scholarly journals Association of Human Immunodeficiency Virus Type 1 Vif with RNA and Its Role in Reverse Transcription

2000 ◽  
Vol 74 (19) ◽  
pp. 8938-8945 ◽  
Author(s):  
Markus Dettenhofer ◽  
Shan Cen ◽  
Bradley A. Carlson ◽  
Lawrence Kleiman ◽  
Xiao-Fang Yu
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Viral Genomic Rna ◽  
Hiv 1

ABSTRACT The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1 vif mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA3 Lyswere additionally present at normal levels in vif mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of Xenopus laevis, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced invif mutant virions. Moreover, purified vifmutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.

scholarly journals Human Immunodeficiency Virus Type 1 Vif Protein Is Packaged into the Nucleoprotein Complex through an Interaction with Viral Genomic RNA

2001 ◽  
Vol 75 (16) ◽  
pp. 7252-7265 ◽  
Author(s):  
Mohammad A. Khan ◽  
Claudia Aberham ◽  
Sandra Kao ◽  
Hirofumi Akari ◽  
Robert Gorelick ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Nucleoprotein Complex ◽  
Vif Protein ◽  
Viral Genomic Rna ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in the production of infectious virions. Previous studies have demonstrated the presence of small amounts of Vif in virus particles. However, Vif packaging was assumed to be nonspecific, and its functional significance has been questioned. We now report that packaging of Vif is dependent on the packaging of viral genomic RNA in both permissive and restrictive HIV-1 target cells. Mutations in the nucleocapsid zinc finger domains that abrogate packaging of viral genomic RNA abolished packaging of Vif. Additionally, an RNA packaging-defective virus exhibited significantly reduced packaging of Vif. Finally, deletion of a putative RNA-interacting domain in Vif abolished packaging of Vif into virions. Virion-associated Vif was resistant to detergent extraction and copurified with components of the viral nucleoprotein complex and functional reverse transcription complexes. Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.

scholarly journals Gag–Pol bearing a reverse transcriptase drug-resistant mutation influences viral genomic RNA incorporation into human immunodeficiency virus type 1 particles

2006 ◽  
Vol 87 (9) ◽  
pp. 2669-2677
Author(s):  
Renato S. Aguiar ◽  
Helena S. Pereira ◽  
Luciana J. Costa ◽  
Rodrigo M. Brindeiro ◽  
Amilcar Tanuri
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Wild Type Counterpart ◽  
Drug Resistant Mutation ◽  
Viral Genomic Rna ◽  
Hiv 1

The unspliced human immunodeficiency virus type 1 (HIV-1) RNA is both the messenger for Gag and Gag–Pol and the viral genomic RNA (vRNA) that is packaged into the virion. Although Gag alone is sufficient for the incorporation of vRNA into virus particles, Gag–Pol molecules play an important role in vRNA dimerization and virion maturation. Here, a cis model for vRNA packaging was demonstrated, in which nascent Gag–Pol molecules were preferentially co-encapsulated with their cognate RNA used as the template. Genome-incorporation frequencies were evaluated for two distinct HIV-1 proviral clones differing in their ability to respond to nevirapine (NVP) treatment in one round of infection. It was shown that, under NVP selection, there was a twofold-higher incorporation of vRNAs and integration of provirus genome carrying NVP resistance when compared with the wild-type counterpart. Although cis incorporation has been already demonstrated for Gag, the novelty of these findings is that newly acquired resistant mutations in Gag–Pol will select their specific genomic RNA during virus replication, thus rapidly increasing the chance of the emergence of resistant viruses during the course of anti-retroviral treatment.

scholarly journals Subunit-Specific Analysis of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo

2004 ◽  
Vol 78 (13) ◽  
pp. 7089-7096 ◽  
Author(s):  
Alok Mulky ◽  
Stefan G. Sarafianos ◽  
Edward Arnold ◽  
Xiaoyun Wu ◽  
John C. Kappes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Coding Region ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer comprised of two structurally distinct subunits (p51 and p66). Since p51 and p66 are derived from the same coding region, subunit-specific structure-function studies of RT have been conducted exclusively by in vitro biochemical approaches. To study RT subunit function in the context of infectious virus, we constructed an LTR-vpr-p51-IRES-p66 expression cassette in which the HIV-1 vpr gene was fused in frame with p51, followed by an internal ribosome entry site (IRES) sequence and the p66 coding region. By coexpression with RT-deficient proviral DNA, we demonstrated that the p66 subunit is specifically and selectively packaged into virions as a Vpr-p51/p66 complex. Our analysis showed that cleavage by the viral protease liberates Vpr and generates functional heterodimeric RT (p51/p66) that supports HIV-1 reverse transcription and virus infection. By exploiting this novel trans-complementation approach, we demonstrated, for the first time with infectious virions, that the YMDD aspartates of p66 are both required and sufficient for RT polymerase function. Mutational analyses of the p51 YMDD aspartates indicated that they play an important structural role in p51 folding and subunit interactions that are required for the formation of an active RT heterodimer within infected cells. Understanding the role of the individual RT subunits in RNA- and DNA-dependent DNA synthesis is integral to our understanding of RT function. Our findings will lead to important new insights into the role of the p51 and p66 subunits in HIV-1 reverse transcription.

