The importance of the A-rich loop in human immunodeficiency virus type 1 reverse transcription and infectivity

Liang, Chen; Li, Xuguang; Liang, Rong; Inouye, Phil; Quan, Yudong; Kleiman, Lawrence; Wainberg, Mark A.

doi:10.1128/jvi.71.8.5750-5757.1997

Cited by 81 publications

(49 citation statements)

References 34 publications

(49 reference statements)

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“…In HIV‐1, the interaction between the A‐rich loop in the 5′‐region upstream of the PBS and the U‐rich anticodon loop of tRNA Lys3 was implicated in the initiation of cDNA synthesis [4–7], and in the positioning of tRNA Lys3 on the PBS [8]. The importance of this interaction was also shown in HIV‐1‐infected cells [9], as well as in in vivo experiments in which HIV‐1 RNA containing a deletion of the A‐rich loop showed decreased reverse transcription [10].…”

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confidence: 99%

Study of HIV‐2 primer–template initiation complex using antisense oligonucleotides

Boulmé

Freund

Gryaznov

et al. 2000

European Journal of Biochemistry

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HIV‐2 reverse transcription is initiated by the retroviral DNA polymerase (reverse transcriptase) from a cellular tRNALys3 partially annealed to the primer binding site in the 5′‐region of viral RNA. The HIV‐2 genome has two A‐rich regions upstream of the primer binding site. In contrast to HIV‐1 RNA, no direct evidence of interactions with the U‐rich anticodon loop of tRNALys3 has been described to date. Here we address the question of the potential role of the interactions between these highly structured regions in the initiation of viral DNA synthesis. To evaluate this we used an antisense approach, first validated in our in vitro HIV‐1 reverse transcription system. Annealing of the antisense oligonucleotides to the pre‐primer binding site (the upstream region contiguous to the HIV‐2 primer binding site) was determined in the presence of native tRNALys3 or synthetic primers. Using natural and chemically modified antisense oligonucleotides we found that interactions between the anticodon of tRNALys3 and an A‐rich loop of viral RNA led to an important destabilization of the pre‐primer binding site; this region became accessible to anti‐pre‐primer binding site oligonucleotides in a cooperative manner. These studies allowed to identify an A‐rich region in HIV‐2ROD RNA capable of interacting with tRNALys3. Better knowledge of these interactions is very important for understanding the primer/template positioning in the early steps of HIV‐2 reverse transcription.

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confidence: 99%

Study of HIV‐2 primer–template initiation complex using antisense oligonucleotides

Boulmé

Freund

Gryaznov

et al. 2000

European Journal of Biochemistry

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“…The second escape pathway is to use a cellular tRNA other than tRNA 3 Lys to prime viral reverse transcription. The genetic barrier for the latter escape pathway might be high, because HIV-1 reverse transcriptase and the viral 5= untranslated region (UTR) also need to change in order to use a new tRNA primer (40,(47)(48)(49)(50)(51), which has rarely occurred in studies where the PBS was changed to match the sequences of different tRNAs (32)(33)(34).…”

