scholarly journals The Nucleocapsid Region of Human Immunodeficiency Virus Type 1 Gag Assists in the Coordination of Assembly and Gag Processing: Role for RNA-Gag Binding in the Early Stages of Assembly

2009 ◽  
Vol 83 (15) ◽  
pp. 7718-7727 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Teresa Shatzer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Protease Activity ◽  
Particle Production ◽  
Rna Binding ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Gag Assembly ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag-RNA interactions are required for virus assembly. However, our prior study found that a defect in particle production exhibited by an HIV-1 proviral mutant with a severe deletion in the RNA-binding nucleocapsid (NC) region of Gag, NX, could be reversed by eliminating its protease activity. While our follow-up study indicated that a secondary RNA-binding site in Gag can also provide the required RNA-binding function, how protease activity inhibits NX virion production is still unclear. Therefore, we tested three possible mechanisms: NX virions are unstable and fall apart after budding; NX Gag assembly is slowed, allowing protease processing to start before particle formation; or the protease region within NX Gag-Pol becomes activated prematurely and processes the assembling Gag. We found that NX particles were as stable as wild-type virions. Furthermore, even a modest slowing of protease activity could rescue NX. Pulse-chase analysis revealed that the initial particle production by NC-deleted Gag was delayed compared to that of wild type Gag, but once started, the rate of production was similar, revealing a defect in the initiation of assembly. Wild-type Gag particle production was not eliminated or decreased in the presence of excess NX Gag-Pol, inconsistent with a premature activation of protease. Overall, these results indicate that the particle formation defect of NX is due to delayed initiation of assembly caused by the absence of NC in Gag, making it vulnerable to protease processing before budding can occur. Therefore, NC plays an important initiating role in Gag assembly.

scholarly journals Elimination of Protease Activity Restores Efficient Virion Production to a Human Immunodeficiency Virus Type 1 Nucleocapsid Deletion Mutant

2003 ◽  
Vol 77 (10) ◽  
pp. 5547-5556 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Elena N. Chertova ◽  
Tracy D. Gagliardi ◽  
Kunio Nagashima ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Protease Activity ◽  
Genomic Rna ◽  
Wild Type ◽  
Virion Production ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four- to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10−6 relative to the wild type) and severely genomic RNA deficient. Immunoblot and high-pressure liquid chromatography analyses showed that all of the mature Gag proteins except NC were present in the mutant virion preparations, although there was a modest decrease in Gag processing. DelNC virions had lower densities and were more heterogeneous than wild-type particles, consistent with a defect in the interaction assembly or I domain. Electron microscopy showed that the DelNC virions displayed a variety of aberrant morphological forms. Inactivating the protease activity of DelNC by mutation or protease inhibitor treatment restored virion production to wild-type levels. DelNC-protease mutants formed immature-appearing particles that were as dense as wild-type virions without incorporating genomic RNA. Therefore, protease activity combined with the absence of NC causes the defect in DelNC virion production, suggesting that premature processing of Gag during assembly causes this effect. These results show that HIV-1 can form particles efficiently without NC.

scholarly journals Redundant Roles for Nucleocapsid and Matrix RNA-Binding Sequences in Human Immunodeficiency Virus Type 1 Assembly

2005 ◽  
Vol 79 (22) ◽  
pp. 13839-13847 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Tracy D. Gagliardi
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Matrix Protein ◽  
Particle Production ◽  
Rna Binding ◽  
Immunodeficiency Virus ◽  
The Matrix ◽  
Hiv 1

ABSTRACT RNA appears to be required for the assembly of retroviruses. This is likely due to binding of RNA by multiple Gags, which in turn organizes and stabilizes the Gag-Gag interactions that form the virion. While the nucleocapsid (NC) domain is the most conspicuous RNA-binding region of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, we have previously shown that NC is not strictly required for efficient particle production. To determine if an RNA requirement for HIV-1 assembly exists, we analyzed virions produced by an NC deletion mutant for the presence of RNA. The results revealed that virions without NC still contained significant amounts of RNA. Since these packaged RNAs are probably incorporated by other RNA-binding sequences in Gag, an RNA-binding site in the matrix protein (MA) of Gag was mutated. While this mutation did not interfere with HIV-1 replication, a construct with both MA and NC mutations (MX/NX) failed to produce particles. The MX/NX mutant was rescued in trans by coassembly with several forms of Gag: wild-type Gag, either of the single-mutant Gags, or Gag truncations that contain MA or NC sequences. Addition of basic sequences to the MX/NX mutant partially restored particle production, consistent with a requirement for Gag-RNA binding in addition to Gag-Gag interactions. Together, these results support an RNA-binding requirement for Gag assembly, which relies on binding of RNA by MA or NC sequences to condense, organize, and stabilize the HIV-1 Gag-Gag interactions that form the virion.

