Differential Effects of Human Immunodeficiency Virus Type 1 Capsid and Cellular Factors Nucleoporin 153 and LEDGF/p75 on the Efficiency and Specificity of Viral DNA Integration

ABSTRACT The core domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) contains a D,D(35)E motif, named for the phylogenetically conserved glutamic acid and aspartic acid residues and the invariant 35 amino acid spacing between the second and third acidic residues. Each acidic residue of the D,D(35)E motif is independently essential for the 3′-processing and strand transfer activities of purified HIV-1 IN protein. Using a replication-defective viral genome with a hygromycin selectable marker, we recently reported that a mutation at any of the three residues of the D,D(35)E motif produces a 103- to 104-fold reduction in infectious titer compared with virus encoding wild-type IN (A. D. Leavitt et al., J. Virol. 70:721–728. 1996). The infectious titer, as measured by the number of hygromycin-resistant colonies formed following infection of cells in culture, was less than a few hundred colonies per μg of p24. To understand the mechanism by which the mutant virions conferred hygromycin resistance, we characterized the integrated viral DNA in cells infected with virus encoding mutations at each of the three residues of the D,D(35)E motif. We found the integrated viral DNA to be colinear with the incoming viral genome. DNA sequencing of the junctions between integrated viral DNA and host DNA showed that (i) the characteristic 5-bp direct repeat of host DNA flanking the HIV-1 provirus was not maintained, (ii) integration often produced a deletion of host DNA, (iii) integration sometimes occurred without the viral DNA first undergoing 3′-processing, (iv) integration sites showed a strong bias for a G residue immediately adjacent to the conserved viral CA dinucleotide, and (v) mutations at each of the residues of the D,D(35)E motif produced essentially identical phenotypes. We conclude that mutations at any of the three acidic residues of the conserved D,D(35)E motif so severely impair IN activity that most, if not all, integration events by virus encoding such mutations are not IN mediated. IN-independent provirus formation may have implications for anti-IN therapeutic agents that target the IN active site.

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Phosphorylation-dependent human immunodeficiency virus type 1 infection and nuclear targeting of viral DNA.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.93.1.367 ◽

1996 ◽

Vol 93 (1) ◽

pp. 367-371 ◽

Cited By ~ 108

Author(s):

A. G. Bukrinskaya ◽

A. Ghorpade ◽

N. K. Heinzinger ◽

T. E. Smithgall ◽

R. E. Lewis ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Nuclear Targeting ◽

Viral Dna ◽

Immunodeficiency Virus

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Probing Interactions between Viral DNA and Human Immunodeficiency Virus Type 1 Integrase Using Dinucleotides

Molecular Pharmacology ◽

10.1124/mol.51.4.567 ◽

1997 ◽

Vol 51 (4) ◽

pp. 567-575 ◽

Cited By ~ 32

Author(s):

Abhijit Mazumder ◽

Hiroyuki Uchida ◽

Nouri Neamati ◽

Sanjay Sunder ◽

Maria Jaworska-Maslanka ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Viral Dna ◽

Immunodeficiency Virus

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Human Immunodeficiency Virus Type 1 (HIV-1) Vpr Enhances Expression from Unintegrated HIV-1 DNA

Journal of Virology ◽

10.1128/jvi.77.7.3962-3972.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 3962-3972 ◽

Cited By ~ 54

Author(s):

Betty Poon ◽

Irvin S. Y. Chen

Keyword(s):

Cell Cycle ◽

Human Immunodeficiency Virus ◽

Cell Cycle Arrest ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Viral Dna ◽

