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 Coding Sequences Upstream of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Domain in Gag-Pol Are Not Essential for Incorporation of the Pr160gag-pol into Virus Particles

2002 ◽  
Vol 76 (7) ◽  
pp. 3221-3231 ◽  
Author(s):  
Hsu-Chen Chiu ◽  
Szu-Yung Yao ◽  
Chin-Tien Wang
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Leukemia Virus ◽  
Deletion Mutants ◽  
Coding Region ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Incorporation of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virions is thought to be mediated by the N-terminal Gag domain via interaction with the Gag precursor. However, one recent study has demonstrated that the murine leukemia virus Pol can be incorporated into virions independently of Gag-Pol expression, implying a possible interaction between the Pol and Gag precursor. To test whether the HIV-1 Pol can be incorporated into virions on removal of the N-terminal Gag domain and to define sequences required for the incorporation of Gag-Pol into virions in more detail, a series of HIV Gag-Pol expression plasmids with various extensive deletions in the region upstream of the reverse transcriptase (RT) domain was constructed, and viral incorporation of the Gag-Pol deletion mutants was examined by cotransfecting 293T cells with a plasmid expressing Pr55 gag . Analysis indicated that deletion of the N-terminal two-thirds of the gag coding region did not significantly affect the incorporation of Gag-Pol into virions. In contrast, Gag-Pol proteins with deletions covering the capsid (CA) major homology regions and the adjacent C-terminal CA regions were impaired with respect to assembly into virions. However, Gag-Pol with sequences deleted upstream of the protease, or of the RT domain but retaining 15 N-terminal gag codons, could still be rescued into virions at a level about 20% of the wild-type level. When assayed in a nonmyristylated Gag-Pol context, all of the Gag-Pol deletion mutants were incorporated into virions at a level comparable to their myristylated counterparts, suggesting that the incorporation of the Gag-Pol deletion mutants into virions is independent of the N-terminal myristylation signal.

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 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 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 Relative Fitness and Replication Capacity of a Multinucleoside Analogue-Resistant Clinical Human Immunodeficiency Virus Type 1 Isolate with a Deletion of Codon 69 in the Reverse Transcriptase Coding Region

2007 ◽  
Vol 81 (9) ◽  
pp. 4713-4721 ◽  
Author(s):  
Cristina Villena ◽  
Julia G. Prado ◽  
Maria Carmen Puertas ◽  
Miguel Ángel Martínez ◽  
Bonaventura Clotet ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Relative Fitness ◽  
Replication Capacity ◽  
Coding Region ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Deletions, insertions, and amino acid substitutions in the β3-β4 hairpin loop-coding region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been associated with resistance to nucleoside RT inhibitors when appearing in combination with other mutations in the RT-coding region. In this work, we have measured the in vivo fitness of HIV-1 variants containing a deletion of 3 nucleotides affecting codon 69 (Δ69) of the viral RT as well as the replication capacity (RC) ex vivo of a series of recombinant HIV-1 variants carrying an RT bearing the Δ69 deletion or the T69A mutation in a multidrug-resistant (MDR) sequence background, including the Q151M complex and substitutions M184V, K103N, Y181C, and G190A. Patient-derived viral clones having RTs with Δ69 together with S163I showed increased RCs under drug pressure. These data were consistent with the viral population dynamics observed in a long-term-treated HIV-1-infected patient. In the absence of drugs, viral clones containing T69A replicated more efficiently than those having Δ69, but only when patient-derived sequences corresponding to RT residues 248 to 527 were present. These effects could be attributed to a functional interaction between the C-terminal domain of the p66 subunit (RNase H domain) and the DNA polymerase domain of the RT. Finally, recombinant HIV-1 clones bearing RTs with MDR-associated mutations, including deletions at codon 69, showed increased susceptibilities to protease inhibitors in phenotypic assays. These effects correlated with impaired Gag cleavage and could be attributed to delayed maturation and decreased production of active protease in those variants.

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 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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