scholarly journals Multimerization of Human Immunodeficiency Virus Type 1 Gag Promotes Its Localization to Barges, Raft-Like Membrane Microdomains

2001 ◽  
Vol 75 (17) ◽  
pp. 7913-7924 ◽  
Author(s):  
O. Wolf Lindwasser ◽  
Marilyn D. Resh
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Membrane Microdomains ◽  
Wild Type ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The Gag polyprotein of human immunodeficiency virus type 1 (HIV-1) organizes the assembly of nascent virions at the plasma membrane of infected cells. Here we demonstrate that a population of Gag is present in distinct raft-like membrane microdomains that we have termed “barges.” Barges have a higher density than standard rafts, most likely due to the presence of oligomeric Gag-Gag assembly complexes. The regions of the Gag protein responsible for barge targeting were mapped by examining the flotation behavior of wild-type and mutant proteins on Optiprep density gradients. N-myristoylation of Gag was necessary for association with barges. Removal of the NC and p6 domains shifted much of the Gag from barges into typical raft fractions. These data are consistent with a model in which multimerization of myristoylated Gag proteins drives association of Gag oligomers into raft-like barges. The functional significance of barge association was revealed by several lines of evidence. First, Gag isolated from virus-like particles was almost entirely localized in barges. Moreover, a comparison of wild-type Gag with Fyn(10)Gag, a chimeric protein containing the N-terminal sequence of Fyn, revealed that Fyn(10)Gag exhibited increased affinity for barges and a two- to fourfold increase in particle production. These results imply that association of Gag with raft-like barge membrane microdomains plays an important role in the HIV-1 assembly process.

scholarly journals Association of Human Immunodeficiency Virus Type 1 Gag with Membrane Does Not Require Highly Basic Sequences in the Nucleocapsid: Use of a Novel Gag Multimerization Assay

2005 ◽  
Vol 79 (22) ◽  
pp. 14131-14140 ◽  
Author(s):  
Akira Ono ◽  
Abdul A. Waheed ◽  
Anjali Joshi ◽  
Eric O. Freed
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Lipid Rafts ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Immunodeficiency Virus ◽  
Gag Assembly ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) particle production, a process driven by the Gag polyprotein precursor, occurs on the plasma membrane in most cell types. The plasma membrane contains cholesterol-enriched microdomains termed lipid rafts, which can be isolated as detergent-resistant membrane (DRM). Previously, we and others demonstrated that HIV-1 Gag is associated with DRM and that disruption of Gag-raft interactions impairs HIV-1 particle production. However, the determinants of Gag-raft association remain undefined. In this study, we developed a novel epitope-based Gag multimerization assay to examine whether Gag assembly is essential for its association with lipid rafts. We observed that membrane-associated, full-length Gag is poorly detected by immunoprecipitation relative to non-membrane-bound Gag. This poor detection is due to assembly-driven masking of Gag epitopes, as denaturation greatly improves immunoprecipitation. Gag mutants lacking the Gag-Gag interaction domain located in the N terminus of the nucleocapsid (NC) were efficiently immunoprecipitated without denaturation, indicating that the epitope masking is caused by higher-order Gag multimerization. We used this assay to examine the relationship between Gag assembly and Gag binding to total cellular membrane and DRM. Importantly, a multimerization-defective NC mutant displayed wild-type levels of membrane binding and DRM association, indicating that NC-mediated Gag multimerization is dispensable for association of Gag with membrane or DRM. We also demonstrate that different properties of sucrose and iodixanol membrane flotation gradients may explain some discrepancies regarding Gag-raft interactions. This report offers new insights into the association of HIV-1 Gag with membrane and with lipid rafts.

scholarly journals Different Abilities of Escape Mutant-Specific Cytotoxic T Cells To Suppress Replication of Escape Mutant and Wild-Type Human Immunodeficiency Virus Type 1 in New Hosts

2007 ◽  
Vol 82 (1) ◽  
pp. 138-147 ◽  
Author(s):  
Mamoru Fujiwara ◽  
Junko Tanuma ◽  
Hirokazu Koizumi ◽  
Yuka Kawashima ◽  
Kazutaka Honda ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Escape Mutant ◽  
Wild Type ◽  
Specific Ctls ◽  
Immunodeficiency Virus ◽  
New Hosts ◽  
Hiv 1 ◽  
Hla Molecules

