:2,自引:1,他引:1
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A chimpanzee (C-499) infected for more than 9 years with two subtype B isolates of human immunodeficiency virus type 1 (HIV-1), one (HIV-1SF2) that replicates poorly and one (HIV-1LAV-1b) that replicates efficiently in chimpanzees, died of AIDS 11 years after initial infection (F. J. Novembre et al., J. Virol. 71:4086–4091, 1997). Nucleotide sequence and phylogenetic analyses of the C2 to V5 region of env (C2-V5env) in proviral DNA from peripheral blood lymphocytes obtained 22 months before death revealed two distinct virus populations. One of these populations appeared to be a recombinant in env, having the V3 loop from HIV-1SF2 and the V4-V5 region from HIV-1LAV-1b; the other population had evolved from HIV-1LAV-1b. In addition to C2-V5env, the entire p17gag and nef genes were sequenced; however, based on nucleotide sequences and phlyogeny, whether the progenitor of the p17gag and nef genes was SF2 or LAV-1b could not be determined. Compared to the two original viruses, the divergence of all clones of C2-V5env ranged from 9.37 to 20.2%, that of p17gag ranged from 3.11 to 9.29%, and that of nef ranged from 4.02 to 7.9%. In contrast, compared to the maximum variation of 20.2% in C2-V5env for C-499, the maximum diversities in C2-V5env in proviruses from two chimpanzees infected with HIV-1LAV-1b for 9 and 10 years were 9.65 and 2.48%, respectively. These results demonstrate that (i) two distinct HIV-1 populations can coexist and undergo extensive diversification in chimpanzees with progressive HIV-1-induced disease and (ii) recombination between two subtype B strains occurred even though the second strain was inoculated 15 months after the first one. Furthermore, evaluation of env genes from three chimpanzees infected with the same strain suggests that the magnitude of HIV-1 diversification could be related to higher viral burdens, manifestations of disease, and/or dual infection. 相似文献
14.
15.
The capsid (CA) and nucleocapsid domains of the human immunodeficiency virus type 1 Gag polyprotein are separated by the p2 spacer peptide, which is essential for virus replication. Previous studies have revealed that p2 has an important role in virus morphogenesis. In this paper, we show that a crucial assembly determinant maps to the highly conserved N terminus of p2, which is predicted to form part of an α-helix that begins in CA. A mutational analysis indicates that the ability of the N terminus of p2 to adopt an α-helical structure is essential for its function during virus assembly. To prevent CA-p2 processing, it was necessary to mutate both the CA-p2 cleavage site and an internal cleavage site within p2. Virions produced by the double mutant lacked a conical core shell and instead contained a thin electron-dense shell about 10 nm underneath the virion membrane. These results suggest that p2 is transiently required for proper assembly, but needs to be removed from the C terminus of CA to weaken CA-CA interactions and allow the rearrangement of the virion core shell during virus maturation.The internal structural proteins of the human immunodeficiency virus type 1 (HIV-1) virion are synthesized in the form of a polyprotein (Pr55gag) which can efficiently form enveloped virus-like particles even when expressed alone (17). Pr55gag is modified by N-terminal myristylation, which is required for its stable association with the inner leaflet of the plasma membrane, where virus assembly occurs (4, 21). During or after the release of an immature particle from the plasma membrane, Pr55gag is cleaved by the viral protease. The major Gag cleavage products are matrix (MA), capsid (CA), nucleocapsid (NC), and p6 (25, 34). MA, which has a crucial role in the incorporation of the viral surface glycoproteins (10, 52), remains associated with the host cell-derived lipid envelope of the virion (16). CA forms the shell of the characteristic cone-shaped core of the mature virion which encloses the viral genomic RNA (16, 27). NC is essential for the encapsidation of the viral genome and is believed to coat the viral RNA within the core of the virion (2, 19, 30). The C-terminal p6 domain of Pr55gag facilitates the release of assembled viral particles from the cell surface (20) and is also needed for the incorporation of the regulatory viral protein Vpr (31, 39).Within the context of Pr55gag, two spacer peptides, p2 and p1, are located between CA and NC and between NC and p6, respectively (24, 25). Cleavage between CA and p2 is much slower than that between p2 and NC or between MA and CA (41). As a consequence, a CA-p2 protein (p25) accumulates in virus-producing cells (34). However, CA-p2 is normally found only in trace amounts in virions. In addition to p2, which comprises 14 amino acids (Ala-363 through Met-376) of the HIV-1HXB2 Gag precursor, a 10-amino-acid p2 fragment which extends from Ser-367 through Met-376 has been isolated from HIV-1 virions, indicating that the viral protease can also cleave within p2 (24, 25).Genetic analyses indicate that the region surrounding the CA-p2 boundary has an important role in particle assembly (21, 28, 50). Within CA, the N-terminal two-thirds forms a domain which appears dispensable for particle assembly but is required for the formation of the cone-shaped core of the mature virion (8, 44, 51). Recent structure determinations have revealed that the N-terminal HIV-1 CA domain is largely α-helical (18, 35). An exposed loop region between two α-helices interacts with the prolyl isomerase cyclophilin A (14), which leads to the incorporation of the cellular enzyme into virions (13, 48). The C-terminal third of CA forms a distinct domain which is essential for Gag oligomerization and particle assembly (8, 12, 44). While genetic and structural