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Determining the Frequency, Size, and Proviral Expression of Infected Cell Clones Harboring Replication-Competent (Intact) HIV-1 Proviruses

Joseph, KW (2023) Determining the Frequency, Size, and Proviral Expression of Infected Cell Clones Harboring Replication-Competent (Intact) HIV-1 Proviruses. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Effective strategies to eliminate human immunodeficiency virus type 1 (HIV-1) reservoirs are likely to require more thorough characterizations of proviruses that persist on current antiretroviral therapy (ART). Infected cells that harbor intact proviruses can persist for long periods of time and clonally expand in response to stimuli, creating populations of infected cells harboring an identical provirus. The rarity of infected CD4+ T-cells and related technical challenges have limited the characterization of integrated proviruses. Current approaches using next-generation sequencing can be inefficient and limited sequencing depth can make it difficult to link proviral sequences to their respective integration sites. Here, we report on an efficient method by which HIV-1 proviruses and their sites of integration are amplified and sequenced, and their clonal frequency determined using integration site-specific qPCR (IS-qPCR). Additionally, this method allows amplification and sequencing of full-length env mRNA transcripts for assessing proviral expression. Across five HIV-1-positive individuals on clinically effective ART, a median of 41.2% (n = 88 of 209) of amplifications yielded near-full-length proviruses and their 5′-host-virus junctions containing a median of 430 bp (range, 18 to 1,363 bp) of flanking host sequence. Unexpectedly, 29.5% (n = 26 of 88) of the sequenced proviruses had structural asymmetries between the 5′ and 3′ long terminal repeats (LTRs), commonly in the form of major 3′ deletions, a novel discovery. Screening of 12,072 proviral endpoint-diluted MDA reactions for detectable env by qPCR yielded 15 sequence-intact proviruses across 3 of the 5 donors. Of the 9 intact proviruses with integration sites identified, all were integrated in introns, and most proviruses (n = 6/9) were in the opposite orientation of the gene of integration. To date, the frequencies of 7 clones with intact proviruses have been determined by IS-qPCR. The frequency of these intact proviruses ranged from 0.03% to 7.48% of all infected cells by LTR qPCR. Sequencing of full-length env mRNA transcripts to assess proviral expression (n = 94) yielded two identical sequences matching one intact provirus integrated in the highly expressed TAB2 gene. These findings demonstrate the large range of sizes of cell clones harboring intact provirus, and the difficulty that exists in characterizing the latent reservoir.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Joseph, KWkej38@pitt.edukej380000-0001-7091-1588
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairMellors, JWjwm1@pitt.edujwm10000-0002-3737-9742
Committee MemberMailliard, RBRBM19@pitt.eduRBM190000-0001-5501-503X
Committee MemberMartinson, JJjmartins@pitt.edujmartins0000-0003-4673-7238
Committee MemberMattila, JTjmattila@pitt.edujmattila0000-0002-6384-1291
Date: 28 June 2023
Date Type: Publication
Defense Date: 17 May 2023
Approval Date: 28 June 2023
Submission Date: 21 June 2023
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 209
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Infectious Diseases and Microbiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: HIV-1, integration, LTR, sequencing, replication-competent provirus, proviral expression
Date Deposited: 28 Jun 2023 15:29
Last Modified: 28 Jun 2023 15:29


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