Specific inhibition of in vitro reverse transcription using antisense oligonucleotides targeted to the TAR regions of HIV-1 and HIV-2

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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A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

Journal of Virology ◽

10.1128/jvi.00809-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8119-8129 ◽

Cited By ~ 4

Author(s):

Eytan Herzig ◽

Nickolay Voronin ◽

Nataly Kucherenko ◽

Amnon Hizi

Keyword(s):

Life Cycle ◽

Viral Replication ◽

Reverse Transcriptase ◽

Dna Polymerase ◽

Reverse Transcription ◽

Polymerase Activity ◽

Rnase H ◽

Strand Transfer ◽

Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

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HIV-1 uncoating occurs via a series of rapid biomechanical changes in the core related to individual stages of reverse transcription

10.1101/2021.01.16.426924 ◽

2021 ◽

Author(s):

Sanela Rankovic ◽

Akshay Deshpande ◽

Shimon Harel ◽

Christopher Aiken ◽

Itay Rousso

Keyword(s):

Reverse Transcription ◽

Viral Genome ◽

Morphological Changes ◽

Viral Capsid ◽

Target Cells ◽

Viral Core ◽

Successful Infection ◽

Capsid Shell ◽

Hiv 1

AbstractThe HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reactions containing the capsid-stabilizing host metabolite IP6. Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10-30, 40-80, and 120-160 minutes after initiation of reverse transcription. Addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, thus establishing that both result from reverse transcription. Using timed addition of efavirenz, and analysis of an RNAseH-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid AFM imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. Our results suggest that reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells.

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The HIV-1 Tat Protein Stimulates Reverse Transcription In Vitro

Current HIV Research ◽

10.2174/157016207781662443 ◽

2007 ◽

Vol 5 (5) ◽

pp. 474-483 ◽

Cited By ~ 21

Author(s):

Ann Apolloni ◽

Luke Meredith ◽

Andreas Suhrbier ◽

Rosemary Kiernan ◽

David Harrich

Keyword(s):

Reverse Transcription ◽

Tat Protein ◽

Transcription In Vitro ◽

Hiv 1

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Dimerization and Template Switching in the 5′ Untranslated Region between Various Subtypes of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.77.5.3020-3030.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3020-3030 ◽

Cited By ~ 26

Author(s):

Ebbe Sloth Andersen ◽

Rienk E. Jeeninga ◽

Christian Kroun Damgaard ◽

Ben Berkhout ◽

Jørgen Kjems

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Untranslated Region ◽

Template Switching ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) particle contains two identical RNA strands, each corresponding to the entire genome. The 5′ untranslated region (UTR) of each RNA strand contains extensive secondary and tertiary structures that are instrumental in different steps of the viral replication cycle. We have characterized the 5′ UTRs of nine different HIV-1 isolates representing subtypes A through G and, by comparing their homodimerization and heterodimerization potentials, found that complementarity between the palindromic sequences in the dimerization initiation site (DIS) hairpins is necessary and sufficient for in vitro dimerization of two subtype RNAs. The 5′ UTR sequences were used to design donor and acceptor templates for a coupled in vitro dimerization-reverse transcription assay. We showed that template switching during reverse transcription is increased with a matching DIS palindrome and further stimulated proportional to the level of homology between the templates. The presence of the HIV-1 nucleocapsid protein NCp7 increased the template-switching efficiency for matching DIS palindromes twofold, whereas the recombination efficiency was increased sevenfold with a nonmatching palindrome. Since NCp7 did not effect the dimerization of nonmatching palindromes, we concluded that the protein most likely stimulates the strand transfer reaction. An analysis of the distribution of template-switching events revealed that it occurs throughout the 5′ UTR. Together, these results demonstrate that the template switching of HIV-1 reverse transcriptase occurs frequently in vitro and that this process is facilitated mainly by template proximity and the level of homology.

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Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase

Journal of Virology ◽

10.1128/jvi.79.16.10356-10368.2005 ◽

2005 ◽

Vol 79 (16) ◽

pp. 10356-10368 ◽

Cited By ~ 55

Author(s):

