A quantitative approach for measuring the reservoir of latent HIV-1 proviruses

Bruner, Katherine M.; Zheng, Wei; Simonetti, Francesco R.; Bender, Alexandra M.; Kwon, Kyungyoon J.; Sengupta, Srona; Fray, Emily J.; Beg, Subul; Antar, Annukka A.R.; Jenike, Katharine M.; Bertagnolli, Lynn N.; Capoferri, Adam A.; Kufera, Joshua T.; Timmons, Andrew; Nobles, Christopher L.; Gregg, John T.; Wada, Nikolas; Ho, Ya-Chi; Zhang, Hao; Margolick, Joseph B.; Blankson, Joel N.; Deeks, Steven G.; Bushman, Frederic D.; Siliciano, Janet D.; Laird, Gregory M.; Siliciano, Robert F.

doi:10.1038/s41586-019-0898-8

Cited by 486 publications

(646 citation statements)

References 36 publications

(64 reference statements)

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“…We then investigated whether human DNA recovered on the nasopharyngeal swab could serve as a molecular marker of specimen collection quality, reasoning that DNA (by virtue of its stability) would be well-preserved in remnant clinical specimens. We employed a sensitive, multiplexed ddPCR protocol for absolute human RPP30 gene copy number quantification (9).…”

Section: Resultsmentioning

confidence: 99%

“…Droplets are assayed at endpoint, after which Poisson statistics are used to calculate input template concentrations. The protocol was originally developed to quantify human genome copy numbers to high accuracy and to estimate DNA shearing in the sample (though the latter is not essential for the present purpose) (9). Briefly, the extract is combined with primer/probe sets targeting two regions in the human RPP30 gene ~9kb apart, ddPCR Supermix for Probes (no dUTPs, BioRad), as part of COVID-19 testing were assessed for human RNAseP RNA levels using the US-CDC protocol on a Roche Lightcycler 480 (7).…”

Section: Methodsmentioning

confidence: 99%

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Suboptimal biological sampling as a probable cause of false-negative COVID-19 diagnostic test results

Kinloch

Ritchie

Brumme

et al. 2020

Preprint

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Suboptimal biological sampling as a probable cause of false-negative COVID-19 diagnostic test results

Kinloch

Ritchie

Brumme

et al. 2020

Preprint

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“…The inability to easily link variation in the provirus to a specific insertion site is an especially pressing problem for the HIV-1 reservoir. Only a small fraction of proviruses (2.4%) in the reservoir are intact, yet these are more than sufficient for the disease to rebound if antiretroviral therapy is removed 33 . As strategies are developed to target these intact proviruses it will be essential to distinguish between the intact and defective proviruses 33 .…”

Section: Discussionmentioning

confidence: 99%

Pooled CRISPR Inverse PCR sequencing (PCIP-seq): simultaneous sequencing of retroviral insertion points and the associated provirus in thousands of cells with long reads

Artesi

Hahaut

Cole

et al. 2019

Preprint

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Retroviral infections create a large population of cells, each defined by a unique proviral insertion site. Methods based on short-read high throughput sequencing can identify thousands of insertion sites, but the proviruses within remain unobserved. We have developed Pooled CRISPR Inverse PCR sequencing (PCIP-seq), a method that leverages long reads on the Oxford Nanopore MinION platform to sequence the insertion site and its associated provirus. We have applied the technique to three exogenous retroviruses, HTLV-1, HIV-1 and BLV, as well as endogenous retroviruses in both cattle and sheep. The long reads of PCIP-seq improved the accuracy of insertion site identification in repetitive regions of the genome. The high efficiency of the method facilitated the identification of tens of thousands of insertion sites in a single sample. We observed thousands of SNPs and dozens of structural variants within proviruses and uncovered evidence of viral hypermutation, recombination and recurrent selection.

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“…Therefore, full genome assays are required to distinguish defective from intact proviruses. These have been incorporated in the measurement of true HIV-1 reservoirs (Bruner et al, 2019). Alternatively, quantitative viral outgrowth assays (qVOA) are required as they detect only replication-competent virus.…”

Section: Introductionmentioning

confidence: 99%

Amplification of Replication Competent HIV-1 by Adoptive Transfer of Human Cells From Infected Humanized Mice

Sravanam

Gorantla

et al. 2020

Front. Cell. Infect. Microbiol.

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Detection of latent human immunodeficiency virus type 1 (HIV-1) in "putative" infectious reservoirs is required for determining treatment efficiency and for viral elimination strategies. Such tests require induction of replication competent provirus and quantitative testing of viral load for validation. Recently, humanized mice were employed in the development of such tests by employing a murine viral outgrowth assay (mVOA). Here blood cells were recovered from virus infected antiretroviral therapy suppressed patients. These cells were adoptively transferred to uninfected humanized mice where replication competent virus was recovered. Prior reports supported the notion that an mVOA assay provides greater sensitivity than cell culture-based quantitative VOA tests for detection of latent virus. In the current study, the mVOA assays was adapted using donor human hematopoietic stem cells-reconstituted mice to affirm research into HIV-1 elimination. We simulated an antiretroviral therapy (ART)-treated virus-infected human by maintaining the infected humanized mice under suppressive treatment. This was operative prior to human cell adoptive transfers. Replication-competent HIV-1 was easily detected in recipient animals from donors with undetectable virus in plasma. Moreover, when the assay was used to investigate viral presence in tissue reservoirs, quantitative endpoints were determined in "putative" viral reservoirs not possible in human sample analyses. We conclude that adoptive transfer of cells between humanized mice is a sensitive and specific assay system for detection of replication competent latent HIV-1.

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A quantitative approach for measuring the reservoir of latent HIV-1 proviruses

Cited by 486 publications

References 36 publications

Suboptimal biological sampling as a probable cause of false-negative COVID-19 diagnostic test results

Suboptimal biological sampling as a probable cause of false-negative COVID-19 diagnostic test results

Pooled CRISPR Inverse PCR sequencing (PCIP-seq): simultaneous sequencing of retroviral insertion points and the associated provirus in thousands of cells with long reads

Amplification of Replication Competent HIV-1 by Adoptive Transfer of Human Cells From Infected Humanized Mice

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