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1.
HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells. 相似文献
2.
The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates. 相似文献
3.
RNA incorporation is critical for retroviral particle integrity after cell membrane assembly of Gag complexes
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The nucleocapsid (NC) domain of retroviruses plays a critical role in specific viral RNA packaging and virus assembly. RNA is thought to facilitate viral particle assembly, but the results described here with NC mutants indicate that it also plays a critical role in particle integrity. We investigated the assembly and integrity of particles produced by the human immunodeficiency virus type 1 M1-2/BR mutant virus, in which 10 of the 13 positive residues of NC have been replaced with alanines and incorporation of viral genomic RNA is virtually abolished. We found that the mutations in the basic residues of NC did not disrupt Gag assembly at the cell membrane. The mutant Gag protein can assemble efficiently at the cell membrane, and viral proteins are detected outside the cell as efficiently as they are for the wild type. However, only approximately 10% of the Gag molecules present in the supernatant of this mutant sediment at the correct density for a retroviral particle. The reduction of positive charge in the NC basic domain of the M1-2/BR virus adversely affects both the specific and nonspecific RNA binding properties of NC, and thus the assembled Gag polyprotein does not bind significant amounts of viral or cellular RNA. We found a direct correlation between the percentage of Gag associated with sedimented particles and the amount of incorporated RNA. We conclude that RNA binding by Gag, whether the RNA is viral or not, is critical to retroviral particle integrity after cell membrane assembly and is less important for Gag-Gag interactions during particle assembly and release. 相似文献
4.
HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. 总被引:1,自引:0,他引:1
Retroviral Gag proteins encode sequences, termed late domains, which facilitate the final stages of particle budding from the plasma membrane. We report here that interactions between Tsg101, a factor involved in endosomal protein sorting, and short peptide motifs in the HIV-1 Gag late domain and Ebola virus matrix (EbVp40) proteins are essential for efficient egress of HIV-1 virions and Ebola virus-like particles. EbVp40 recruits Tsg101 to sites of particle assembly and a short, EbVp40-derived Tsg101-binding peptide sequence can functionally substitute for the HIV-1 Gag late domain. Notably, recruitment of Tsg101 to assembling virions restores budding competence to a late-domain-defective HIV-1 in the complete absence of viral late domain. These studies define an essential virus-host interaction that is conserved in two unrelated viruses. Because the Tsg101 is recruited by small, conserved viral sequence motifs, agents that mimic these structures are potential inhibitors of the replication of these lethal human pathogens. 相似文献
5.
Ternois F Sticht J Duquerroy S Kräusslich HG Rey FA 《Nature structural & molecular biology》2005,12(8):678-682
Immature HIV particles bud from infected cells after assembly at the cytoplasmic side of cellular membranes. This assembly is driven by interactions between Gag polyproteins. Mature particles, each containing a characteristic conical core, are later generated by proteolytic maturation of Gag in the virion. The C-terminal domain of the HIV-1 capsid protein (C-CA) has been shown to contain oligomerization determinants essential for particle assembly. Here we report the 1.7-A-resolution crystal structure of C-CA in complex with a peptide capable of inhibiting immature- and mature-like particle assembly in vitro. The peptide inserts as an amphipathic alpha-helix into a conserved hydrophobic groove of C-CA, resulting in formation of a compact five-helix bundle with altered dimeric interactions. This structure thus reveals the details of an allosteric site in the HIV capsid protein that can be targeted for antiviral therapy. 相似文献
6.
7.
Many pathological processes are driven by RNA-protein interactions, making such interactions promising targets for molecular interventions. HIV-1 assembly is one such process, in which the viral genomic RNA interacts with the viral Gag protein and serves as a scaffold to drive Gag multimerization that ultimately leads to formation of a virus particle. Here, we develop self-assembled RNA nanostructures that can inhibit HIV-1 virus assembly, achieved through hybridization of multiple artificial small RNAs with a stem–loop structure (STL) that we identify as a prominent ligand of Gag that can inhibit virus particle production via STL-Gag interactions. The resulting STL-decorated nanostructures (double and triple stem–loop structures denoted as Dumbbell and Tribell, respectively) can elicit more pronounced viral blockade than their building blocks, with the inhibition arising as a result of nanostructures interfering with Gag multimerization. These findings could open up new avenues for RNA-based therapy. 相似文献
8.
9.
AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly 总被引:17,自引:0,他引:17
Dong X Li H Derdowski A Ding L Burnett A Chen X Peters TR Dermody TS Woodruff E Wang JJ Spearman P 《Cell》2005,120(5):663-674
Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway. 相似文献
10.