Section: Discussionmentioning

confidence: 99%

HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

Wang

Cui

et al. 2018

J Virol

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The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology has been used to inactivate viral DNA as a new strategy to eliminate chronic viral infections, including HIV-1. This utility of CRISPR-Cas9 is challenged by the high heterogeneity of HIV-1 sequences, which requires the design of the single guide RNA (sgRNA; utilized by the CRISPR-Cas9 system to recognize the target DNA) to match a specific HIV-1 strain in an HIV patient. One solution to this challenge is to target the viral primer binding site (PBS), which HIV-1 copies from cellular tRNA in each round of reverse transcription and is thus conserved in almost all HIV-1 strains. In this study, we demonstrate that PBS-targeting sgRNA directs Cas9 to cleave the PBS DNA, which evokes deletions or insertions (indels) and strongly diminishes the production of infectious HIV-1. While HIV-1 escapes from PBS-targeting Cas9/sgRNA, unique resistance mechanisms are observed that are dependent on whether the plus or the minus strand of the PBS DNA is bound by sgRNA. Characterization of these viral escape mechanisms will inform future engineering of Cas9 variants that can more potently and persistently inhibit HIV-1 infection. The results of this study demonstrate that the gene-editing complex Cas9/sgRNA can be programmed to target and cleave HIV-1 PBS DNA, and thus, inhibit HIV-1 infection. Given that almost all HIV-1 strains have the same PBS, which is copied from the cellular tRNA during reverse transcription, PBS-targeting sgRNA can be used to inactivate HIV-1 DNA of different strains. We also discovered that HIV-1 uses different mechanisms to resist Cas9/sgRNAs, depending on whether they target the plus or the minus strand of PBS DNA. These findings allow us to predict that a Cas9 variant that uses the CCA sequence as the protospacer adjacent motif (PAM) should more strongly and persistently suppress HIV-1 replication. Together, these data have identified the PBS as the target DNA of Cas9/sgRNA and have predicted how to improve Cas9/sgRNA to achieve more efficient and sustainable suppression of HIV-1 infection, therefore improving the capacity of Cas9/sgRNA in curing HIV-1 infection.

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“…A similar model has been proposed for HIV-2 [9]. Precisely how these RNA interactions function in RTN is not known, but RNA stem-loop structures are associated with pausing by RT and by RNA polymerase II during synthesis [14,[18][19][20], which makes it somewhat surprising that mutations in the A-rich loop which prevent kissing-loop base pairing result in decreased initiation efficiency and reduced virus replication kinetics [17]. Likewise, subtle changes in RNA sequences upstream of the PBS inhibit proviral DNA synthesis [21][22][23][24][25], but the precise role of these sequences in RTN is also unknown.…”

Section: Introductionmentioning

confidence: 89%

“…In vitro RTN reactions using HIV-1 RNA templates are less efficient and show reduced initiation levels when synthetic oligonucleotides are used to prime the reaction than when natural tRNA lys3 is used [8,46,47]. Viruses deleted or mutated in the A-rich region are also defective for DNA synthesis and have reduced replication kinetics [17,48,49]. How an RNA structure directly adjacent to the initiation site stimulates the RTN initiation reaction is unknown.…”

Section: The Reverse Transcription Initiation Complexmentioning

confidence: 99%

“…It is generally observed that virions contain very low amounts of DNA, an observation attributed to the low nucleotide levels in supernatants [10][11][12][13] and more recently suspected to be an intrinsic property of the RTNIC [14,15]. In HIV-1, a kissing-loop structure formed by the tRNA lys3 anticodon loop and an HIV A-rich loop (nt+169 to +172, HXB2D) is required for efficient RTN initiation and virus replication [16,17]. A similar model has been proposed for HIV-2 [9].…”

Section: Introductionmentioning

confidence: 99%

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Mechanistic aspects of HIV‐1 reverse transcription initiation

Harrich

Hooker

2002

Reviews in Medical Virology

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During reverse transcription, the positive-strand HIV-1 RNA genome is converted into a double-stranded DNA copy which can be permanently integrated into the host cell genome. Recent analyses show that HIV-1 reverse transcription is a highly regulated process. The initiation reaction can be distinguished from a subsequent elongation reaction carried out by a reverse transcription complex composed of (at least) heterodimeric reverse transcriptase, cellular tRNA(lys3) and HIV-1 genomic RNA sequences. In addition, viral factors including Tat, Nef, Vif, Vpr, IN and NCp7, cellular proteins, and TAR RNA and other RNA stem-loop structures appear to influence this complex and contribute to the efficiency of the initiation reaction. As viral resistance to many antiretroviral compounds is a continuing problem, understanding the ways in which these factors influence the reverse transcription complex will likely lead to novel antiretroviral strategies.

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The importance of the A-rich loop in human immunodeficiency virus type 1 reverse transcription and infectivity

Cited by 81 publications

References 34 publications

Study of HIV‐2 primer–template initiation complex using antisense oligonucleotides

Study of HIV‐2 primer–template initiation complex using antisense oligonucleotides

HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

Mechanistic aspects of HIV‐1 reverse transcription initiation

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