HIV-1 Reverse Transcription

AbstractSAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

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High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs

Nature Communications ◽

10.1038/s41467-021-22628-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Betty Ha ◽

Kevin P. Larsen ◽

Jingji Zhang ◽

Ziao Fu ◽

Elizabeth Montabana ◽

...

Keyword(s):

Reverse Transcriptase ◽

Viral Entry ◽

Reverse Transcription ◽

Molecular Mechanisms ◽

Dissociation Kinetics ◽

Structural Mechanism ◽

Medium Resolution ◽

Kinetics Of ◽

Hiv 1

AbstractReverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC–nevirapine, and RTIC–efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA–tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

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Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

International Journal of Molecular Sciences ◽

10.3390/ijms22010058 ◽

2020 ◽

Vol 22 (1) ◽

pp. 58

Author(s):

Thomas Gremminger ◽

Zhenwei Song ◽

Juan Ji ◽

Avery Foster ◽

Kexin Weng ◽

...

Keyword(s):

Reverse Transcription ◽

Potential Interaction ◽

Viral Rna ◽

Primer Binding Site ◽

Rna Integrity ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Extended Interaction ◽

Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

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An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1

Methods ◽

10.1016/j.ymeth.2021.03.006 ◽

2021 ◽

Author(s):

Samreen Falak ◽

Rainer Macdonald ◽

Eloise J. Busby ◽

Denise M.O'Sullivan ◽

Mojca Milavec ◽

...

Keyword(s):

Reverse Transcription ◽

Digital Pcr ◽

Hiv 1

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NMR and biochemical characterization of recombinant human tRNA3 Lys expressed in Escherichia coli: Identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription

RNA ◽

10.1017/s1355838200000947 ◽

2000 ◽

Vol 6 (10) ◽

pp. 1403-1412 ◽

Cited By ~ 44

Author(s):

CARINE TISNÉ ◽

MICKAËL RIGOURD ◽

ROLAND MARQUET ◽

CHANTAL EHRESMANN ◽

FRÉDÉRIC DARDEL

Keyword(s):

Escherichia Coli ◽

Reverse Transcription ◽

Biochemical Characterization ◽

Hiv 1

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Mechanism of HIV-1 NC Protein-Induced Condensation of Double Stranded DNA as a Model for DNA Compaction during Reverse Transcription

Biophysical Journal ◽

10.1016/j.bpj.2020.11.1403 ◽

2021 ◽

Vol 120 (3) ◽

pp. 206a

Author(s):

Helena Gien ◽

Michael Morse ◽

Jonathan Kitzrow ◽

Ioulia F. Rouzina ◽

Karin Musier-Forsyth ◽

...

Keyword(s):

Reverse Transcription ◽

Dna Compaction ◽

Double Stranded Dna ◽

Hiv 1

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In Vivo Analysis of Human T-Cell Leukemia Virus Type 1 Reverse Transcription Accuracy

Journal of Virology ◽

10.1128/jvi.74.20.9525-9531.2000 ◽

2000 ◽

Vol 74 (20) ◽

pp. 9525-9531 ◽

Cited By ~ 58

Author(s):

Louis M. Mansky

Keyword(s):

T Cell ◽

Mutation Rate ◽

Virus Type ◽

Reverse Transcription ◽

Leukemia Virus ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Hiv 1

ABSTRACT Several studies have indicated that the genetic diversity of human T-cell leukemia virus type 1 (HTLV-1), a virus associated with adult T-cell leukemia, is significantly lower than that of other retroviruses, including that of human immunodeficiency virus type 1 (HIV-1). To test whether HTLV-1 variation is lower than other retroviruses, a tractable vector system has been developed to measure reverse transcription accuracy in one round of HTLV-1 replication. This system consists of a HTLV-1 vector that contains a cassette with the neomycin phosphotransferase (neo) gene, a bacterial origin of DNA replication, and the lacZα peptide gene region (the mutational target). The vector was replicated bytrans-complementation with helper plasmids. The in vivo mutation rate for HTLV-1 was determined to be 7 × 10−6 mutations per target base pair per replication cycle. The majority of the mutations identified were base substitution mutations, namely, G-to-A and C-to-T transitions, frameshift mutations, and deletion mutations. Mutation of the methionine residue in the conserved YMDD motif of the HTLV-1 reverse transcriptase to either alanine or valine (i.e., M188A or M188V) led to a factor of two increase in the rate of mutation, indicating the role of this motif in enzyme accuracy. The HTLV-1 in vivo mutation rate is comparable to that of bovine leukemia virus (BLV), another member of the HTLV/BLV genus of retroviruses, and is about fourfold lower than that of HIV-1. These observations indicate that while the mutation rate of HTLV-1 is significantly lower than HIV-1, this lower rate alone would not explain the low diversity in HTLV-1 isolates, supporting the hypothesis that HTLV-1 replicates primarily as a provirus during cellular DNA replication rather than as a virus via reverse transcription.

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HIV-1 Exploits a Dynamic Multi-aminoacyl-tRNA Synthetase Complex To Enhance Viral Replication

Journal of Virology ◽

10.1128/jvi.01240-17 ◽

2017 ◽

Vol 91 (21) ◽

Cited By ~ 15

Author(s):

Alice A. Duchon ◽

Corine St. Gelais ◽

Nathan Titkemeier ◽

Joshua Hatterschide ◽

Li Wu ◽

...

