SJP-L-5 inhibits HIV-1 polypurine tract primed plus-strand DNA elongation, indicating viral DNA synthesis initiation at multiple sites under drug pressure

ABSTRACTIt is important to develop new anti-HIV drugs that are effective against the existing drug-resistant mutants. Because the excision mechanism is an important pathway for resistance to nucleoside analogs, we are preparing analogs that retain a 3′-OH and can be extended after they are incorporated by the viral reverse transcriptase. We show that 4′-C-alkyl-deoxyadenosine (4′-C-alkyl-dA) compounds can be phosphorylated in cultured cells and can inhibit the replication of HIV-1 vectors: 4′-C-methyl- and 4′-C-ethyl-dA show both efficacy and selectivity against HIV-1. The compounds are also effective against viruses that replicate using reverse transcriptases (RTs) that carry nucleoside reverse transcriptase inhibitor resistance mutations, with the exception of the M184V mutant. Analysis of viral DNA synthesis in infected cells showed that viral DNA synthesis is blocked by the incorporation of either 4′-C-methyl- or 4′-C-ethyl-2′-deoxyadenosine.In vitroexperiments with purified HIV-1 RT showed that 4′-C-methyl-2′-dATP can compete with dATP and that incorporation of the analog causes pausing in DNA synthesis. The 4′-C-ethyl compound also competes with dATP and shows a differential ability to block DNA synthesis on RNA and DNA templates. Experiments that measure the ability of the compounds to block DNA synthesis in infected cells suggest that this differential block to DNA synthesis also occurs in infected cells.

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A Highly Defective HIV-1 Group O Provirus: Evidence for the Role of Local Sequence Determinants in G → A Hypermutation during Negative-Strand Viral DNA Synthesis

Virology ◽

10.1006/viro.1995.1191 ◽

1995 ◽

Vol 208 (2) ◽

pp. 601-609 ◽

Cited By ~ 39

Author(s):

Andrew M. Borman ◽

Caroline Quillent ◽

Pierre Charneau ◽

Katherine M. Kean ◽

François Clavel

Keyword(s):

Dna Synthesis ◽

Viral Dna ◽

Negative Strand ◽

Local Sequence ◽

Hiv 1

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Compensatory Role of Human Immunodeficiency Virus Central Polypurine Tract Sequence in Kinetically Disrupted Reverse Transcription

Journal of Virology ◽

10.1128/jvi.00120-08 ◽

2008 ◽

Vol 82 (15) ◽

pp. 7716-7720 ◽

Cited By ~ 14

Author(s):

Mark Skasko ◽

Baek Kim

Keyword(s):

Human Immunodeficiency Virus ◽

Dna Synthesis ◽

Lung Fibroblasts ◽

Polypurine Tract ◽

Immunodeficiency Virus ◽

Deoxynucleoside Triphosphate ◽

Central Polypurine Tract ◽

Hiv 1 ◽

Vector Transduction

ABSTRACT We tested whether the additional positive-strand DNA synthesis initiation of human immunodeficiency virus type 1 (HIV-1) from the central polypurine tract (cPPT) facilitates efficient completion of kinetically disturbed proviral DNA synthesis induced by dysfunctional reverse transcriptase (RT) mutants or limited cellular deoxynucleoside triphosphate (dNTP) pools. Indeed, the cPPT enabled the HIV-1 vectors harboring RT mutants with reduced dNTP binding affinity to transduce human lung fibroblasts (HLFs), without which these mutant vectors normally fail to transduce. The cPPT showed little effect on wild-type HIV-1 vector transduction in HLF, whereas it significantly enhanced vector transduction in HLFs engineered to contain reduced dNTP pools, suggesting a novel compensatory role for cPPT in viruses harboring kinetically impaired RT.

