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

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Zhen Wang ◽  
Wenzhou Wang ◽  
Ya Cheng Cui ◽  
Qinghua Pan ◽  
Weijun Zhu ◽  
...  
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Gene Editing ◽  
Viral Escape ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Guide Rna ◽  
Target Dna ◽  
Almost All ◽  
Hiv 1

ABSTRACTThe 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 tRNA3Lysin 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.IMPORTANCEThe 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 tRNA3Lysduring 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.

How the HIV-1 Nucleocapsid Protein Binds and Destabilises the (−)Primer Binding Site During Reverse Transcription

2008 ◽  
Vol 383 (5) ◽  
pp. 1112-1128 ◽  
Author(s):  
Sarah Bourbigot ◽  
Nick Ramalanjaona ◽  
Christian Boudier ◽  
Gilmar F.J. Salgado ◽  
Bernard P. Roques ◽  
...  
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Primer Binding Site ◽  
Hiv 1

scholarly journals Spelling changes and fluorescent tagging with prime editing vectors for plants

2020 ◽  
Author(s):  
Li Wang ◽  
Hilal Betul Kaya ◽  
Ning Zhang ◽  
Rhitu Rai ◽  
Matthew R. Willmann ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Binding Site ◽  
Nicotiana Benthamiana ◽  
Primer Binding Site ◽  
First Report ◽  
Guide Rna ◽  
Insertions And Deletions ◽  
Target Dna ◽  
Fluorescent Tagging

AbstractPrime editing (PE) is a recent adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase (RT) fusion and a guide RNA (pegRNA) amended with template and primer binding site (PBS) sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of PE in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for PE in dicots and monocots, their validation in Nicotiana benthamiana, rice and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

scholarly journals Spelling Changes and Fluorescent Tagging With Prime Editing Vectors for Plants

2021 ◽  
Vol 3 ◽  
Author(s):  
Li Wang ◽  
Hilal Betul Kaya ◽  
Ning Zhang ◽  
Rhitu Rai ◽  
Matthew R. Willmann ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Binding Site ◽  
Nicotiana Benthamiana ◽  
Primer Binding Site ◽  
First Report ◽  
Guide Rna ◽  
Insertions And Deletions ◽  
Target Dna ◽  
Fluorescent Tagging

Prime editing is an adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase fusion and a guide RNA amended with template and primer binding site sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of prime editing in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for prime editing in dicots as well as monocots, their validation in Nicotiana benthamiana, rice, and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

scholarly journals Stimulation of HIV-1 Minus Strand Strong Stop DNA Transfer by Genomic Sequences 3′ of the Primer Binding Site

2006 ◽  
Vol 281 (34) ◽  
pp. 24227-24235 ◽  
Author(s):  
Min Song ◽  
Mini Balakrishnan ◽  
Yan Chen ◽  
Bernard P. Roques ◽  
Robert A. Bambara
Keyword(s):  
Binding Site ◽  
Dna Transfer ◽  
Genomic Sequences ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Hiv 1 ◽  
Strong Stop ◽  
Stimulation Of

Polyamide Nucleic Acid Targeted to the Primer Binding Site of the HIV-1 RNA Genome Blocksin VitroHIV-1 Reverse Transcription†

Biochemistry ◽  
1998 ◽  
Vol 37 (3) ◽  
pp. 900-910 ◽  
Author(s):  
Reaching Lee ◽  
Neerja Kaushik ◽  
Mukund J. Modak ◽  
Ravi Vinayak ◽  
Virendra N. Pandey
Keyword(s):  
Nucleic Acid ◽  
Binding Site ◽  
Reverse Transcription ◽  
Primer Binding Site ◽  
Rna Genome ◽  
Hiv 1

scholarly journals Multiplexed tat-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1223
Author(s):  
Youdiil Ophinni ◽  
Sayaka Miki ◽  
Yoshitake Hayashi ◽  
Masanori Kameoka
Keyword(s):  
T Cells ◽  
Viral Escape ◽  
Escape Mutant ◽  
Knock Out ◽  
Guide Rna ◽  
Acute Hiv ◽  
Almost All ◽  
First Time ◽  
Hiv 1

HIV-1 cure strategy by means of proviral knock-out using CRISPR-Cas9 has been hampered by the emergence of viral resistance against the targeting guide RNA (gRNA). Here, we proposed multiple, concentrated gRNA attacks against HIV-1 regulatory genes to block viral escape. The T cell line were transduced with single and multiple gRNAs targeting HIV-1 tat and rev using lentiviral-based CRISPR-Cas9, followed by replicative HIV-1NL4-3 challenge in vitro. Viral p24 rebound was observed for almost all gRNAs, but multiplexing three tat-targeting gRNAs maintained p24 suppression and cell viability, indicating the inhibition of viral escape. Multiplexed tat gRNAs inhibited acute viral replication in the 2nd round of infection, abolished cell-associated transmission to unprotected T cells, and maintained protection through 45 days, post-infection (dpi) after a higher dose of HIV-1 infection. Finally, we describe here for the first time the assembly of all-in-one lentiviral vectors containing three and six gRNAs targeting tat and rev. A single-vector tat-targeting construct shows non-inferiority to the tat-targeting multi-vector in low-dose HIV-1 infection. We conclude that Cas9-induced, DNA repair-mediated mutations in tat are sufficiently deleterious and deplete HIV-1 fitness, and multiplexed disruption of tat further limits the possibility of an escape mutant arising, thus elevating the potential of CRISPR-Cas9 to achieve a long-term HIV-1 cure.

scholarly journals Alternative tRNA Priming of Human Immunodeficiency Virus Type 1 Reverse Transcription Explains Sequence Variation in the Primer-Binding Site That Has Been Attributed to APOBEC3G Activity

2005 ◽  
Vol 79 (5) ◽  
pp. 3179-3181 ◽  
Author(s):  
Atze T. Das ◽  
Monique Vink ◽  
Ben Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Sequence Variation ◽  
Primer Binding Site ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT It is generally assumed that human immunodeficiency virus type 1 (HIV-1) uses exclusively the cellular \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} molecule as a primer for reverse transcription. We demonstrate that HIV-1 uses not only \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} but also an alternative tRNA primer. This tRNA was termed \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{5}^{Lys}\) \end{document} , and the near completion of the human genome project has allowed the identification of four \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{5}^{Lys}\) \end{document} encoding genes. Priming with \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{5}^{Lys}\) \end{document} results in a single nucleotide polymorphism in the viral primer-binding site that is present in multiple natural and laboratory HIV isolates. This sequence variation was recently attributed to APOBEC3G activity. However, our results show that alternative tRNA priming can cause this mutation in the absence of APOBEC3G.

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

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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