Datta SA Heinrich F Raghunandan S Krueger S Curtis JE Rein A Nanda H 《Journal of molecular biology》2011,406(2):205-564
The retroviral Gag polyprotein mediates viral assembly. The Gag protein has been shown to interact with other Gag proteins, with the viral RNA, and with the cell membrane during the assembly process. Intrinsically disordered regions linking ordered domains make characterization of the protein structure difficult. Through small-angle scattering and molecular modeling, we have previously shown that monomeric human immunodeficiency virus type 1 (HIV-1) Gag protein in solution adopts compact conformations. However, cryo-electron microscopic analysis of immature virions shows that in these particles, HIV-1 Gag protein molecules are rod shaped. These differing results imply that large changes in Gag conformation are possible and may be required for viral formation. By recapitulating key interactions in the assembly process and characterizing the Gag protein using neutron scattering, we have identified interactions capable of reversibly extending the Gag protein. In addition, we demonstrate advanced applications of neutron reflectivity in resolving Gag conformations on a membrane. Several kinds of evidence show that basic residues found on the distal N- and C-terminal domains enable both ends of Gag to bind to either membranes or nucleic acid. These results, together with other published observations, suggest that simultaneous interactions of an HIV-1 Gag molecule with all three components (protein, nucleic acid, and membrane) are required for full extension of the protein. 相似文献
11.
Identification of a Conserved Residue of Foamy Virus Gag Required for Intracellular Capsid Assembly
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In contrast to all retroviruses but similar to the hepatitis B virus, foamy viruses (FV) require expression of the envelope protein for budding of intracellular capsids from the cell, suggesting a specific interaction between the Gag and Env proteins. Capsid assembly occurs in the cytoplasm of infected cells in a manner similar to that for the B- and D-type viruses; however, in contrast to these retroviruses, FV Gag lacks an N-terminal myristylation signal and capsids are not targeted to the plasma membrane (PM). We have found that mutation of an absolutely conserved arginine (Arg) residue at position 50 to alanine (R50A) of the simian foamy virus SFV cpz(hu) inhibits proper capsid assembly and abolishes viral budding even in the presence of the envelope (Env) glycoproteins. Particle assembly and extracellular release of virus can be restored to this mutant with the addition of an N-terminal Src myristylation signal (Myr-R50A), presumably by providing an alternate site for assembly to occur at the PM. In addition, the strict requirement of Env expression for capsid budding can be bypassed by addition of a PM-targeting signal to Gag. These results suggest that intracellular capsid assembly may be mediated by a signal akin to the cytoplasmic targeting and retention signal CTRS found in Mason-Pfizer monkey virus and that FV Gag has the inherent ability to assemble capsids at multiple sites like conventional retroviruses. The necessity of Env expression for particle egress is most probably due to the lack of a membrane-targeting signal within FV Gag to direct capsids to the PM for release and indicates that Gag-Env interactions are essential to drive particle budding. 相似文献
12.
Pregenomic RNA is required for efficient incorporation of pol polyprotein into foamy virus capsids
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Heinkelein M Leurs C Rammling M Peters K Hanenberg H Rethwilm A 《Journal of virology》2002,76(19):10069-10073
The foamy virus (FV) Pol polyprotein is translated independently of Gag from a spliced mRNA. This method of expression raises the question of how Pol is associated with the viral particle. Using a transient FV vector transfection system, it is shown that pregenomic RNA is required for efficient virion incorporation of functionally active Pol and that protein-protein interactions of Pol with Gag are not sufficient to complete particle assembly. 相似文献
13.
A novel fluorescence resonance energy transfer assay demonstrates that the human immunodeficiency virus type 1 Pr55Gag I domain mediates Gag-Gag interactions 总被引:3,自引:0,他引:3
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Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55(Gag) 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 Pr55(Gag), 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. 相似文献
14.
In Vitro Assembly Properties of Human Immunodeficiency Virus Type 1 Gag Protein Lacking the p6 Domain 总被引:21,自引:17,他引:4
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Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Δp6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Δp6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Δp6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Δp6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Δp6 and nucleic acid are not sufficient for the formation of normal-sized particles. 相似文献
15.