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IP6 is an HIV pocket factor that prevents capsid collapse and promotes DNA synthesis

eLife ◽

10.7554/elife.35335 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 59

Author(s):

Donna L Mallery ◽

Chantal L Márquez ◽

William A McEwan ◽

Claire F Dickson ◽

David A Jacques ◽

...

Keyword(s):

Dna Synthesis ◽

Single Molecule ◽

Reverse Transcription ◽

Isotopic Labelling ◽

Viral Dna ◽

Infectious Virion ◽

Capsid Stability ◽

Hiv 1 ◽

Transcription Assays

The HIV capsid is semipermeable and covered in electropositive pores that are essential for viral DNA synthesis and infection. Here, we show that these pores bind the abundant cellular polyanion IP6, transforming viral stability from minutes to hours and allowing newly synthesised DNA to accumulate inside the capsid. An arginine ring within the pore coordinates IP6, which strengthens capsid hexamers by almost 10°C. Single molecule measurements demonstrate that this renders native HIV capsids highly stable and protected from spontaneous collapse. Moreover, encapsidated reverse transcription assays reveal that, once stabilised by IP6, the accumulation of new viral DNA inside the capsid increases >100 fold. Remarkably, isotopic labelling of inositol in virus-producing cells reveals that HIV selectively packages over 300 IP6 molecules per infectious virion. We propose that HIV recruits IP6 to regulate capsid stability and uncoating, analogous to picornavirus pocket factors. HIV-1/IP6/capsid/co-factor/reverse transcription.

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Negative effect of the M184V mutation in HIV-1 reverse transcriptase on initiation of viral DNA synthesis

Virology ◽

10.1016/s0042-6822(03)00173-9 ◽

2003 ◽

Vol 311 (1) ◽

pp. 202-212 ◽

Cited By ~ 36

Author(s):

Xin Wei ◽

Chen Liang ◽

Matthias Götte ◽

Mark A Wainberg

Keyword(s):

Reverse Transcriptase ◽

Dna Synthesis ◽

Viral Dna ◽

Negative Effect ◽

Hiv 1 ◽

M184v Mutation

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Quantification of the Effects on Viral DNA Synthesis of Reverse Transcriptase Mutations Conferring Human Immunodeficiency Virus Type 1 Resistance to Nucleoside Analogues

Journal of Virology ◽

10.1128/jvi.79.2.812-822.2005 ◽

2005 ◽

Vol 79 (2) ◽

pp. 812-822 ◽

Cited By ~ 10

Author(s):

Francine Bouchonnet ◽

Elisabeth Dam ◽

Fabrizio Mammano ◽

Vaea de Soultrait ◽

Gaëlle Henneré ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Dna Synthesis ◽

Nucleoside Analogues ◽

Resistance Mutations ◽

Viral Dna ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1 ◽

M184v Mutation

ABSTRACT Human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) resistance mutations reduce the susceptibility of the virus to nucleoside analogues but may also impair viral DNA synthesis. To further characterize the effect of nucleoside analogue resistance mutations on the efficiency and kinetics of HIV-1 DNA synthesis and to evaluate the impact of the depletion of deoxynucleoside triphosphates (dNTP) on this process, DNA synthesis was evaluated by allowing DNA synthesis to proceed with natural HIV-1 templates and primers, either within permeabilized viral particles or in newly infected cells, and quantifying the products by real-time PCR. Three recombinant viruses derived from three pNL4-3 molecular clones expressing mutations associated with resistance to zidovudine: a clone expressing RT mutation M184V, a clone expressing mutations M41L plus T215Y (M41L+T215Y), and clinical isolate BV34 (carrying seven resistance mutations). Following infection of P4 cells, the BV34 mutant, but not viruses expressing the M184V mutation or M41L+T215Y, exhibited a defect in DNA synthesis. Importantly, however, for mutants carrying the M184V mutation or M41L+T215Y mutations, a defect could be detected by using target cells in which dATP pools had been reduced by pretreatment with hydroxyurea. Based on these observations, we developed a recombinant-virus assay to assess the effects of hydroxyurea pretreatment on infectivity of viruses carrying plasma-derived RT sequences from patients with nucleoside resistance. Using this assay, we found that many, but not all, viruses carrying RT resistance mutations display an increased sensitivity to hydroxyurea, suggesting that the impact of RT resistance mutations on viral replication may be more profound in cell populations characterized by smaller dNTP pools.

