Investigation of HERV-K (HML-2) expression during HIV-1 infection.
endogenous retrovirus group K (HERV-K) proviruses are among the limited number of human
endogenous retroviral elements to retain coding sequence. Of interest, expression from
the Human Mouse Mammary Tumor Virus-like 2 (HML-2) subgroup of HERV-K proviruses has
been widely associated with disease, including different types of cancers as well as in
HIV-1 infection. In particular, ... read morerecent studies have suggested that HML-2 expression may
play a role in the pathogenesis of HIV-1 infection. RNA from the HML-2 subgroup was
reported to be highly expressed at the cellular level and detectable in the plasma of
HIV-1 infected patients, suggestive of virion production and potentially replication. In
order to investigate this phenomenon, an HML-2 specific quantitative PCR assay was
developed, which detects 51 of the >90 known HML-2 proviruses in the human genome.
Plasma and peripheral blood mononuclear cells (PBMCs) from HIV- negative controls and
HIV-1 infected patients were collected for analysis of HML-2 RNA expression. Contrary to
previous reports, high levels of HML-2 RNA were not detected in the plasma of HIV-1
infected patients, but a significant increase of HML-2 RNA in total PBMCs was observed.
The level of HML-2 expression in PBMCs did not appear to be related to patient use of
antiretrovirals or to HIV-1 plasma RNA, cellular RNA or cellular DNA levels. To
investigate the source of expressed HML-2 RNA, patient PBMCs were sorted into CD3+CD4+ T
cells, CD3+CD8+ T cells, CD3-CD14+ monocytes and CD3-CD20+ B cell subsets and then
analyzed for HML-2 RNA levels using the quantitative PCR assay. No single cell subset
was enriched for HML-2 RNA expression in HIV-1 infected patients, but there was
substantial variability in the level of HML-2 expression dependent on the cell type. In
order to understand the potential impact of HML-2 expression, an RNAseq methodology was
developed to annotate expression at the proviral level. Prior to use on the HIV-1
infected and uninfected populations, this RNASeq strategy was implemented using the
teratocarcinoma cell line Tera-1, known to express high levels of cellular HML- 2 RNA
and produce non-infectious HML-2 virions. RNASeq effectively discerned the proviral
expression pattern of this cell line and was capable of identifying the core expressed
transcripts, which originated from two proviruses located on chromosome 22 (chr 22q11.21
and chr 22q11.23). Interestingly, only one of these proviral transcripts appeared to be
packaged into virions at a high level, suggesting a preference for recently integrated
proviruses to be packaged into virions over those from other highly expressed but older
elements. The validated RNASeq approach was used to investigate the HML-2 profile of
PBMCs collected from HIV-1 infected and uninfected subjects. Bioinformatic analysis
revealed that HML-2 expression profiles between these populations are remarkably similar
in composition, though not in magnitude of expression. Increased overall HML-2
expression detected in the HIV-1 population appears to be driven by the higher
expression of the provirus on chr 1q22. However, as this provirus does not maintain open
reading frames for retroviral genes gag, pro, pol and env, it is unlikely that it leads
to production of retroviral particles or participates in productive recombination events
that could lead to replicating HML-2 virus. Thus, based on these studies, HML-2
expression during HIV-1 infection is not predicted to have a direct pathogenic
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Molecular Microbiology.
Advisors: John Coffin, and Naomi Rosenberg.
Committee: Michael Jordan, Carol Kumamoto, Honorine Ward, and Welkin Johnson.
Keywords: Virology, Bioinformatics, and Microbiology.read less
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