Inhibition of HIV-1 reverse transcription by triple-helix forming oligonucleotides with viral RNA

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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HIV-1 Nucleocapsid Protein and the Secondary Structure of the Binary Complex Formed between tRNALys.3and Viral RNA Template Play Different Roles during Initiation of (−) Strand DNA Reverse Transcription

Journal of Biological Chemistry ◽

10.1074/jbc.m105124200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47725-47732 ◽

Cited By ~ 15

Author(s):

Liwei Rong ◽

Chen Liang ◽

Mayla Hsu ◽

Xiaofeng Guo ◽

Bernard P. Roques ◽

...

Keyword(s):

Reverse Transcriptase ◽

Zinc Finger ◽

Reverse Transcription ◽

Viral Rna ◽

Primer Binding Site ◽

Binary Complex ◽

Nucleotide Position ◽

Gag Protein ◽

Hiv 1 ◽

Stem Structure

In human immunodeficiency virus type 1 (HIV-1), the tRNALys.3primer and viral RNA template can form a specific complex that is characterized by extensive inter- and intramolecular interactions. Initiation of reverse transcription from this complex has been shown to be distinguished from subsequent elongation by early pausing events, such as at the +1 and +3 nucleotide positions. One major concern regarding the biological relevance of these results is that most kinetic studies of HIV-1 reverse transcription have been performed using tRNALys.3-viral (v) RNA complexes that were formed by heat annealing. In contrast, tRNALys.3in viruses is placed onto the primer binding site by nucleocapsid (NC) sequences of the Gag protein. In this study, we have further characterized the initiation features of reverse transcription in the presence of HIV-1 NC protein. In contrast to results obtained with a heat-annealed tRNALys.3·vRNA complex, we found that polymerization reactions catalyzed by HIV-1 reverse transcriptase did not commonly pause at the +1 nucleotide position when a NC-annealed RNA complex was used, and that this was true regardless whether NC was actually still present during reverse transcription. This activity of NC required both zinc finger motifs, as demonstrated by experiments that employed zinc finger-mutated forms of NC protein (H23C NC and ddNC), supporting the involvement of the zinc fingers in the RNA chaperone activity of NC. However, NC was not able to help reverse transcriptase to escape the +3 pausing event. Mutagenesis of a stem structure within the tRNALys.3. vRNA complex led to disappearance of the +3 pausing event as well as to significantly reduced rates of reverse transcription. Thus, this stem structure is essential for optimal reverse transcription, despite its role in promotion of the +3 pausing event.

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Allosteric HIV-1 Integrase Inhibitors Lead to Premature Degradation of the Viral RNA Genome and Integrase in Target Cells

Journal of Virology ◽

10.1128/jvi.00821-17 ◽

2017 ◽

Vol 91 (17) ◽

Cited By ~ 15

Author(s):

Michaela K. Madison ◽

Dana Q. Lawson ◽

Jennifer Elliott ◽

Ayşe Naz Ozantürk ◽

Pratibha C. Koneru ◽

...

Keyword(s):