Richard Lu ◽

Hina Z. Ghory ◽

Alan Engelman

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcription ◽

Class Ii ◽

Class I ◽

In Vitro Assays ◽

Mutant Proteins ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Results of in vitro assays identified residues in the C-terminal domain (CTD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) important for IN-IN and IN-DNA interactions, but the potential roles of these residues in virus replication were mostly unknown. Sixteen CTD residues were targeted here, generating 24 mutant viruses. Replication-defective mutants were typed as class I (blocked at integration) or class II (additional reverse transcription and/or assembly defects). Most defective viruses (15 of 17) displayed reverse transcription defects. In contrast, replication-defective HIV-1E246K synthesized near-normal cDNA levels but processing of Pr55 g ag was largely inhibited in virus-producing cells. Because single-round HIV-1E246K.Luc(R-) transduced cells at approximately 8% of the wild-type level, we concluded that the late-stage processing defect contributed significantly to the overall replication defect of HIV-1E246K. Results of complementation assays revealed that the CTD could function in trans to the catalytic core domain (CCD) in in vitro assays, and we since determined that certain class I and class II mutants defined a novel genetic complementation group that functioned in cells independently of IN domain boundaries. Seven of eight novel Vpr-IN mutant proteins efficiently trans-complemented class I active-site mutant virus, demonstrating catalytically active CTD mutant proteins during infection. Because most of these mutants inefficiently complemented a class II CCD mutant virus, the majority of CTD mutants were likely more defective for interactions with cellular and/or viral components that affected reverse transcription and/or preintegration trafficking than the catalytic activity of the IN enzyme.

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The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

Retrovirology ◽

10.1186/1742-4690-7-40 ◽

2010 ◽

Vol 7 (1) ◽

pp. 40 ◽

Cited By ~ 7

Author(s):

Jinwoo Ahn ◽

In-Ja L Byeon ◽

Sanjeewa Dharmasena ◽

Kelly Huber ◽

Jason Concel ◽

...

Keyword(s):

Reverse Transcriptase ◽

Reverse Transcription ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Transcription In Vitro ◽

Hiv 1

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The tRNA Primer Activation Signal in the Human Immunodeficiency Virus Type 1 Genome Is Important for Initiation and Processive Elongation of Reverse Transcription

Journal of Virology ◽

10.1128/jvi.76.5.2329-2339.2002 ◽

2002 ◽

Vol 76 (5) ◽

pp. 2329-2339 ◽

Cited By ~ 57

Author(s):

Nancy Beerens ◽

Ben Berkhout

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Sequence Motif ◽

Immunodeficiency Virus ◽

Trna Primer ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed by the cellular tRNA3 Lys molecule, which binds, with its 3"-terminal 18 nucleotides (nt), to a complementary sequence in the viral genome, the primer-binding site (PBS). Besides PBS-anti-PBS pairing, additional interactions between viral RNA sequences and the tRNA primer are thought to regulate the process of reverse transcription. We previously identified a novel 8-nt sequence motif in the U5 region of the HIV-1 RNA genome that is critical for tRNA3 Lys-mediated initiation of reverse transcription in vitro. This motif activates initiation from the natural tRNA3 Lys primer but is not involved in tRNA placement and was therefore termed primer activation signal (PAS). It was proposed that the PAS interacts with the anti-PAS motif in the TΨC arm of tRNA3 Lys. In this study, we analyzed several PAS-mutated viruses and performed reverse transcription assays with virion-extracted RNA-tRNA complexes. Mutation of the PAS reduced the efficiency of tRNA-primed reverse transcription. In contrast, mutations in the opposing leader sequence that trigger release of the PAS from base pairing stimulated reverse transcription. These results are similar to the reverse transcription effects observed in vitro. We also selected revertant viruses that partially overcome the reverse transcription defect of the PAS deletion mutant. Remarkably, all revertants acquired a single nucleotide substitution that does not restore the PAS sequence but that stimulates elongation of reverse transcription. These combined results indicate that the additional PAS-anti-PAS interaction is needed to assemble an initiation-competent and processive reverse transcription complex.

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Targeting Human Immunodeficiency Virus (HIV) Type 2 Integrase Protein into HIV Type 1

Journal of Virology ◽

10.1128/jvi.73.10.8831-8836.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8831-8836 ◽

Cited By ~ 10

Author(s):

Hongmei Liu ◽

Xiaoyun Wu ◽

Hongling Xiao ◽

John C. Kappes

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcription ◽

Wild Type Virus ◽

Strand Transfer ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell’s chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3′ processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN2) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN2 into IN mutant HIV-1 virions by expression intrans as a Vpr-IN fusion protein.Trans-complementation with IN2 markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous trans-IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126–2135, 1999), cells infected with IN2-complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous trans-IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, thetrans-IN2 protein was shown to support integration up to a level of 55% compared with that of the homologoustrans-IN protein. The delivery of heterologous IN protein into HIV-1 particles in trans offers a novel approach to understand IN protein function in vivo.

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