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(Unpublished)
Abstract
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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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| 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 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/45015 |
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