Reverse Transcriptase ◽

Reverse Transcription ◽

Hexagonal Lattice ◽

Viral Rna ◽

Productive Infection ◽

Target Cells ◽

Integrase Inhibitors ◽

Ribonucleoprotein Complexes ◽

Rna Genome ◽

Hiv 1

ABSTRACT Recent evidence indicates that inhibition of HIV-1 integrase (IN) binding to the viral RNA genome by allosteric integrase inhibitors (ALLINIs) or through mutations within IN yields aberrant particles in which the viral ribonucleoprotein complexes (vRNPs) are eccentrically localized outside the capsid lattice. These particles are noninfectious and are blocked at an early reverse transcription stage in target cells. However, the basis of this reverse transcription defect is unknown. Here, we show that the viral RNA genome and IN from ALLINI-treated virions are prematurely degraded in target cells, whereas reverse transcriptase remains active and stably associated with the capsid lattice. The aberrantly shaped cores in ALLINI-treated particles can efficiently saturate and be degraded by a restricting TRIM5 protein, indicating that they are still composed of capsid proteins arranged in a hexagonal lattice. Notably, the fates of viral core components follow a similar pattern in cells infected with eccentric particles generated by mutations within IN that inhibit its binding to the viral RNA genome. We propose that IN-RNA interactions allow packaging of both the viral RNA genome and IN within the protective capsid lattice to ensure subsequent reverse transcription and productive infection in target cells. Conversely, disruption of these interactions by ALLINIs or mutations in IN leads to premature degradation of both the viral RNA genome and IN, as well as the spatial separation of reverse transcriptase from the viral genome during early steps of infection. IMPORTANCE Recent evidence indicates that HIV-1 integrase (IN) plays a key role during particle maturation by binding to the viral RNA genome. Inhibition of IN-RNA interactions yields aberrant particles with the viral ribonucleoprotein complexes (vRNPs) eccentrically localized outside the conical capsid lattice. Although these particles contain all of the components necessary for reverse transcription, they are blocked at an early reverse transcription stage in target cells. To explain the basis of this defect, we tracked the fates of multiple viral components in infected cells. Here, we show that the viral RNA genome and IN in eccentric particles are prematurely degraded, whereas reverse transcriptase remains active and stably associated within the capsid lattice. We propose that IN-RNA interactions ensure the packaging of both vRNPs and IN within the protective capsid cores to facilitate subsequent reverse transcription and productive infection in target cells.

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Identification of Capsid Mutations That Alter the Rate of HIV-1 Uncoating in Infected Cells

Journal of Virology ◽

10.1128/jvi.03043-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 643-651 ◽

Cited By ~ 32

Author(s):

Amy E. Hulme ◽

Z Kelley ◽

Eneniziaogochukwu A. Okocha ◽

Thomas J. Hope

Keyword(s):

Cell Lines ◽

Reverse Transcription ◽

Viral Rna ◽

Wild Type Virus ◽

Wild Type ◽

Cellular Environment ◽

Cellular Factors ◽

Infected Cells ◽

Kinetics Of ◽

Hiv 1

ABSTRACTAfter viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. We recently utilized the cyclosporine (CsA) washout assay, in which TRIM-CypA-mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of uncoating in HIV-1-infected cells. Here we have extended this analysis to examine the effects of p24 capsid protein (p24CA) mutations and cellular environment on the kinetics of uncoating in infected cells. We found that p24CAmutations can significantly increase (A92E), delay (E45A and N74D), or have no effect (G94D) on the rate of uncoating and that these alterations are not due to changes in reverse transcription. Inhibition of reverse transcription delayed uncoating kinetics to an extent similar to that of the wild-type virus with all the p24CAmutant viruses tested. In addition, we observed differences in uncoating in two cell lines, which suggests that the cellular environment can differentially impact the disassembly of wild-type and mutant capsids. Collectively, these experiments suggest that viral and cellular factors are important for the process of uncoating. Finally, these data support the model whereby early steps in reverse transcription facilitate HIV-1 uncoating.IMPORTANCEThe HIV-1 capsid is a cone-shaped structure, composed of the HIV-1-encoded protein p24CA, which contains the viral RNA and other proteins needed for infection. After the virus enters a target cell, this capsid must disassemble by a process called uncoating. Uncoating is required for HIV-1 infection to progress, but the details of how this process occurs is not known. In this study, we used anin vivoassay to examine the uncoating process in HIV-1-infected cells. We determined that p24CAmutations could increase or decrease the rate of uncoating and that this rate varied in different cell lines. We also found that reverse transcription of the viral RNA altered the process of uncoating before the p24CAmutations. Collectively, these experiments provide a better understanding of how viral and cellular factors are involved with a poorly understood step in HIV-1 infection.