Cycle Arrest ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Retroviral DNA synthesized prior to integration, termed unintegrated viral DNA, is classically believed to be transcriptionally inert and to serve only as a precursor to the transcriptionally active integrated proviral DNA form. However, it has recently been found to be expressed under some circumstances during human immunodeficiency virus type 1 (HIV-1) replication and may play a significant role in HIV-1 pathogenesis. HIV-1 Vpr is a virion-associated accessory protein that is critical for HIV-1 replication in nondividing cells and induces cell cycle arrest and apoptosis. We find that Vpr, either expressed de novo or released from virions following viral entry, is essential for unintegrated viral DNA expression. HIV-1 mutants defective for integration in either the integrase catalytic domain or the cis-acting att sites can express unintegrated viral DNA at levels similar to that of wild-type HIV-1, but only in the presence of Vpr. In the absence of Vpr, the expression of unintegrated viral DNA decreases 10- to 20-fold. Vpr does not affect the efficiency of integration from integrase-defective HIV-1. Vpr-mediated enhancement of expression from integrase-defective HIV-1 requires that the viral DNA be generated in cells through infection and is mediated via a template that declines over time. Vpr activation of expression does not require exclusive nuclear localization of Vpr nor does it correlate with Vpr-mediated cell cycle arrest. These results attribute a new function to HIV-1 Vpr and implicate Vpr as a critical component in expression from unintegrated HIV-1 DNA.

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The Naphthyridinone GSK364735 Is a Novel, Potent Human Immunodeficiency Virus Type 1 Integrase Inhibitor and Antiretroviral

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01218-07 ◽

2007 ◽

Vol 52 (3) ◽

pp. 901-908 ◽

Cited By ~ 46

Author(s):

Edward P. Garvey ◽

Brian A. Johns ◽

Margaret J. Gartland ◽

Scott A. Foster ◽

Wayne H. Miller ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Antiviral Activity ◽

Metal Binding ◽

Integrase Inhibitor ◽

Viral Dna ◽

Dna Integration ◽

Cellular Assays ◽

Immunodeficiency Virus ◽

Viral Dna Integration

ABSTRACT The naphthyridinone GSK364735 potently inhibited recombinant human immunodeficiency virus type 1 (HIV-1) integrase in a strand transfer assay (mean 50% inhibitory concentration ± standard deviation, 8 ± 2 nM). As expected based on the structure of the drug, it bound competitively with another two-metal binding inhibitor (K d [binding constant], 6 ± 4 nM). In a number of different cellular assays, GSK364735 inhibited HIV replication with potency at nanomolar concentrations (e.g., in peripheral blood mononuclear cells and MT-4 cells, 50% effective concentrations were 1.2 ± 0.4 and 5 ± 1 nM, respectively), with selectivity indexes of antiviral activity versus in-assay cytotoxicity of at least 2,200. When human serum was added, the antiviral potency decreased (e.g., a 35-fold decrease in the presence of 100% human serum was calculated by extrapolation from the results of the MT-4 cell assay). In cellular assays, GSK364735 blocked viral DNA integration, with a concomitant increase in two-long-terminal-repeat circles. As expected, this integrase inhibitor was equally active against wild-type viruses and mutant viruses resistant to approved drugs targeting either reverse transcriptase or protease. In contrast, some but not all viruses resistant to other integrase inhibitors were resistant to GSK364735. When virus was passaged in the presence of the inhibitor, we identified resistance mutations within the integrase active site that were the same as or similar to mutations arising in response to other two-metal binding inhibitors. Finally, either additive or synergistic effects were observed when GSK364735 was tested in combination with approved antiretrovirals (i.e., no antagonistic effects were seen). Thus, based on all the data, GSK364735 exerted potent antiviral activity through the inhibition of viral DNA integration by interacting at the two-metal binding site within the catalytic center of HIV integrase.

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Inhibition of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document}-Primed Reverse Transcription by Human APOBEC3G during Human Immunodeficiency Virus Type 1 Replication

Journal of Virology ◽

10.1128/jvi.01038-06 ◽

2006 ◽

Vol 80 (23) ◽

pp. 11710-11722 ◽

Cited By ~ 147

Author(s):