An assembly domain of the Rous sarcoma virus Gag protein required late in budding. 总被引:29,自引:27,他引:2
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J W Wills C E Cameron C B Wilson Y Xiang R P Bennett J Leis 《Journal of virology》1994,68(10):6605-6618
The Gag protein of Rous sarcoma virus has the ability to direct particle assembly at the plasma membrane in the absence of all the other virus-encoded components. An extensive deletion analysis has revealed that very large regions of this protein can be deleted without impairing budding and has suggested that the essential functions map to three discrete regions. In the studies reported here, we establish the location of assembly domain 2 (AD2) within the proline-rich p2b sequence of this Gag protein. AD2 mutants lacking the p2b sequence were completely defective for particle release even though their Gag proteins were tightly associated with the membrane fraction and exhibited high levels of protease activity. Mutations that inactivate the viral protease did not restore budding to wild-type levels for these mutants, indicating that the defect is not due simply to a loss of protease regulation. AD2 mutants could be rescued into dense particles in genetic complementation assays, indicating that their defect is not due to a gross alteration of the overall conformation of the protein and that the assembly function is not needed on every Gag molecule in the population. Several mutants with amino acid substitutions in the p2b sequence were found to have an intermediate capacity for budding. Inactivation of the protease of these mutants stabilized the Gag polyprotein within the cells and allowed an increase in particle release; however, the rate of budding remained slow. We favor the idea that AD2 is a dynamic region of movement, perhaps serving as a molecular hinge to allow the particle to emerge from the surface of the cell during budding. 相似文献
16.
17.
Incorporation of human immunodeficiency virus type 1 Gag proteins into murine leukemia virus virions. 总被引:13,自引:10,他引:3
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The retroviral Gag polyprotein is necessary and sufficient for assembly and budding of viral particles. However, the exact inter- and intramolecular interactions of the Gag polyproteins during this process are not known. To locate functional domains within Gag, we generated chimeric proviruses between human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV). In these chimeric proviruses, the matrix or capsid proteins of MuLV were precisely replaced with the matrix or capsid proteins of HIV-1. Although the chimeric proviruses were unable to efficiently assemble into mature viral particles by themselves, coexpression of wild-type MuLV Gag rescued the HIV proteins into virions. The specificity of the rescue of HIV proteins into MuLV virions shows that specific interactions involving homologous matrix or capsid regions of Gag are necessary for retroviral particle formation. 相似文献
18.
The Gag Domains Required for Avian Retroviral RNA Encapsidation Determined by Using Two Independent Assays
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Eun-gyung Lee Ashly Yeo Brian Kraemer Marvin Wickens Maxine L. Linial 《Journal of virology》1999,73(8):6282-6292
The Rous sarcoma virus (RSV) Gag precursor polyprotein is the only viral protein which is necessary for specific packaging of genomic RNA. To map domains within Gag which are important for packaging, we constructed a series of Gag mutations in conjunction with a protease (PR) active-site point mutation in a full-length viral construct. We found that deletion of either the matrix (MA), the capsid (CA), or the protease (PR) domain did not abrogate packaging, although the MA domain is likely to be required for proper assembly. A previously characterized deletion of both Cys-His motifs in RSV nucleocapsid protein (NC) reduced both the efficiency of particle release and specific RNA packaging by 6- to 10-fold, consistent with previous observations that the NC Cys-His motifs played a role in assembly and RNA packaging. Most strikingly, when amino acid changes at Arg 549 and 551 immediately downstream of the distal NC Cys-His box were made, RNA packaging was reduced by more than 25-fold with no defect in particle release, demonstrating the importance of this basic amino acid region in packaging. We also used the yeast three-hybrid system to study avian retroviral RNA-Gag interactions. Using this assay, we found that the interactions of the minimal packaging region (Mpsi) with Gag are of high affinity and specificity. Using a number of Mpsi and Gag mutants, we have found a clear correlation between a reporter gene activation in a yeast three-hybrid binding system and an in vivo packaging assay. Our results showed that the binding assay provides a rapid genetic assay of both RNA and protein components for specific encapsidation. 相似文献
19.
Viruses have to encapsidate their own genomes during the assembly process. For most RNA viruses, there are sequences within the viral RNA and virion proteins needed for high efficiency of genome encapsidation. However, the roles of host proteins in this process are not understood. Here we find that the cellular DEAD-box RNA helicase DDX6 is required for efficient genome packaging of foamy virus, a spumaretrovirus. After infection, a significant amount of DDX6, normally concentrated in P bodies and stress granules, re-localizes to the pericentriolar site where viral RNAs and Gag capsid proteins are concentrated and capsids are assembled. Knockdown of DDX6 by siRNA leads to a decreased level of viral nucleic acids in extracellular particles, although viral protein expression, capsid assembly and release, and accumulation of viral RNA and Gag protein at the assembly site are little affected. DDX6 does not interact stably with Gag proteins nor is it incorporated into particles. However, we find that the ATPase/helicase motif of DDX6 is essential for viral replication. This suggests that the ATP hydrolysis and/or the RNA unwinding activities of DDX6 function in moderating the viral RNA conformation and/or viral RNA-Gag ribonucleoprotein complex in a transient manner to facilitate incorporation of the viral RNA into particles. These results reveal a unique role for a highly conserved cellular protein of RNA metabolism in specifically re-locating to the site of viral assembly for its function as a catalyst in retroviral RNA packaging. 相似文献
20.
HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly. 相似文献