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The Interaction of APOBEC3G with Human Immunodeficiency Virus Type 1 Nucleocapsid Inhibits tRNA3Lys Annealing to Viral RNA

Journal of Virology ◽

10.1128/jvi.00162-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 11322-11331 ◽

Cited By ~ 90

Author(s):

Fei Guo ◽

Shan Cen ◽

Meijuan Niu ◽

Yiliang Yang ◽

Robert J. Gorelick ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Transcription Initiation ◽

Rna Binding ◽

Viral Rna ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) containing human APOBEC3G (hA3G) has a reduced ability to produce viral DNA in newly infected cells. At least part of this hA3G-facilitated inhibition is due to a cytidine deamination-independent reduction in the ability to initiate reverse transcription. HIV-1 nucleocapsid (NCp7) is required both for the incorporation of hA3G into virions and for the annealing between viral RNA and tRNA3 Lys, the primer tRNA for reverse transcription. Herein we present evidence that the interaction of hA3G with nucleocapsid is required for the inhibition of reverse transcription initiation. A tRNA3 Lys priming complex was produced in vitro by the NCp7-facilitated annealing of tRNA3 Lys to synthetic viral RNA in the absence or presence of hA3G. The effect of hA3G on the annealing of tRNA3 Lys to viral RNA and the ability of tRNA3 Lys to initiate reverse transcription was measured. Our results show the following. (i) Electrophoretic band shift and primer binding site assays show that hA3G reduces the annealing of tRNA3 Lys 44 and 60%, respectively, but does not disrupt the annealed complex once formed. (ii) hA3G inhibits tRNA3 Lys priming 70 to 80%. (iii) Inhibition of tRNA3 Lys priming by hA3G requires an interaction between hA3G and NCp7 during annealing. Thus, annealing of tRNA3 Lys is insensitive to hA3G inhibition when facilitated by a zinc finger mutant of NCp7 unable to interact with hA3G. NCp7-independent annealing of DNA to viral RNA also is insensitive to hA3G inhibition. These results indicate that hA3G does not sterically block tRNA3 Lys annealing by binding to viral RNA. Annealing and priming are not affected by another RNA binding protein, QKI-6.

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The R263K Dolutegravir Resistance-Associated Substitution Progressively Decreases HIV-1 Integration

mBio ◽

10.1128/mbio.00157-17 ◽

2017 ◽

Vol 8 (2) ◽

Cited By ~ 8

Author(s):

Thibault Mesplède ◽

Jing Leng ◽

Hanh Thi Pham ◽

Jiaming Liang ◽

Yudong Quan ◽

...

Keyword(s):

Tissue Culture ◽

Quantitative Pcr ◽

Reverse Transcription ◽

Integrase Inhibitor ◽

Jurkat Cells ◽

Viral Rna ◽

Viral Dna ◽

Hiv Dna ◽

Hiv 1 ◽

Over Time

ABSTRACTHuman immunodeficiency virus (HIV) infection persists despite decades of active antiretroviral therapy (ART), effectively preventing viral eradication. Treatment decreases plasma viral RNA, but viral DNA persists, mostly integrated within the genome of nucleated blood cells. Viral DNA blood levels correlate with comorbidities and the rapidity of viral rebound following treatment interruption. To date, no intervention aiming at decreasing HIV DNA levels below those attained through ART has been successful. This includes use of some integrase inhibitors either as part of ART or in treatment intensification studies. We have argued that using the integrase inhibitor dolutegravir (DTG) in similar studies may yield better results, but this remains to be studied. In treatment-experienced individuals, the most frequent substitution associated with failure with dolutegravir is R263K in integrase. R263K decreases integration both in cell-free and tissue culture assays. We investigated here how integrated DNA levels evolve over time during prolonged infections with R263K viruses. To investigate a potential defect in reverse transcription with R263K, the levels of reverse transcripts were measured by quantitative PCR. We measured HIV type 1 (HIV-1) integration in Jurkat cells over the course of 4-week infections using Alu-mediated quantitative PCR. The results show that R263K did not decrease reverse transcription. Prolonged infections with R263K mutant viruses led to less HIV-1 integrated DNA over time compared to wild-type viruses. These tissue culture results help to explain the absence of the R263K substitution in most individuals experiencing failure with DTG and support studies aiming at longitudinally measuring the levels of integrated DNA in individuals treated with this drug.IMPORTANCEAntiretroviral treatment decreases plasma viral RNA, but HIV DNA persists for decades within infected cells. Studies of nonhuman primates have suggested that reducing retroviral DNA levels might represent a path to eradication. The integrase inhibitor dolutegravir is less susceptible than any other anti-HIV drug to the emergence of resistance in treatment-naive individuals. In treatment-experienced individuals, in contrast, rare cases of treatment failure were commonly associated with emergence of an R263K integrase substitution that confers low-level resistance to dolutegravir. It is unclear why this substitution is not more common in individuals experiencing failure with dolutegravir. We report here that R263K progressively diminishes the levels of integrated HIV-1 DNA in tissue culture over multiple cycles of infection. Our results help to explain aspects of the clinical efficacy of dolutegravir and suggest that this drug may be able to reduce HIV DNA levels within infected individuals compared to other drugs.