scholarly journals Amino-Terminal Region of the Human Immunodeficiency Virus Type 1 Nucleocapsid Is Required for Human APOBEC3G Packaging

2004 ◽  
Vol 78 (21) ◽  
pp. 11841-11852 ◽  
Author(s):  
Kun Luo ◽  
Bindong Liu ◽  
Zuoxiang Xiao ◽  
Yunkai Yu ◽  
Xianghui Yu ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Terminal Region ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Viral Genomic Rna ◽  
Hiv 1 ◽  
Human Apobec3g

ABSTRACT 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.

scholarly journals Requirement for Integrase during Reverse Transcription of Human Immunodeficiency Virus Type 1 and the Effect of Cysteine Mutations of Integrase on Its Interactions with Reverse Transcriptase

2004 ◽  
Vol 78 (10) ◽  
pp. 5045-5055 ◽  
Author(s):  
Kai Zhu ◽  
Charles Dobard ◽  
Samson A. Chow
Keyword(s):  
Human Immunodeficiency Virus ◽  
Life Cycle ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retroviral integrase catalyzes the essential step of integrating a double-stranded DNA copy of the viral genome into a host cell chromosome. Mutational studies have revealed that integrase is involved in additional steps of viral replication, but the mechanism for the pleiotropic effect is not well characterized. Since Cys residues generally play crucial roles in protein structure and function, we introduced Cys-to-Ser substitutions at positions 56, 65, and 130 of human immunodeficiency virus type 1 (HIV-1) integrase to determine their effects on integration activity and viral replication. None of the substitutions significantly affected the enzymatic activities in vitro. When introduced into the NL4-3 molecular clone of HIV-1, mutant viruses encoding Cys mutations at positions 56 and 65 of integrase replicated similarly to the wild-type virus in CD4+-T-cell lines, whereas the C130S-containing virus was noninfectious. The entry and postintegration steps of the viral life cycle for all mutant viruses were normal, and all had particle-associated reverse transcriptase (RT) activity. However, early reverse-transcribed DNA products were absent in the lysate of cells infected with the C130S mutant virus, indicating that the mutation abolished the ability of the virus to initiate endogenous reverse transcription. Coimmunoprecipitation using purified integrase and RT showed that the C-terminal domain of wild-type HIV-1 integrase interacted with RT. The interaction between integrase and RT was not affected in the presence of a reducing or alkylating agent, suggesting that the interaction did not involve a disulfide linkage. The C130S substitution within the core region may disrupt the protein recognition interface of the C-terminal domain and abolish its ability to interact with RT. Our results indicate that integrase plays an important role during the reverse-transcription step of the viral life cycle, possibly through physical interactions with RT.

scholarly journals Diminished RNA Primer Usage Associated with the L74V and M184V Mutations in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Provides a Possible Mechanism for Diminished Viral Replication Capacity

