Browsing by Subject "Human Immunodeficiency Virus Type-1 (HIV-1)"
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Item CHARACTERIZATION OF THE HIV-1 PROVIRAL LANDSCAPE IN PERINATALLY INFECTED EARLY TREATED CHILDREN ON LONG-TERM ANTIRETROVIRAL THERAPY(2022) Hasson, Jenna; Kearney, Mary; Patro, Sean; Boyd, Ann; Hood College Biomedical ScienceAntiretroviral therapy (ART) prevents disease progression in people living with Human Immunodeficiency Virus (HIV) (PLWH). However, off-target effects can result in liver disease, heart disease, diabetes, and more, and there is no accessible cure for the virus. Therefore, understanding the mechanisms for HIV persistence on ART is important to inform strategies for the development of future potential curative inventions. Studies investigating the genetics of integrated HIV genomes (proviruses) in adults revealed the sources of low-level viremia and informed new targets for possible curative strategies. However, little is known about the effect of HIV and long-term ART on the developing immune systems of infants and children. Differences between the immune systems of adults and children may influence mechanisms of viral persistence and immune control, which may be reflected by differences in the HIV proviral genetics. The aim of this thesis research was to characterize the HIV proviral genetic landscape in children on long-term ART and compare it to infants on short-term ART, children on short-term ART, and adults on long-term ART to elucidate any potential differences in the proviral populations that may inform new targets for future cure interventions.Item IDENTICAL HIV-1 PROVIRUSES ORIGINATE FROM CELL PROLIFERATION OR INFECTION WITH A COMMON VIRAL ANCESTORNiyongabo, Aurelie; Kearney, MaryUnderstanding the mechanisms for HIV-1 persistence during antiretroviral therapy (ART) is crucial for developing curative strategies. Due to the high intra-patient genetic diversity of HIV-1, I hypothesized that integrated HIV-1 DNA (proviruses) with identical sub-genomic sequences are sustained during ART through cellular proliferation. To test this hypothesis, a method called “Multiple-Displacement Amplification Single-Genome Sequencing” (MDA-SGS) wherein the site of proviral integration in the host genome and the full-length HIV-1 sequence can be determined, was applied to 34 sets of proviruses with identical P6, protease, reverse transcriptase (P6-PR-RT) sequences in a single donor with viremia suppressed on ART reported by Musick, et al(1). The MDA-SGS workflow includes the isothermal amplification of DNA from cells containing single proviruses of interest within their sites of host integration followed by integration site analysis on the products to determine if proviruses identical in P6-PR-RT also have identical integration sites and, therefore, result from the proliferation of a single infected cell. If different sites of integration are observed, then I conclude that the identical proviruses result from infection of two or more different cells by a closely related viral ancestor. Of the 20 populations of proviruses with identical P6-PR-RT sequences successfully assayed by MDA-SGS, I found 9 to contain only identical integration sites (cell clones), 6 to have only unique integration sites (infection with a common ancestor), and 5 to contain a combination. The finding that proviruses identical in P6-PR-RT often have different sites of integration suggests infection of multiple cells prior to ART or during an ART interruption with a common viral ancestor, each establishing a latent infection allowing them to survive and, likely, divide. Targeting such long-lived, infected, proliferating cells is necessary to achieve HIV-1 remission without ART.Item A MAJORITY OF CLONALLY-EXPANDED T CELLS CONTAINING REPLICATION-COMPETENT HIV-1 PROVIRUSES ARE TRANSCRIPTIONALLY SILENT(2019) Musick, Andrew; Kearney, Mary; Coffin, John; Boyd, Ann; Biomedical Science Master's Program; Biomedical Science Master's ProgramCurrent antiretroviral therapy (ART) is highly effective at blocking HIV-1 replication but does not cure the infection due to the persistence of latently-infected cells that are able to undergo cellular proliferation (1). The majority of HIV-1 proviruses that persist during ART are defective. Of the minority that are intact and replication competent, it is not known what fraction are transcriptionally active in vivo versus those that are transcriptionally silent (latently infected). To address this question, I determined the fraction of HIV-1 proviruses in populations of expanded cell clones that express unspliced, cell-associated RNA during ART in one individual. In total, 34 different cell clones carrying either intact or defective proviruses in “Patient 1” from Maldarelli, et al. (1) were assessed. We found that a median of 2.3% of cells within clones harboring replication-competent proviruses contained unspliced HIV-1 RNA. Highest levels of HIV-1 RNA were found in the effector memory T cell subset, including for the replication-competent AMBI-1 clone, which was the source of persistent viremia on ART. The fraction of cells within a clone that contained HIV-1 RNA was not different in clones with replication-competent vs. defective proviruses. However, higher fractions and levels of RNA were found in cells with proviruses containing multiple drug resistance mutations, including those contributing to rebound viremia. These findings suggest that the vast majority of HIV-1 proviruses in persistently-infected cells, including replication-competent proviruses, are transcriptionally silent at any given time. This silence, if maintained over time, may allow infected cells to persist and expand during effective ART.