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The Tat Protein of Human Immunodeficiency Virus Type 1 (HIV-1) Can Promote Placement of tRNA Primer onto Viral RNA and Suppress Later DNA Polymerization in HIV-1 Reverse Transcription

Journal of Virology ◽

10.1128/jvi.76.8.3637-3645.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 3637-3645 ◽

Cited By ~ 31

Author(s):

Masanori Kameoka ◽

Max Morgan ◽

Marc Binette ◽

Rodney S. Russell ◽

Liwei Rong ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Viral Rna ◽

Dna Polymerization ◽

Functional Homology ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type-1 Tat has been proposed to play a role in the regulation of reverse transcription. We previously demonstrated that wild-type Tat can augment viral infectivity by suppressing the reverse transcriptase (RT) reaction at late stages of the viral life cycle in order to prevent the premature synthesis of potentially deleterious viral DNA products. Here we have performed a detailed analysis of the cell-free reverse transcription reaction to elucidate the mechanism(s) whereby Tat can affect this process. Our results show that Tat can suppress nonspecific DNA elongation while moderately affecting the specific initiation stage of reverse transcription. In addition, Tat has an RNA-annealing activity and can promote the placement of tRNA onto viral RNA. This points to a functional homology between Tat and the viral nucleocapsid (NC) protein that is known to be directly involved in this process. Experiments using a series of mutant Tat proteins revealed that the cysteine-rich and core domains of Tat are responsible for suppression of DNA elongation, while each of the cysteine-rich, core, and basic domains, as well as a glutamine-rich region in the C-terminal domain, are important for the placement of tRNA onto the viral RNA genome. These results suggest that Tat can play at least two different roles in the RT reaction, i.e., suppression of DNA polymerization and placement of tRNA onto viral RNA. We believe that the first of these activities of Tat may contribute to the overall efficiency of reverse transcription of the viral genome during a new round of infection as well as to enhanced production of infectious viral particles. We hypothesize that the second activity, illustrating functional homology between Tat and NC, suggests a potential role for NC in the displacement of Tat during viral maturation.

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Clustering and reverse transcription of HIV-1 genomes in nuclear niches of macrophages

10.1101/2020.04.12.038067 ◽

2020 ◽

Author(s):

Elena Rensen ◽

Florian Mueller ◽

Viviana Scoca ◽

Jyotsana J. Parmar ◽

Philippe Souque ◽

...

Keyword(s):

Reverse Transcription ◽

Replication Cycle ◽

Viral Rna ◽

Target Cells ◽

Nuclear Speckles ◽

Viral Dna ◽

Immunodeficiency Virus ◽

Early Replication ◽

Key Events ◽

Hiv 1

SummaryIn order to replicate, the Human Immunodeficiency Virus (HIV-1) reverse transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes. These two key events of the viral life cycle are commonly viewed as separate not only in time but also in cellular space, since reverse transcription (RT) is thought to be completed in the cytoplasm before nuclear import and integration. However, the spatiotemporal organization of the early replication cycle in macrophages, natural non-dividing target cells that constitute reservoirs of HIV-1 and an obstacle to curing AIDS, remains unclear. Here, we demonstrate that infected macrophages display large nuclear foci of viral DNA and viral RNA, in which multiple genomes cluster together. These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6 and localize to nuclear speckles. Strikingly, we show that viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where RT is pharmacologically suppressed and in untreated cells. We demonstrate that, after temporary inhibition, RT can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear RT can result in transcription competent viral DNA. These findings change our understanding of the early HIV-1 replication cycle, and may have implications for understanding HIV-1 persistence.

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