2003 ◽  
Vol 77 (16) ◽  
pp. 8621-8632 ◽  
Author(s):  
Karidia Diallo ◽  
Bruno Marchand ◽  
Xin Wei ◽  
Luciano Cellai ◽  
Matthias Götte ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Replication Capacity ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The emergence of drug resistance-conferring mutations can severely compromise the success of chemotherapy directed against human immunodeficiency virus type 1 (HIV-1). The M184V and/or L74V mutation in the reverse transcriptase (RT) gene are frequently found in viral isolates from patients treated with the nucleoside RT inhibitors lamivudine (3TC), abacavir (ABC), and didanosine (ddI). However, the effectiveness of combination therapy with regimens containing these compounds is often not abolished in the presence of these mutations; it has been conjectured that diminished fitness of HIV-1 variants containing L74V and M184V may contribute to sustained antiviral effects in such cases. We have determined that viruses containing both L74V and M184V are more impaired in replication capacity than viruses containing either mutation alone. To understand the biochemical mechanisms responsible for this diminished fitness, we generated a series of recombinant mutated enzymes containing either or both of the L74V and M184V substitutions. These enzymes were tested for their abilities to bypass important rate-limiting steps during the complex process of reverse transcription. We studied both the initiation of minus-strand DNA synthesis with the cognate replication primer human tRNA3 Lys and the initiation of plus-strand DNA synthesis, using a short RNA primer derived from the viral polypurine tract. We observed that the efficiencies of both reactions were diminished with enzymes containing either L74V or M184V and that these effects were significantly amplified with the double mutant. We also show that release from intrinsic pausing sites during reverse transcription appears to be a major obstacle that cannot be efficiently bypassed. Our data suggest that the efficiency of RNA-primed DNA synthesis represents an important consideration that can affect viral replication kinetics.

scholarly journals Novel Nonnucleoside Inhibitors That Select Nucleoside Inhibitor Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

2006 ◽  
Vol 50 (8) ◽  
pp. 2772-2781 ◽  
Author(s):  
Zhijun Zhang ◽  
Michelle Walker ◽  
Wen Xu ◽  
Jae Hoon Shim ◽  
Jean-Luc Girardet ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Immunodeficiency Virus ◽  
Nonnucleoside Inhibitors ◽  
Rt Inhibitors ◽  
Hiv 1 ◽  
M184v Mutation

ABSTRACT Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies.

scholarly journals Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Nuclear Localization Mediates Early Viral mRNA Expression

2002 ◽  
Vol 76 (20) ◽  
pp. 10444-10454 ◽  
Author(s):  
Jielin Zhang ◽  
Clyde S. Crumpacker
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mrna Expression ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nucleocapsid Protein ◽  
Viral Mrna ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT An important aspect of the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection is the ability of the virus to replicate in the host vigorously without a latent phase and to kill cells with a dynamic turnover of 1.8 × 109 cells/day and 10.3 × 109 virions/24 h. The transcription of HIV-1 RNA in acute infection occurs at two stages; the transcription of viral spliced mRNA occurs early, and the transcription of viral genomic RNA occurs later. The HIV-1 Tat protein is translated from the early spliced mRNA and is critical for HIV-1 genomic RNA expression. The cellular transcription factors are important for HIV-1 early spliced mRNA expression. In this study we show that virion nucleocapsid protein (NC) has a role in expression of HIV-1 early spliced mRNA. The HIV-1 NC migrates from the cytoplasm to the nucleus and accumulates in the nucleus at 18 h postinfection. Mutations on HIV-1 NC zinc fingers change the pattern of early viral spliced mRNA expression and result in a delayed expression of early viral mRNA in HIV-infected cells. This delayed HIV-1 early spliced mRNA expression occurs after proviral DNA has been integrated into the cellular genome, as shown by a quantitative integration assay. These results show that virion NC plays an important role in inducing HIV-1 early mRNA expression and contributes to the rapid viral replication that occurs during HIV-1 infection.

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.

An Approach towards the Synthesis of Potential Metal-Chelating TSAO-T Derivatives as Bidentate Inhibitors of Human Immunodeficiency virus Type 1 Reverse Transcriptase

1998 ◽  
Vol 9 (5) ◽  
pp. 412-421 ◽  
Author(s):  
C Chamorro ◽  
M-J Camarasa ◽  
M-J Pérez-Pérez ◽  
E de Clercq ◽  
J Balzarini ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Parent Compound ◽  
Immunodeficiency Virus ◽  
Anti Hiv ◽  
Hiv 1

Novel derivatives of the potent human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor TSAO-T have been designed, synthesized and tested for their in vitro antiretro-viral activity against HIV. These TSAO-T derivatives have been designed as potential bidentate inhibitors of HIV-1 RT, which combine in their structure the functionality of a non-nucleoside RT inhibitor (TSAO-T) and a bivalent ion-chelating moiety (a β-diketone moiety) linked through an appropriate spacer to the N-3 of thymine of TSAO-T . Some of the new compounds have an anti-HIV-1 activity comparable to that of the parent compound TSAO-T, but display a markedly increased antiviral selectivity. There was a clear relationship between antiviral activity and the length of the spacer group that links the TSAO molecule with the chelating moiety. A shorter spacer invariably resulted in increased antiviral potency. None of the TSAO-T derivatives were endowed with anti-HIV-2 activity.

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