studies indicate that the N-terminal boundary of the CA assembly domain coincides with a uniquely conserved sequence, termed the major homology region (8, 15, 18, 32), its C-terminal boundary remains less well defined.The replacement of the scissile dipeptide Leu-Ala at the CA-p2 boundary with Ser-Arg in a mutant designated SVC-C2 led to the formation of grossly distorted capsid structures and caused a significant reduction in particle yield, indicating that the very C terminus of CA and/or p2 is crucial for HIV-1 morphogenesis (21). The possibility that the CA assembly domain extends into p2 is also suggested by the finding that the precise deletion of p2 from Pr55gag markedly reduced particle production (28). Electron microscopy revealed an accumulation of large electron-dense plaques underneath the plasma membrane in the absence of p2 (28), a phenotype which is similar to that observed for the SVC-C2 cleavage site mutant (21). However, the role of p2 in virus assembly remains controversial, because its removal appeared to have no effect on particle release in another study (41).In the present study, we focused on the N-terminal portion of p2, since it is considerably more conserved than the C terminus and because it is predicted to be part of an α-helix which begins in CA. The analysis of a panel of single-amino-acid changes shows that the conserved N terminus of p2 is essential for virus replication and indicates that its predicted α-helical conformation is crucial for virus assembly. In contrast, a deletion which removed 5 out of 10 amino acids between a previously reported cleavage site within p2 and NC delayed but did not abolish virus replication, demonstrating that this relatively variable region of p2 has no essential function in the viral life cycle. We also show that processing of CA-p2 can be essentially prevented by disrupting both the CA-p2 cleavage site and the reported Met-Ser site (25) within p2. Interestingly, the mutant particles often contained a prominent circular structure underneath the viral membrane, indicating that the presence of p2 at the C terminus of CA prevented the rearrangement of the core into a conical tube. 相似文献
16.
Christy R. Gaines Emre Tkacik Amalia Rivera-Oven Phoebe Somani Alecia Achimovich Tawakalitou Alabi Angela Zhu Noel Getachew Ae Lim Yang Matthew McDonough Tarik Hawkins Zoe Spadaro Michael F. Summers 《Journal of molecular biology》2018,430(14):2113-2127
The N-terminally myristoylated matrix (MA) domain of the HIV-1 Gag polyprotein promotes virus assembly by targeting Gag to the inner leaflet of the plasma membrane. Recent studies indicate that, prior to membrane binding, MA associates with cytoplasmic tRNAs (including tRNALys3), and in vitro studies of tRNA-dependent MA interactions with model membranes have led to proposals that competitive tRNA interactions contribute to membrane discrimination. We have characterized interactions between native, mutant, and unmyristylated (myr-) MA proteins and recombinant tRNALys3 by NMR spectroscopy and isothermal titration calorimetry. NMR experiments confirm that tRNALys3 interacts with a patch of basic residues that are also important for binding to the plasma membrane marker, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Unexpectedly, the affinity of MA for tRNALys3 (Kd = 0.63 ± 0.03 μM) is approximately 1 order of magnitude greater than its affinity for PI(4,5)P2-enriched liposomes (Kd(apparent) = 10.2 ± 2.1 μM), and NMR studies indicate that tRNALys3 binding blocks MA association with liposomes, including those enriched with PI(4,5)P2, phosphatidylserine, and cholesterol. However, the affinity of MA for tRNALys3 is diminished by mutations or sample conditions that promote myristate exposure. Since Gag–Gag interactions are known to promote myristate exposure, our findings support virus assembly models in which membrane targeting and genome binding are mechanistically coupled. 相似文献
17.
18.
19.
Viraj Kulkarni Antonio Valentin Margherita Rosati Morgane Rolland James I. Mullins George N. Pavlakis Barbara K. Felber 《PloS one》2014,9(10)
To target immune responses towards invariable regions of the virus, we engineered DNA-based immunogens encoding conserved elements (CE) of HIV-1 p24gag. This conserved element vaccine is designed to avoid decoy epitopes by focusing responses to critical viral elements. We previously reported that vaccination of macaques with p24CE DNA induced robust cellular immune responses to CE that were not elicited upon wild type p55gag DNA vaccination. p24CE DNA priming followed by p55gag DNA boost provided a novel strategy to increase the magnitude and breadth of the cellular immune responses to HIV-1 Gag, including the induction of strong, multifunctional T-cell responses targeting epitopes within CE. Here, we examined the humoral responses induced upon p24CE DNA or p55gag DNA vaccination in macaques and found that although both vaccines induced robust p24gag binding antibody responses, the responses induced by p24CE DNA showed a unique broad range of linear epitope recognition. In contrast, antibodies elicited by p55gag DNA vaccine failed to recognize p24CE protein and did not recognize linear epitopes spanning the CE. Interestingly, boosting of p24CE DNA primed animals with p55gag DNA resulted in augmentation of antibodies able to recognize p24gag as well as the p24CE proteins, thereby inducing broadest immunity. Our results indicate that an effectively directed vaccine strategy that includes priming with the conserved element vaccine followed by boost with the complete immunogen induces broad cellular and humoral immunity focused on the conserved regions of the virus. This novel and effective strategy to broaden responses could be applied against other antigens of highly diverse pathogens. 相似文献