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1.
We have recently demonstrated that alteration of the human immunodeficiency virus type 1 (HIV-1) Gag/Gag-Pol ratio in virus-producing cells reduces the infectivity of progeny viruses and hinders the formation of stable virion RNA dimers without impairing virion packaging of the viral genomic RNA. In addition, we have previously shown that the expression of GagPol mediates the selective packaging of tRNA Lys3 . In this study we report that overexpression of uncleaved GagPol in the virus-producing cell did not alter the packaging levels of tRNA Lys3 . Similarly, altering the virion-associated Gag/GagPol ratio did not affect the virion packaging of the HIV-1 envelope protein nor cyclophilin A. Thin section electron microscopy analysis of the cells overexpressing protease-defective [PR(-)] GagPol revealed immature virions but no mature virions. These immature virions were seen both extracellularly and in membrane-bound cytoplasmic vacuoles. Furthermore, an accumulation of electron-dense material was occasionally found at the plasma membrane and associated with intracytoplasmic membranous vacuoles in cells expressing excess PR(–) GagPol. No intracellular HIV was seen in the wild-type control. Density gradient analysis showed that the overall density of these mutant virions with excess PR(–) GagPol was identical to that of the wild-type HIV-1. The findings indicate that overexpression of PR(–) GagPol, in the presence of Gag synthesis, promotes intracellular budding of the mutant virions and inhibits virus maturation.  相似文献   

2.
Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.  相似文献   

3.
Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism.  相似文献   

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Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in cis, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself.  相似文献   

8.
X Wu  H Liu  H Xiao  J A Conway    J C Kappes 《Journal of virology》1996,70(6):3378-3384
The human immunodeficiency virus type I (HIV-1) Vpr and HIV-2 Vpx proteins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have been exploited to incorporate foreign proteins into virions by expression as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995). To explore the possibility of utilizing Vpx and Vpr to target dominant negative mutants of the HIV Pol proteins into virions, we fused HIV-2 Vpx with an enzymatically defective protease (PR) mutant. Using a vector system to facilitate transient coexpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein was expressed and packaged efficiently into HIV-2 and simian immunodeficiency virus virions. Immunoblot analysis of purified virions demonstrated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-characterized active-site PR inhibitor. The incomplete processing of Gag and Gag/Pol was consistent with a 25-fold reduction in virion infectivity. The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions. Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were normal with respect to the processing of Gag protein and the ability to infect and replicate in vitro. These results indicate that VpxPR(M) specifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign proteins into virions via fusion with Vpx can inhibit HIV replication. The use of accessory proteins as vehicles to deliver deleterious proteins to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antiretroviral strategies. The ability to package PR by expression in trans, independent of the Gag/Pol precursor, also represents a novel approach that may be exploited to study the function of the Pol proteins.  相似文献   

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In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag–gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5′-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein–protein interactions.  相似文献   

11.
A single protein, termed Gag, is responsible for retrovirus particle assembly. After the assembled virion is released from the cell, Gag is cleaved at several sites by the viral protease (PR). The cleavages catalyzed by PR bring about a wide variety of physical changes in the particle, collectively termed maturation, and convert the particle into an infectious virion. In murine leukemia virus (MLV) maturation, Gag is cleaved at three sites, resulting in formation of the matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. We introduced mutations into MLV that inhibited cleavage at individual sites in Gag. All mutants had lost the intensely staining ring characteristic of immature particles; thus, no single cleavage event is required for this feature of maturation. Mutant virions in which MA was not cleaved from p12 were still infectious, with a specific infectivity only approximately 10-fold below that of the wild type. Particles in which p12 and CA could not be separated from each other were noninfectious and lacked a well-delineated core despite the presence of dense material in their interiors. In both of these mutants, the dimeric viral RNA had undergone the stabilization normally associated with maturation, suggesting that this change may depend upon the separation of CA from NC. Alteration of the C-terminal end of CA blocked CA-NC cleavage but also reduced the efficiency of particle formation and, in some cases, severely disrupted the ability of Gag to assemble into regular structures. This observation highlights the critical role of this region of Gag in assembly.  相似文献   

12.
Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus.  相似文献   

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During human immunodeficiency virus type 1 (HIV-1) assembly in HIV-1-transfected COS7 cells, almost all steady-state Gag/Gag and Gag/GagPol complexes are membrane bound. However, exposure to 1% Triton X-100 gives results indicating that while all Gag/GagPol complexes remain associated with the detergent-resistant membrane (DRM), only 30% of Gag/Gag complexes are associated with the DRM. Analysis of the localization of newly synthesized Gag/Gag and Gag/GagPol to the membrane indicates that after a 10-min pulse with radioactive [(35)S]Cys-[(35)S]Met, all newly synthesized Gag/GagPol is found at the DRM. Only 30% of newly synthesized Gag/Gag moves to the membrane, and at 0 min of chase, only 38% of this membrane-bound Gag/Gag is associated with the DRM. During the first 30 min of chase, most membrane-bound Gag/Gag moves to the DRM, while between 30 and 60 min of chase, there is a significant decrease in membrane-bound Gag/Gag and Gag/GagPol. Since the localization of newly synthesized Gag/Gag to the DRM and the interaction of GagPol with Gag both depend upon Gag multimerization, the rapid localization of GagPol to the DRM probably reflects the interaction of all newly synthesized GagPol with the first newly synthesized polymeric Gag to associate with the DRM.  相似文献   