ABSTRACT There is much evidence that in human immunodeficiency virus type 1 (HIV-1)-infected individuals, strong cytotoxic T lymphocyte (CTL)-mediated immune pressure results in the selection of HIV-1 mutants that have escaped from wild-type-specific CTLs. If escape mutant-specific CTLs are not elicited in new hosts sharing donor HLA molecules, the transmission of these mutants results in the accumulation of escape mutants in the population. However, whether escape mutant-specific CTLs are definitively not elicited in new hosts sharing donor HLA molecules still remains unclear. A previous study showed that a Y-to-F substitution at the second position (2F) of the Nef138-10 epitope is significantly detected in HLA-A*2402+ hemophilic donors. Presently, we confirmed that this 2F mutant was an escape mutant by demonstrating strong and weak abilities of Nef138-10-specific CTL clones to suppress replication of the wild-type and 2F mutant viruses, respectively. We demonstrated the existence of the 2F-specific CTLs in three new hosts who had been primarily infected with the 2F mutant. The 2F-specific CTL clones suppressed the replication of both wild-type and mutant viruses. However, the abilities of these clones to suppress replication of the 2F virus were much weaker than those of wild-type-specific and the 2F-specific ones to suppress replication of the wild-type virus. These findings indicate that the 2F mutant is conserved in HIV-1-infected donors having HLA-A*2402, because the 2F-specific CTLs failed to completely suppress the 2F mutant replication and effectively prevented viral reversion in new hosts carrying HLA-A*2402.

scholarly journals Mutation of Dileucine-Like Motifs in the Human Immunodeficiency Virus Type 1 Capsid Disrupts Virus Assembly, Gag-Gag Interactions, Gag-Membrane Binding, and Virion Maturation

2006 ◽  
Vol 80 (16) ◽  
pp. 7939-7951 ◽  
Author(s):  
Anjali Joshi ◽  
Kunio Nagashima ◽  
Eric O. Freed
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Virus Assembly ◽  
Membrane Binding ◽  
Single Amino Acid ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein Pr55Gag drives the assembly and release of virus-like particles in the infected cell. The capsid (CA) domain of Gag plays an important role in these processes by promoting Gag-Gag interactions during assembly. The C-terminal domain (CTD) of CA contains two dileucine-like motifs (L189/L190 and I201/L202) implicated in regulating the localization of Gag to multivesicular bodies (MVBs). These dileucine-like motifs are located in the vicinity of the CTD dimer interface, a region of CA critical for Gag-Gag interactions during virus assembly and CA-CA interactions during core formation. To study the importance of the CA dileucine-like motifs in various aspects of HIV-1 replication, we introduced a series of mutations into these motifs in the context of a full-length, infectious HIV-1 molecular clone. CA mutants LL189,190AA and IL201,202AA were both severely impaired in virus particle production because of a variety of defects in the binding of Gag to membrane, Gag multimerization, and CA folding. In contrast to the model suggesting that the CA dileucine-like motifs regulate MVB targeting, the IL201,202AA mutation did not alter Gag localization to the MVB in either HeLa cells or macrophages. Revertants of single-amino-acid substitution mutants were obtained that no longer contained dileucine-like motifs but were nevertheless fully replication competent. The varied phenotypes of the mutants reported here provide novel insights into the interplay among Gag multimerization, membrane binding, virus assembly, CA dimerization, particle maturation, and virion infectivity.

scholarly journals Mutational Analysis of the Hydrophobic Tail of the Human Immunodeficiency Virus Type 1 p6Gag Protein Produces a Mutant That Fails To Package Its Envelope Protein

1999 ◽  
Vol 73 (1) ◽  
pp. 19-28 ◽  
Author(s):  
David E. Ott ◽  
Elena N. Chertova ◽  
Laura K. Busch ◽  
Lori V. Coren ◽  
Tracy D. Gagliardi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hydrophobic Tail ◽  
Carboxyl Terminal ◽  
Hiv 1

ABSTRACT The p6Gag protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6Gag between Y36 and P37, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6Gag proteins in HIV-1MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6Gag, site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6Gag, Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41TM cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6Gag mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.

scholarly journals The Thiocarboxanilide Nonnucleoside Inhibitor UC781 Restores Antiviral Activity of 3′-Azido-3′-Deoxythymidine (AZT) against AZT-Resistant Human Immunodeficiency Virus Type 1

