Comparative nucleic acid chaperone properties of the nucleocapsid protein NCp7 and Tat protein of HIV-1

Godet, Julien; Boudier, Christian; Humbert, Nicolas; Ivanyi‐Nagy, Roland; Darlix, Jean‐Luc; Mély, Yves

doi:10.1016/j.virusres.2012.06.021

Cited by 25 publications

(25 citation statements)

References 180 publications

(230 reference statements)

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“…Tat is a virion protein (13) that has reported paradoxical abilities to positively (7,8,11,12,(40)(41)(42)(43) or negatively (9,44,45) affect DNA synthesis by, or molecular components important for, reverse transcription. The opposing effects of Tat on reverse transcription leave open possibilities that Nullbasic may be either a TDNi form of Tat or a better Tat-based inhibitor of reverse transcription.…”

Section: Discussionmentioning

confidence: 99%

A Mutant Tat Protein Inhibits HIV-1 Reverse Transcription by Targeting the Reverse Transcription Complex

Lin

Apolloni

Cutillas

et al. 2015

J Virol

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Previously, we reported that a mutant of Tat referred to as Nullbasic inhibits HIV-1 reverse transcription although the mechanism of action is unknown. Here we show that Nullbasic is a reverse transcriptase (RT) binding protein that targets the reverse transcription complex rather than directly inhibiting RT activity. An interaction between Nullbasic and RT was observed by using coimmunoprecipitation and pulldown assays, and a direct interaction was measured by using a biolayer interferometry assay. Mixtures of recombinant 6؋His-RT and Nullbasic-FLAG-V5-6؋His at molar ratios of up to 1:20,000 did not inhibit RT activity in standard homopolymer primer template assays. An analysis of virus made by cells that coexpressed Nullbasic showed that Nullbasic copurified with virus particles, indicating that it was a virion protein. In addition, analysis of reverse transcription complexes (RTCs) isolated from cells infected with wild type or Nullbasic-treated HIV-1 showed that Nullbasic reduced the levels of viral DNA in RTC fractions. In addition, a shift in the distribution of viral DNA and CAp24 to less-dense non-RTC fractions was observed, indicating that RTC activity from Nullbasic-treated virus was impaired. Further analysis showed that viral cores isolated from Nullbasic-treated HIV undergo increased disassembly in vitro compared to untreated HIV-1. To our knowledge, this is the first description of an antiviral protein that inhibits reverse transcription by targeting the RTC and affecting core stability. Like all retroviruses, HIV-1 has a single positive-sense strand of RNA genome that is converted into double-strand proviral DNA by a hallmark process called reverse transcription. Proviral DNA is subsequently integrated into the host chromosomes and is transcribed by RNA polymerase II producing viral mRNA. The mechanisms regulating reverse HIV-1 transcription have been described in detail elsewhere (1). Briefly, the viral mRNA genome annealed to host cell tRNA Lys3 form a ribonucleoprotein complex with viral proteins, including reverse transcriptase (RT), integrase (IN), and nucleocapsid to form a prototypical reverse transcription complex (RTC) (2). The initiation of reverse transcription by the RTC begins shortly after cell infection after cytoplasmic nucleotides become available. Using tRNA Lys3 as a primer, DNA synthesis by RT produces a short strand of DNA called negativestrand strong stop DNA (ϪsssDNA). Degradation of the viral RNA strand by RT RNase H activity liberates ϪsssDNA that is transferred to the 3= end of the viral RNA by annealing of complementary nucleotide sequences, a step called first-strand transfer. The synthesis of the remaining negative-strand DNA can then be completed by RT. The complete synthesis of double-strand proviral DNA follows additional DNA synthesis following additional priming reactions and strand displacement DNA synthesis by RT. Cellular factors, including eEF1A, associate with the RTC and play an important role in the reverse transcription process (3, 4).Many virion ...

