Integrated Human Immunodeficiency Virus Type 1 Sequence in J-Lat 10.6

Chung, Ching‐Hu; Mele, Anthony R.; Allen, Alex; Costello, R; Dampier, Will; Nonnemacher, Michael R.; Wigdahl, Brian

doi:10.1128/mra.00179-20

Cited by 13 publications

(14 citation statements)

References 14 publications

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“…a PCS: Predicted cleavage sites; b HXB2: Whether the absolute gRNA sequence perfectly matched with HXB2 reference sequence. Note that the NL4-3 and R7/E-/eGFP in J-Lat (Chung C.-H. et al, 2020b) also showed the same results as HXB2 (Table S1); c# OT: number of potential off-target sites with CFD above 0.569 in human genome. d g8D9BC2: Previously published as gTatRev in (Wang et al, 2016a;Wang et al, 2016b;Darcis et al, 2019); e g01A4C4: Previously published as gGag3 in (Wang et al, 2016a;Zhao et al, 2017).…”

Section: Low Diversity Target Sites With High Global Patient Coveragementioning

confidence: 61%

Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy

Chung

Allen

Atkins

et al. 2021

Front. Cell. Infect. Microbiol.

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Clustered regularly interspaced short palindromic repeats (CRISPR)-based HIV-1 genome editing has shown promising outcomes in in vitro and in vivo viral infection models. However, existing HIV-1 sequence variants have been shown to reduce CRISPR-mediated efficiency and induce viral escape. Two metrics, global patient coverage and global subtype coverage, were used to identify guide RNA (gRNA) sequences that account for this viral diversity from the perspectives of cross-patient and cross-subtype gRNA design, respectively. Computational evaluation using these parameters and over 3.6 million possible 20-bp sequences resulted in nine lead gRNAs, two of which were previously published. This analysis revealed the benefit and necessity of considering all sequence variants for gRNA design. Of the other seven identified novel gRNAs, two were of note as they targeted interesting functional regions. One was a gRNA predicted to induce structural disruption in the nucleocapsid binding site (Ψ), which holds the potential to stop HIV-1 replication during the viral genome packaging process. The other was a reverse transcriptase (RT)-targeting gRNA that was predicted to cleave the subdomain responsible for dNTP incorporation. CRISPR-mediated sequence edits were predicted to occur on critical residues where HIV-1 has been shown to develop resistance against antiretroviral therapy (ART), which may provide additional evolutionary pressure at the DNA level. Given these observations, consideration of broad-spectrum gRNAs and cross-subtype diversity for gRNA design is not only required for the development of generalizable CRISPR-based HIV-1 therapy, but also helps identify optimal target sites.

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Section: Low Diversity Target Sites With High Global Patient Coveragementioning

confidence: 61%

Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy

Chung

Allen

Atkins

et al. 2021

Front. Cell. Infect. Microbiol.

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“…To assess the extent of nucleotide misincorporation (polymerase errors) that may occur during the PRLS assay, we activated J-Lat 10.3 cells ( 40 ) with phorbol 12-myristate 13-acetate (PMA) and extracted the supernatant before amplifying the cDNA with the gag -3′ primers. We compared 94 individual gag -3′ sequences (828,705 total nucleotides) to the published sequence of the proviral genome found in J-Lat cells ( 41 ) and determined the overall error rate of the assay per nucleotide to be 0.0076% (95% confidence interval [CI], 0.0054%, 0.0098%), or approximately 0.63 nucleotides/ gag -3′ genome.…”

Section: Resultsmentioning

confidence: 99%

“…Latency reversal was induced by incubating 5 × 10 4 cells with 2 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 h. The culture supernatant from the activated cells was harvested and stored at −80°C until used for RNA extraction. Following RNA extraction and cDNA synthesis, 94 genomes were amplified and sequenced using the PRLS assay, and SNPs or frameshift mutations were identified in the genomes that were genetically different from the reference sequence for the J-Lat cell line ( 41 ). The number of nucleotides involved in these changes was quantified and divided by the total number of nucleotides sequenced (828,705 total nucleotides) to determine the mean and 95% CI for the error rate of the assay.…”