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Anti-HIV-1 activity of Trim 37

Journal of General Virology ◽

10.1099/vir.0.057653-0 ◽

2014 ◽

Vol 95 (4) ◽

pp. 960-967 ◽

Cited By ~ 12

Author(s):

Azah A. Tabah ◽

Keith Tardif ◽

Louis M. Mansky

Keyword(s):

Dna Synthesis ◽

Viral Dna ◽

Virion Incorporation ◽

Immunodeficiency Virus ◽

Transient Overexpression ◽

Anti Hiv ◽

Antiviral Mechanism ◽

Hiv 1 ◽

Antiretroviral Activity

Trim 5α was the first member of the tripartite motif (TRIM) family of proteins that was identified to potently restrict human immunodeficiency virus type 1 (HIV-1) replication. The breadth of antiretroviral activity of TRIM family members is an active area of investigation. In this study, we demonstrate that human Trim 37 possesses anti-HIV-1 activity. This antiretroviral activity and the manner in which it was displayed were implicated by (1) decreased viral replication upon Trim 37 transient overexpression in virus-producing cells, (2) correlation of the reduction of viral infectivity with Trim 37 virion incorporation, (3) increased HIV-1 replication during siRNA depletion of Trim 37 expression, and (4) reduction in viral DNA synthesis upon Trim 37 transient overexpression. Our findings provide the first demonstration, to our knowledge, of the potent antiviral activity of human Trim 37, and implicate an antiviral mechanism whereby Trim 37 interferes with viral DNA synthesis.

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Human Immunodeficiency Virus Type 1 cDNAs Produced in the Presence of APOBEC3G Exhibit Defects in Plus-Strand DNA Transfer and Integration

Journal of Virology ◽

10.1128/jvi.00272-07 ◽

2007 ◽

Vol 81 (13) ◽

pp. 7099-7110 ◽

Cited By ~ 211

Author(s):

Jean L. Mbisa ◽

Rebekah Barr ◽

James A. Thomas ◽

Nick Vandegraaff ◽

Irene J. Dorweiler ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Dna Synthesis ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Dna Transfer ◽

Viral Dna ◽

Trna Processing ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Encapsidation of host restriction factor APOBEC3G (A3G) into vif-deficient human immunodeficiency virus type 1 (HIV-1) blocks virus replication at least partly by C-to-U deamination of viral minus-strand DNA, resulting in G-to-A hypermutation. A3G may also inhibit HIV-1 replication by reducing viral DNA synthesis and inducing viral DNA degradation. To gain further insight into the mechanisms of viral inhibition, we examined the metabolism of A3G-exposed viral DNA. We observed that an overall 35-fold decrease in viral infectivity was accompanied by a five- to sevenfold reduction in viral DNA synthesis. Wild-type A3G induced an additional fivefold decrease in the amount of viral DNA that was integrated into the host cell genome and similarly reduced the efficiency with which HIV-1 preintegration complexes (PICs) integrated into a target DNA in vitro. The A3G C-terminal catalytic domain was required for both of these antiviral activities. Southern blotting analysis of PICs showed that A3G reduced the efficiency and specificity of primer tRNA processing and removal, resulting in viral DNA ends that are inefficient substrates for integration and plus-strand DNA transfer. However, the decrease in plus-strand DNA transfer did not account for all of the observed decrease in viral DNA synthesis associated with A3G. These novel observations suggest that HIV-1 cDNA produced in the presence of A3G exhibits defects in primer tRNA processing, plus-strand DNA transfer, and integration.

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