16.
Luo K  Liu B  Xiao Z  Yu Y  Yu X  Gorelick R  Yu XF 《Journal of virology》2004,78(21):11841-11852
APOBEC3G exerts its antiviral activity by targeting to retroviral particles and inducing viral DNA hypermutations in the absence of Vif. However, the mechanism by which APOBEC3G is packaged into virions remains unclear. We now report that viral genomic RNA enhances but is not essential for human APOBEC3G packaging into human immunodeficiency virus type 1 (HIV-1) virions. Packaging of APOBEC3G was also detected in HIV-1 Gag virus-like particles (VLP) that lacked all the viral genomic RNA packaging signals. Human APOBEC3G could be packaged efficiently into a divergent subtype HIV-1, as well as simian immunodeficiency virus, strain mac, and murine leukemia virus Gag VLP. Cosedimentation of human APOBEC3G and intracellular Gag complexes was detected by equilibrium density and velocity sucrose gradient analysis. Interaction between human APOBEC3G and HIV-1 Gag was also detected by coimmunoprecipitation experiments. This interaction did not require p6, p1, or the C-terminal region of NCp7. However, the N-terminal region, especially the first 11 amino acids, of HIV-1 NCp7 was critical for HIV-1 Gag and APOBEC3G interaction and virion packaging. The linker region flanked by the two active sites of human APOBEC3G was also important for efficient packaging into HIV-1 Gag VLP. Association of human APOBEC3G with RNA-containing intracellular complexes was observed. These results suggest that the N-terminal region of HIV-1 NC, which is critical for binding to RNA and mediating Gag-Gag oligomerization, plays an important role in APOBEC3G binding and virion packaging.  相似文献   

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18.
J Luban  C Lee    S P Goff 《Journal of virology》1993,67(6):3630-3634
We have expressed the human immunodeficiency virus type 1 (HIV-1) protease (PR) in bacteria as a Gag-PR polyprotein (J. Luban and S.P. Goff, J. Virol. 65:3203-3212, 1991). The protein displays enzymatic activity, cleaving the Gag polyprotein precursor Pr55gag to the expected products. The PR enzyme is only active as a dimer, and we hypothesized that PR activation might be used as an indicator of polyprotein multimerization. We constructed 25 linker insertion mutations throughout gag and assessed the PR activity of mutant Gag-PR polyproteins by the appearance of Gag cleavage products in bacterial lysates. All mutant constructs produced stable protein in bacteria. PR activity of the majority of the Gag-PR mutants was indistinguishable from that of the wild type. Six mutants, one with an insertion in the matrix (MA), four with insertions in the capsid (CA), and one with insertions in the nucleocapsid (NC), globally disrupted polyprotein processing. When PR was provided in trans on a separate plasmid, the Gag proteins were cleaved with wild-type efficiency. These results suggest that the gag mutations identified as disruptive of polyprotein processing did not conceal the scissile bonds of the polyprotein. Rather, the mutations prevented PR activation in the context of a Gag-PR polyprotein, perhaps by preventing polyprotein dimerization.  相似文献   

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J Colgan  H E Yuan  E K Franke    J Luban 《Journal of virology》1996,70(7):4299-4310
The cellular peptidyl-prolyl isomerase cyclophilin A (CyPA) is incorporated into human immunodeficiency virus type 1 (HIV-1) virions via direct contacts with the HIV-1 Gag polyprotein. Disruption of the Gag-CyPA interaction leads to the production of HIV-1 particles lacking CyPA; these virions are noninfectious, indicating that contacts between CyPA and Gag are necessary for HIV-1 replication. Here, we have used the yeast two-hybrid system in conjunction with an in vitro binding assay to identify the minimal domain of Gag required for binding to CyPA. Analysis of a panel of gag deletion mutants in the two-hybrid system indicated that a region spanning the central portion of the capsid (CA) domain was sufficient for interactions with CyPA, but discrepancies between results obtained in different fusion protein contexts suggested that multimerization of Gag might also be necessary for binding to CyPA. Consistent with a requirement for multimerization, the binding of Gag to CyPA in vitro required a region within the nucleocapsid (NC) domain shown previously to be important for Gag self-association. Substitution of a heterologous dimerization motif for the region from NC also promoted specific binding to CyPA, confirming that interactions with CyPA are dependent on Gag multimerization. Fusion of the heterologous dimerization motif to a 100-amino-acid domain from CA was sufficient for binding to CyPA in vitro. These results define the minimal CyPA-binding domain within Gag and provide insight into the mechanism by which CyPA is incorporated into HIV-1 virions.  相似文献   

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