Section: Methodsmentioning

confidence: 99%

Plasma-Derived HIV-1 Virions Contain Considerable Levels of Defective Genomes

Fisher

Wang

Lee

et al. 2022

J Virol

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Genetically-characterising full-length HIV-1 RNA is critical for identifying genetically-intact genomes, and comparing these RNA genomes to proviral DNA. We have developed a method for sequencing plasma-derived RNA using long-range sequencing (PRLS assay; ∼8.3 kb from gag to the 3’, or ∼5 kb from integrase to the 3’ end). We employed the gag -3’ PRLS assay to sequence HIV-1 RNA genomes from ART-naïve participants during acute/early infection (n=6) or chronic infection (n=2). On average only 65% of plasma-derived genomes were genetically-intact. Defects were found in all genomic regions, but were concentrated in env and pol . We compared these genomes to near-full-length proviral sequences from paired PBMC samples for the acute/early group, and found that near-identical (>99.98% identical) sequences were identified only during acute infection. For three participants who initiated therapy during acute infection, we used the int -3’ PRLS assay to sequence plasma-derived genomes from an analytical treatment interruption, and identified 100% identical genomes between pre-therapy and rebound timepoints. The PRLS assay provides a new level of sensitivity for understanding the genetic composition of plasma-derived HIV-1 RNA from viremic individuals either pre-therapy or after treatment interruption, which will be invaluable in assessing possible HIV-1 curative strategies. Importance: We developed novel plasma-derived RNA using long-range sequencing assays (PRLS assay; gag -3’ 8.3 kb and int -3’ 5.0 kb). Employing the gag -3’ PRLS assay, we found that 26-51% of plasma-derived genomes are genetically-defective, largely as a result of frameshift mutations and deletions. These genetic defects were concentrated in the env region compared to gag and pol , likely a reflection of viral immune escape in env during untreated HIV-1 infection. Employing the int -3’ PRLS assay, we found that analytical treatment interruption (ATI) plasma-derived sequences were identical and genetically-intact. Several sequences from the ATI plasma samples were identical to viral sequences from pre-therapy plasma and PBMC samples, indicating that HIV-1 reservoirs established prior to therapy contribute to viral rebound during an ATI. Therefore, near-full-length sequencing of HIV-1 particles is required to gain an accurate picture of the genetic landscape of plasma HIV-1 virions in studies of HIV-1 replication and persistence.

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“…To construct a reference reflecting integration of HIV-1 into J-Lat cells, we started with the UCSC hg38 human reference sequence from the Illumina iGenomes collection (https://support.illumina.com/sequencing/sequencing_software/igenome.html). Sequence for the integrated HIV-1 genome, including flanking human sequence, was obtained from accession MN989412.1 [84]. Accounting for flanking human sequence, we excised chr9:136468439-136468594 from the reference Fasta file and inserted MN989412.1 in its place.…”

Section: Methodsmentioning

confidence: 99%

“…Sequence for the integrated HIV-1 genome, including flanking human sequence, was obtained from accession MN989412.1 [78]. Accounting for flanking human sequence, we excised chr9:136468439-136468594 from the reference Fasta file and inserted MN989412.1 in its place.…”

Section: Automated Cutandtag Profilingmentioning

confidence: 99%

A modular CRISPR screen identifies individual and combination pathways contributing to HIV-1 latency

Hsieh

Janssens

Paddison

et al. 2022

Preprint

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Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms and are a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR, which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent, AZD5582, an activator of the non-canonical NFkB pathway, to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR, and the combination of ING3 knockout with the activation of non-canonical NFkB via AZD5582 act together to dramatically increase initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters.

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Integrated Human Immunodeficiency Virus Type 1 Sequence in J-Lat 10.6

Cited by 13 publications

References 14 publications

Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy

Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy

Plasma-Derived HIV-1 Virions Contain Considerable Levels of Defective Genomes

A modular CRISPR screen identifies individual and combination pathways contributing to HIV-1 latency

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