scholarly journals Role of Vif in Stability of the Human Immunodeficiency Virus Type 1 Core

2000 ◽  
Vol 74 (23) ◽  
pp. 11055-11066 ◽  
Author(s):  
Åsa Öhagen ◽  
Dana Gabuzda
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Synthesis ◽  
Virus Entry ◽  
Wild Type ◽  
The Core ◽  
Immunodeficiency Virus ◽  
The Stability ◽  
Hiv 1

ABSTRACT The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity. Previous studies have shown thatvif-defective virions exhibit structural abnormalities in the virus core and are defective in the ability to complete proviral DNA synthesis in acutely infected cells. We developed novel assays to assess the relative stability of the core in HIV-1 virions. Using these assays, we examined the role of Vif in the stability of the HIV-1 core. The integrity of the core was examined following virion permeabilization or removal of the lipid envelope and treatment with various triggers, including S100 cytosol, deoxynucleoside triphosphates, detergents, NaCl, and buffers of different pH to mimic aspects of the uncoating and disassembly process which occurs after virus entry but preceding or during reverse transcription.vif mutant cores were more sensitive to disruption by all triggers tested than wild-type cores, as determined by endogenous reverse transcriptase (RT) assays, biochemical analyses, and electron microscopy. RT and the p7 nucleocapsid protein were released more readily from vif mutant virions than from wild-type virions, suggesting that the internal nucleocapsid is less stably packaged in the absence of Vif. Purified cores could be isolated from wild-type but not vif mutant virions by sedimentation through detergent-treated gradients. These results demonstrate that Vif increases the stability of virion cores. This may permit efficient viral DNA synthesis by preventing premature degradation or disassembly of viral nucleoprotein complexes during early events after virus entry.

scholarly journals Role of Human Immunodeficiency Virus Type 1 Integrase in Uncoating of the Viral Core

2010 ◽  
Vol 84 (10) ◽  
pp. 5181-5190 ◽  
Author(s):  
Marisa S. Briones ◽  
Charles W. Dobard ◽  
Samson A. Chow
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Core Stability ◽  
The Core ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT After membrane fusion with a target cell, the core of human immunodeficiency virus type 1 (HIV-1) enters into the cytoplasm, where uncoating occurs. The cone-shaped core is composed of the viral capsid protein (CA), which disassembles during uncoating. The underlying factors and mechanisms governing uncoating are poorly understood. Several CA mutations can cause changes in core stability and a block at reverse transcription, demonstrating the requirement for optimal core stability during viral replication. HIV-1 integrase (IN) catalyzes the insertion of the viral cDNA into the host genome, and certain IN mutations are pleiotropic. Similar to some CA mutants, two IN mutants, one with a complete deletion of IN (NL-ΔIN) and the other with a Cys-to-Ser substitution (NL-C130S), were noninfectious, with a replication block at reverse transcription. Compared to the wild type (WT), the cytoplasmic CA levels of the IN mutants in infected cells were reduced, suggesting accelerated uncoating. The role of IN during uncoating was examined by isolating and characterizing cores from NL-ΔIN and NL-C130S. Both IN mutants could form functional cores, but the core yield and stability were decreased. Also, virion incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to CA, was decreased in the IN mutants. Cores isolated from WT virus depleted of CypA had an unstable-core phenotype, confirming a role of CypA in promoting optimal core stability. Taken together, our results indicate that IN is required during uncoating for maintaining CypA-CA interaction, which promotes optimal stability of the viral core.

scholarly journals Subunit Stoichiometry of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trimers during Virus Entry into Host Cells

2006 ◽  
Vol 80 (9) ◽  
pp. 4388-4395 ◽  
Author(s):  
Xinzhen Yang ◽  
Svetla Kurteva ◽  
Xinping Ren ◽  
Sandra Lee ◽  
Joseph Sodroski
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Glycoprotein ◽  
Virus Entry ◽  
Envelope Glycoproteins ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a homotrimer of gp120/gp41 heterodimers to support virus entry. During the process of virus entry, an individual HIV-1 envelope glycoprotein trimer binds the cellular receptors CD4 and CCR5/CXCR4 and mediates the fusion of the viral and the target cellular membranes. By studying the function of heterotrimers between wild-type and nonfunctional mutant envelope glycoproteins, we found that two wild-type subunits within an envelope glycoprotein trimer are required to support virus entry. Complementation between HIV-1 envelope glycoprotein mutants defective in different functions to allow virus entry was not evident. These results assist our understanding of the mechanisms whereby the HIV-1 envelope glycoproteins mediate virus entry and membrane fusion and guide attempts to inhibit these processes.

scholarly journals A Conserved Tryptophan-Rich Motif in the Membrane-Proximal Region of the Human Immunodeficiency Virus Type 1 gp41 Ectodomain Is Important for Env-Mediated Fusion and Virus Infectivity

1999 ◽  
Vol 73 (3) ◽  
pp. 2469-2480 ◽  
Author(s):  
Karl Salzwedel ◽  
John T. West ◽  
Eric Hunter
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Envelope Glycoprotein ◽  
Virus Entry ◽  
Immunodeficiency Virus ◽  
Tryptophan Residues ◽  
Hiv 1

ABSTRACT Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an α-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.

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 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 Removal of a Single N-Linked Glycan in Human Immunodeficiency Virus Type 1 gp120 Results in an Enhanced Ability To Induce Neutralizing Antibody Responses

2007 ◽  
Vol 82 (2) ◽  
pp. 638-651 ◽  
Author(s):  
Yun Li ◽  
Bradley Cleveland ◽  
Igor Klots ◽  
Bruce Travis ◽  
Barbra A. Richardson ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Pronounced Increase ◽  
Enhanced Sensitivity ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Glycans on human immunodeficiency virus (HIV) envelope protein play an important role in infection and evasion from host immune responses. To examine the role of specific glycans, we introduced single or multiple mutations into potential N-linked glycosylation sites in hypervariable regions (V1 to V3) of the env gene of HIV type 1 (HIV-1) 89.6. Three mutants tested showed enhanced sensitivity to soluble CD4. Mutant N7 (N197Q) in the carboxy-terminal stem of the V2 loop showed the most pronounced increase in sensitivity to broadly neutralizing antibodies (NtAbs), including those targeting the CD4-binding site (IgG1b12) and the V3 loop (447-52D). This mutant is also sensitive to CD4-induced NtAb 17b in the absence of CD4. Unlike the wild-type (WT) Env, mutant N7 mediates CD4-independent infection in U87-CXCR4 cells. To study the immunogenicity of mutant Env, we immunized pig-tailed macaques with recombinant vaccinia viruses, one expressing SIVmac239 Gag-Pol and the other expressing HIV-1 89.6 Env gp160 in WT or mutant forms. Animals were boosted 14 to 16 months later with simian immunodeficiency virus gag DNA and the cognate gp140 protein before intrarectal challenge with SHIV89.6P-MN. Day-of-challenge sera from animals immunized with mutant N7 Env had significantly higher and broader neutralizing activities than sera from WT Env-immunized animals. Neutralizing activity was observed against SHIV89.6, SHIV89.6P-MN, HIV-1 SF162, and a panel of subtype B primary isolates. Compared to control animals, immunized animals showed significant reduction of plasma viral load and increased survival after challenge, which correlated with prechallenge NtAb titers. These results indicate the potential advantages for glycan modification in vaccine design, although the role of specific glycans requires further examination.

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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