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

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.

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

Scientific Reports ◽

10.1038/s41598-018-20954-5 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 1

Author(s):

Xing-Jie Zhang ◽

Rui-Rui Wang ◽

Huan Chen ◽

Rong-Hua Luo ◽

Liu-Meng Yang ◽

...

Keyword(s):

Dna Synthesis ◽

Drug Pressure ◽

Viral Dna ◽

Polypurine Tract ◽

Hiv 1

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4′-C-Methyl-2′-Deoxyadenosine and 4′-C-Ethyl-2′-Deoxyadenosine Inhibit HIV-1 Replication

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01290-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2379-2389 ◽

Cited By ~ 6

Author(s):

B. Christie Vu ◽

Paul L. Boyer ◽

Maqbool A. Siddiqui ◽

Victor E. Marquez ◽

Stephen H. Hughes

Keyword(s):

Reverse Transcriptase ◽

Dna Synthesis ◽

Cultured Cells ◽

Nucleoside Analogs ◽

Transcriptase Inhibitor ◽

Resistance Mutations ◽

Viral Dna ◽

Infected Cells ◽

Hiv 1

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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The role of progeny viral DNA in the regulation of enzyme and DNA synthesis

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(64)90367-5 ◽

1964 ◽

Vol 16 (5) ◽

pp. 410-415 ◽

Cited By ~ 11

Author(s):

Tomoya Kamiya ◽

Tamar Ben-Porat ◽

Albert S. Kaplan

Keyword(s):

Dna Synthesis ◽

Viral Dna

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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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Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis

Journal of Molecular Biology ◽

10.1016/j.jmb.2007.11.021 ◽

2008 ◽

Vol 375 (5) ◽

pp. 1234-1248 ◽

Cited By ~ 28

Author(s):

Tania Matamoros ◽

Baek Kim ◽

Luis Menéndez-Arias

Keyword(s):

Reverse Transcriptase ◽

Dna Synthesis ◽

Hiv 1

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Binding to DCAF1 distinguishes TASOR and SAMHD1 degradation by HIV-2 Vpx

PLoS Pathogens ◽

10.1371/journal.ppat.1009609 ◽

2021 ◽

Vol 17 (10) ◽

pp. e1009609

Author(s):

Michaël M. Martin ◽

Roy Matkovic ◽

Pauline Larrous ◽

Marina Morel ◽

Angélique Lasserre ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Restriction Factors ◽

Viral Dna ◽

Host Restriction ◽

Viral Expression ◽

Host Restriction Factors ◽

Human Immunodeficiency Viruses ◽

Hiv 1

Human Immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) succeed to evade host immune defenses by using their viral auxiliary proteins to antagonize host restriction factors. HIV-2/SIVsmm Vpx is known for degrading SAMHD1, a factor impeding the reverse transcription. More recently, Vpx was also shown to counteract HUSH, a complex constituted of TASOR, MPP8 and periphilin, which blocks viral expression from the integrated viral DNA. In a classical ubiquitin ligase hijacking model, Vpx bridges the DCAF1 ubiquitin ligase substrate adaptor to SAMHD1, for subsequent ubiquitination and degradation. Here, we investigated whether the same mechanism is at stake for Vpx-mediated HUSH degradation. While we confirm that Vpx bridges SAMHD1 to DCAF1, we show that TASOR can interact with DCAF1 in the absence of Vpx. Nonetheless, this association was stabilized in the presence of Vpx, suggesting the existence of a ternary complex. The N-terminal PARP-like domain of TASOR is involved in DCAF1 binding, but not in Vpx binding. We also characterized a series of HIV-2 Vpx point mutants impaired in TASOR degradation, while still degrading SAMHD1. Vpx mutants ability to degrade TASOR correlated with their capacity to enhance HIV-1 minigenome expression as expected. Strikingly, several Vpx mutants impaired for TASOR degradation, but not for SAMHD1 degradation, had a reduced binding affinity for DCAF1, but not for TASOR. In macrophages, Vpx R34A-R42A and Vpx R42A-Q47A-V48A, strongly impaired in DCAF1, but not in TASOR binding, could not degrade TASOR, while being efficient in degrading SAMHD1. Altogether, our results highlight the central role of a robust Vpx-DCAF1 association to trigger TASOR degradation. We then propose a model in which Vpx interacts with both TASOR and DCAF1 to stabilize a TASOR-DCAF1 complex. Furthermore, our work identifies Vpx mutants enabling the study of HUSH restriction independently from SAMHD1 restriction in primary myeloid cells.

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Binding to DCAF1 distinguishes TASOR and SAMHD1 degradation by HIV-2 Vpx

10.1101/2021.05.06.442913 ◽

2021 ◽

Author(s):

Michaël M Martin ◽

Roy Matkovic ◽

Pauline Larrous ◽

Marina Morel ◽

Angélique Lasserre ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Restriction Factors ◽

Viral Dna ◽

Host Restriction ◽

Viral Expression ◽

Host Restriction Factors ◽

Human Immunodeficiency Viruses ◽

Hiv 1

Human Immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) succeed to evade host immune defenses by using their viral auxiliary proteins to antagonize host restriction factors. HIV-2/SIVsmm Vpx is known for degrading SAMHD1, a factor impeding the reverse transcription. More recently, Vpx was also shown to counteract HUSH, a complex constituted of TASOR, MPP8 and periphilin, which blocks viral expression from the integrated viral DNA. In a classical ubiquitin ligase hijacking model, Vpx bridges the DCAF1 ubiquitin ligase substrate adaptor to SAMHD1, for subsequent ubiquitination and degradation. Here, we investigated whether the same mechanism is at stake for Vpx-mediated HUSH degradation. While we confirm that Vpx bridges SAMHD1 to DCAF1, we show that TASOR can interact with DCAF1 in the absence of Vpx. Nonetheless, this association was stabilized in the presence of Vpx, suggesting the existence of a ternary complex. The N-terminal PARP-like domain of TASOR is involved in DCAF1 binding, but not in Vpx binding. We also characterized a series of HIV-2 Vpx point mutants impaired in TASOR degradation, while still degrading SAMHD1. Vpx mutants ability to degrade TASOR correlated with their capacity to enhance HIV-1 minigenome expression as expected. Strikingly, several Vpx mutants impaired for TASOR degradation, but not for SAMHD1 degradation, had a reduced binding affinity for DCAF1, but not for TASOR. In macrophages, Vpx R34A-R42A and Vpx R42A-Q47A-V48A, strongly impaired in DCAF1, but not in TASOR binding, could not degrade TASOR, while being efficient in degrading SAMHD1. Altogether, our results highlight the central role of a robust Vpx-DCAF1 association to trigger TASOR degradation. We then propose a model in which Vpx interacts with both TASOR and DCAF1 to stabilize a TASOR-DCAF1 complex. Furthermore, our work identifies Vpx mutants enabling the study of HUSH restriction independently from SAMHD1 restriction in primary myeloid cells.

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