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Section: Discussionmentioning

confidence: 99%

A Mutant Tat Protein Inhibits HIV-1 Reverse Transcription by Targeting the Reverse Transcription Complex

Lin

Apolloni

Cutillas

et al. 2015

J Virol

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“…The mature NC protein, which is formed upon protease-mediated cleavage of the Gag polyprotein, facilitates encapsidation of the nucleic acids and is found in tight association with viral RNA in the viral core. During viral replication, NC chaperones the reverse transcriptase (RT) mediated synthesis of viral DNA and cooperates with the integrase for maintenance and integration of viral DNA (reviewed in (Godet et al, 2012; Sleiman et al, 2012)).…”

Section: Introductionmentioning

confidence: 99%

Advances in targeting nucleocapsid–nucleic acid interactions in HIV-1 therapy

Garg

Torbett

2014

Virus Research

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The continuing challenge of HIV-1 treatment resistance in patients creates a need for the development of new antiretroviral inhibitors. The HIV nucleocapsid (NC) protein is a potential therapeutic target. NC is necessary for viral RNA packaging and in the early stages of viral infection. The high level of NC amino acid conservation among all HIV-1 clades suggests a low tolerance for mutations. Thus, NC mutations that could arise during inhibitor treatment to provide resistance may render the virus less fit. Disruption of NC function provides a unique opportunity to strongly dampen replication at multiple points during the viral life cycle with a single inhibitor. Although NC exhibits desirable features for a potential antiviral target, the structural flexibility, size, and the presence of two zinc fingers makes small molecule targeting of NC a challenging task. In this review, we discuss the recent advances in strategies to develop inhibitors of NC function and present a perspective on potential novel approaches that may help to overcome some of the current challenges in the field.

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“…In contrast to the zinc fingers, the N-terminal domain is less conserved, but the positions of the basic residues are mainly conserved; also the basic linker between the two zinc fingers is highly conserved [36,37]. Early studies have demonstrated that disruption of the zinc fingers by removal of the zinc led to a loss of viral replication [38].…”

Section: Resultsmentioning

confidence: 99%

A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions

et al. 2012

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BackgroundSexual acquisition of the human immunodeficiency virus (HIV) through mucosal transmission may be prevented by using topically applied agents that block HIV transmission from one individual to another. Therefore, virucidal agents that inactivate HIV virions may be used as a component in topical microbicides.ResultsHere, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a low-molecular-weight molecule that inactivates HIV particles. Both HIV-1 and HIV-2 virions pretreated with this compound were unable to infect permissive cells. Moreover, WDO-217 was able to inhibit infections of a wide spectrum of wild-type and drug-resistant HIV-1, including clinical isolates, HIV-2 and SIV strains. Whereas the capture of virus by DC-SIGN was unaffected by the compound, it efficiently prevented the transmission of DC-SIGN-captured virus to CD4+ T-lymphocytes. Interestingly, exposure of virions to WDO-217 reduced the amount of virion-associated genomic RNA as measured by real-time RT-qPCR. Further mechanism-of-action studies demonstrated that WDO-217 efficiently ejects zinc from the zinc fingers of the retroviral nucleocapsid protein NCp7 and inhibits the cTAR destabilization properties of this protein. Importantly, WDO-217 was able to eject zinc from both zinc fingers, even when NCp7 was bound to oligonucleotides, while no covalent interaction between NCp7 and WDO-217 could be observed.ConclusionThis compound is a new lead structure that can be used for the development of a new series of NCp7 zinc ejectors as candidate topical microbicide agents.

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Comparative nucleic acid chaperone properties of the nucleocapsid protein NCp7 and Tat protein of HIV-1

Cited by 25 publications

References 180 publications

A Mutant Tat Protein Inhibits HIV-1 Reverse Transcription by Targeting the Reverse Transcription Complex

A Mutant Tat Protein Inhibits HIV-1 Reverse Transcription by Targeting the Reverse Transcription Complex

Advances in targeting nucleocapsid–nucleic acid interactions in HIV-1 therapy

A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions

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