Fei Guo ◽

Shan Cen ◽

Meijuan Niu ◽

Jenan Saadatmand ◽

Lawrence Kleiman

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Viral Infectivity ◽

Viral Dna ◽

Immunodeficiency Virus ◽

Dna Production ◽

Hiv 1

ABSTRACT Cells are categorized as being permissive or nonpermissive according to their ability to produce infectious human immunodeficiency virus type 1 (HIV-1) lacking the viral protein Vif. Nonpermissive cells express the human cytidine deaminase APOBEC3G (hA3G), and Vif has been shown to bind to APOBEC3G and facilitate its degradation. Vif-negative HIV-1 virions produced in nonpermissive cells incorporate hA3G and have a severely reduced ability to produce viral DNA in newly infected cells. While it has been proposed that the reduction in DNA production is due to hA3G-facilitated deamination of cytidine, followed by DNA degradation, we provide evidence here that a decrease in the synthesis of the DNA by reverse transcriptase may account for a significant part of this reduction. During the infection of cells with Vif-negative HIV-1 produced from 293T cells transiently expressing hA3G, much of the inhibition of early (≥50% reduction) and late (≥95% reduction) viral DNA production, and of viral infectivity (≥95% reduction), can occur independently of DNA deamination. The inhibition of the production of early minus-sense strong stop DNA is also correlated with a similar inability of tRNA3 Lys to prime reverse transcription. A similar reduction in tRNA3 Lys priming and viral infectivity is also seen in the naturally nonpermissive cell H9, albeit at significantly lower levels of hA3G expression.

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Characterization of the Behavior of Functional Viral Genomes during the Early Steps of Human Immunodeficiency Virus Type 1 Infection

Journal of Virology ◽

10.1128/jvi.00429-09 ◽

2009 ◽

Vol 83 (15) ◽

pp. 7524-7535 ◽

Cited By ~ 43

Author(s):

Vanessa Arfi ◽

Julia Lienard ◽

Xuan-Nhi Nguyen ◽

Gregory Berger ◽

Dominique Rigal ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Target Cells ◽

Viral Dna ◽

Immunodeficiency Virus ◽

Nucleoprotein Complexes ◽

Kinetics Of

ABSTRACT Infectious viral DNA constitutes only a small fraction of the total viral DNA produced during retroviral infection, and as such its exact behavior is largely unknown. In the present study, we characterized in detail functional viral DNA produced during the early steps of human immunodeficiency virus type 1 infection by analyzing systematically their kinetics of synthesis and integration in different target cells. In addition, we have compared the functional stability of viral nucleoprotein complexes arrested at their pre-reverse transcription state, and we have attempted to measure the kinetics of loss of capsid proteins from viral complexes through the susceptibility of the early phases of infection to cyclosporine, a known inhibitor of the interaction between viral capsid and cyclophilin A. Overall, our data suggest a model in which loss of capsid proteins from viral complexes and reverse transcription occur concomitantly and in which the susceptibility of target cells to infection results from a competition between the ability of the cellular environment to quickly destabilize viral nucleoprotein complexes and the capability of the virus to escape such targeting by engaging the reverse transcription reaction.

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Reinfection results in accumulation of unintegrated viral DNA in cytopathic and persistent human immunodeficiency virus type 1 infection of CEM cells.

Journal of Experimental Medicine ◽

10.1084/jem.172.4.1035 ◽

1990 ◽

Vol 172 (4) ◽

pp. 1035-1042 ◽

Cited By ~ 67

Author(s):

C D Pauza ◽

J E Galindo ◽

D D Richman

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Virus Production ◽

Neutralizing Antibody ◽

Viral Dna ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Hiv 1

High levels of unintegrated viral DNA accumulate during human immunodeficiency virus type 1 (HIV-1) infection of CEM T cells. Reinfection of already infected cells is required to attain these levels and reinfection also promotes the development of HIV-induced cytopathology. Rates of virus production, however, are independent of the accumulation of unintegrated viral DNA. Neutralizing antibody added soon after infection reduced viral DNA levels without appreciably affecting the production of cell-free viral p24 antigen or reverse transcriptase activity. Only 50 pM AZT were required to reduce the accumulation of unintegrated viral DNA by 50% in contrast to the 25 nM required to inhibit virus production by 50%. Cytopathology, as measured by number of syncytia in infected cell cultures, was correlated with highly elevated levels of unintegrated viral DNA. The minimal levels of unintegrated viral DNA present constitutively in the persistently infected HCEM cell line were consonant with the absence of cytopathic effects in these cells. These data demonstrate that inhibiting the reinfection of already infected cells modulates cytopathic HIV-1 infection to a form that is persistent and noncytopathic.

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