Keyword(s):

Viral Replication ◽

Nuclear Localization ◽

Reverse Transcription ◽

Transcriptase Inhibitor ◽

Trna Synthetase ◽

Mitogen Activated Protein Kinase ◽

Heat Inactivation ◽

Target Cells ◽

Aminoacyl Trna Synthetase ◽

Hiv 1

ABSTRACT A hallmark of retroviruses such as human immunodeficiency virus type 1 (HIV-1) is reverse transcription of genomic RNA to DNA, a process that is primed by cellular tRNAs. HIV-1 recruits human tRNALys3 to serve as the reverse transcription primer via an interaction between lysyl-tRNA synthetase (LysRS) and the HIV-1 Gag polyprotein. LysRS is normally sequestered in a multi-aminoacyl-tRNA synthetase complex (MSC). Previous studies demonstrated that components of the MSC can be mobilized in response to certain cellular stimuli, but how LysRS is redirected from the MSC to viral particles for packaging is unknown. Here, we show that upon HIV-1 infection, a free pool of non-MSC-associated LysRS is observed and partially relocalized to the nucleus. Heat inactivation of HIV-1 blocks nuclear localization of LysRS, but treatment with a reverse transcriptase inhibitor does not, suggesting that the trigger for relocalization occurs prior to reverse transcription. A reduction in HIV-1 infection is observed upon treatment with an inhibitor to mitogen-activated protein kinase that prevents phosphorylation of LysRS on Ser207, release of LysRS from the MSC, and nuclear localization. A phosphomimetic mutant of LysRS (S207D) that lacked the capability to aminoacylate tRNALys3 localized to the nucleus, rescued HIV-1 infectivity, and was packaged into virions. In contrast, a phosphoablative mutant (S207A) remained cytosolic and maintained full aminoacylation activity but failed to rescue infectivity and was not packaged. These findings suggest that HIV-1 takes advantage of the dynamic nature of the MSC to redirect and coopt cellular translation factors to enhance viral replication. IMPORTANCE Human tRNALys3, the primer for reverse transcription, and LysRS are essential host factors packaged into HIV-1 virions. Previous studies found that tRNALys3 packaging depends on interactions between LysRS and HIV-1 Gag; however, many details regarding the mechanism of tRNALys3 and LysRS packaging remain unknown. LysRS is normally sequestered in a high-molecular-weight multi-aminoacyl-tRNA synthetase complex (MSC), restricting the pool of free LysRS-tRNALys. Mounting evidence suggests that LysRS is released under a variety of stimuli to perform alternative functions within the cell. Here, we show that HIV-1 infection results in a free pool of LysRS that is relocalized to the nucleus of target cells. Blocking this pathway in HIV-1-producing cells resulted in less infectious progeny virions. Understanding the mechanism by which LysRS is recruited into the viral assembly pathway can be exploited for the development of specific and effective therapeutics targeting this nontranslational function.

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PF74 Inhibits HIV-1 Integration by Altering the Composition of the Preintegration Complex

Journal of Virology ◽

10.1128/jvi.01741-18 ◽

2018 ◽

Vol 93 (6) ◽

Cited By ~ 11

Author(s):

Muthukumar Balasubramaniam ◽

Jing Zhou ◽

Amma Addai ◽

Phillip Martinez ◽

Jui Pandhare ◽

...

Keyword(s):

Reverse Transcription ◽

Integrase Inhibitor ◽

Antiviral Effect ◽

Inhibitory Effect ◽

Clear Understanding ◽

Nuclear Entry ◽

Preintegration Complex ◽

Gradient Based ◽

Hiv 1

ABSTRACTThe HIV-1 capsid protein (CA) facilitates reverse transcription and nuclear entry of the virus. However, CA’s role in post-nuclear entry steps remains speculative. We describe a direct link between CA and integration by employing the capsid inhibitor PF74 as a probe coupled with the biochemical analysis of HIV-1 preintegration complexes (PICs) isolated from acutely infected cells. At a low micromolar concentration, PF74 potently inhibited HIV-1 infection without affecting reverse transcription. Surprisingly, PF74 markedly reduced proviral integration owing to inhibition of nuclear entry and/or integration. However, a 2-fold reduction in nuclear entry by PF74 did not quantitatively correlate with the level of antiviral activity. Titration of PF74 against the integrase inhibitor raltegravir showed an additive antiviral effect that is dependent on a block at the post-nuclear entry step. PF74’s inhibitory effect was not due to the formation of defective viral DNA ends or a delay in integration, suggesting that the compound inhibits PIC-associated integration activity. Unexpectedly, PICs recovered from cells infected in the presence of PF74 exhibited elevated integration activity. PF74’s effect on PIC activity is CA specific since the compound did not increase the integration activity of PICs of a PF74-resistant HIV-1 CA mutant. Sucrose gradient-based fractionation studies revealed that PICs assembled in the presence of PF74 contained lower levels of CA, suggesting a negative association between CA and PIC-associated integration activity. Finally, the addition of a CA-specific antibody or PF74 inhibited PIC-associated integration activity. Collectively, our results demonstrate that PF74’s targeting of PIC-associated CA results in impaired HIV-1 integration.IMPORTANCEAntiretroviral therapy (ART) that uses various combinations of small molecule inhibitors has been highly effective in controlling HIV. However, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. The HIV-1 CA plays critical roles in the virus life cycle and is an attractive therapeutic target. While currently there is no CA-based therapy, highly potent CA-specific inhibitors are being developed as a new class of antivirals. Efforts to develop a CA-targeted therapy can be aided through a clear understanding of the role of CA in HIV-1 infection. CA is well established to coordinate reverse transcription and nuclear entry of the virus. However, the role of CA in post-nuclear entry steps of HIV-1 infection is poorly understood. We show that a CA-specific drug PF74 inhibits HIV-1 integration revealing a novel role of this multifunctional viral protein in a post-nuclear entry step of HIV-1 infection.

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