1999 ◽  
Vol 43 (2) ◽  
pp. 259-263 ◽  
Author(s):  
Gadi Borkow ◽  
Dominique Arion ◽  
Mark A. Wainberg ◽  
Michael A. Parniak
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT N-[4-Chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbothioamide (UC781) is an exceptionally potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. We found that a 1:1 molar combination of UC781 and 3′-azido-3′-deoxythymidine (AZT) showed high-level synergy in inhibiting the replication of AZT-resistant virus, implying that UC781 can restore antiviral activity to AZT against AZT-resistant HIV-1. Neither the nevirapine plus AZT nor the 2′,5′-bis-O-(t-butyldimethylsilyl)-3′-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide plus AZT combinations had this effect. Studies with purified HIV-1 reverse transcriptase (from a wild type and an AZT-resistant mutant) showed that UC781 was a potent inhibitor of the pyrophosphorolytic cleavage of nucleotides from the 3′ end of the DNA polymerization primer, a process that we have proposed to be critical for the phenotypic expression of AZT resistance. Combinations of UC781 plus AZT did not act in synergy to inhibit the replication of either wild-type virus or UC781-resistant HIV-1. Importantly, the time to the development of viral resistance to combinations of UC781 plus AZT is significantly delayed compared to the time to the development of resistance to either drug alone.

scholarly journals Activity against Human Immunodeficiency Virus Type 1, Intracellular Metabolism, and Effects on Human DNA Polymerases of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

2007 ◽  
Vol 51 (8) ◽  
pp. 2701-2708 ◽  
Author(s):  
Hirotomo Nakata ◽  
Masayuki Amano ◽  
Yasuhiro Koh ◽  
Eiichi Kodama ◽  
Guangwei Yang ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Transcriptase Inhibitor ◽  
Multidrug Resistant ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT We examined the intracytoplasmic anabolism and kinetics of antiviral activity against human immunodeficiency virus type 1 (HIV-1) of a nucleoside reverse transcriptase inhibitor, 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), which has potent activity against wild-type and multidrug-resistant HIV-1 strains. When CEM cells were exposed to 0.1 μM [3H]EFdA or [3H]3′-azido-2′,3′-dideoxythymidine (AZT) for 6 h, the intracellular EFdA-triphosphate (TP) level was 91.6 pmol/109 cells, while that of AZT was 396.5 pmol/109 cells. When CEM cells were exposed to 10 μM [3H]EFdA, the amount of EFdA-TP increased by 22-fold (2,090 pmol/109 cells), while the amount of [3H]AZT-TP increased only moderately by 2.4-fold (970 pmol/109 cells). The intracellular half-life values of EFdA-TP and AZT-TP were ∼17 and ∼3 h, respectively. When MT-4 cells were cultured with 0.01 μM EFdA for 24 h, thoroughly washed to remove EFdA, further cultured without EFdA for various periods of time, exposed to HIV-1NL4-3, and cultured for an additional 5 days, the protection values were 75 and 47%, respectively, after 24 and 48 h with no drug incubation, while those with 1 μM AZT were 55 and 9.2%, respectively. The 50% inhibitory concentration values of EFdA-TP against human polymerases α, β, and γ were >100 μM, >100 μM, and 10 μM, respectively, while those of ddA-TP were >100 μM, 0.2 μM, and 0.2 μM, respectively. These data warrant further development of EFdA as a potential therapeutic agent for those patients who harbor wild-type HIV-1 and/or multidrug-resistant variants.

scholarly journals Multiple human immunodeficiency virus type 1 Nef functions contribute to efficient replication in primary human macrophages

2004 ◽  
Vol 85 (6) ◽  
pp. 1463-1469 ◽  
Author(s):  
Amanda Brown ◽  
Shaghayegh Moghaddam ◽  
Thomas Kawano ◽  
Cecilia Cheng-Mayer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Single Point ◽  
Wild Type Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Efficient Replication ◽  
Hiv 1

The human immunodeficiency virus type 1 (HIV-1) Nef protein has been shown to accelerate viral growth kinetics in primary human T-lymphocytes and macrophages; however, the specific function(s) of Nef responsible for this phenotype in macrophages is unknown. To address this issue, mutants of a molecularly cloned macrophage-tropic isolate, HIV-1SF162, were generated expressing single point mutations that abrogate the ability of Nef to interact with cellular kinases or mediate CD4 down-regulation. Infection of primary monocyte-derived macrophages (MDM) with these mutant viruses revealed that residues in the PXXP motif contribute to efficient replication. Interestingly, viruses expressing alleles of Nef defective in CD4 down-modulation activity retain wild-type levels of infectivity in single-round assays but exhibited delayed replication kinetics and grew to lower titres compared to the wild-type virus in MDM. These data suggest that efficient HIV-1 replication is dependent on the ability of Nef to interact with cellular kinases and remove CD4 from the surface of infected macrophages.

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