The influence of specific HIV integration-sites on host gene regulation and T cell clonal expansion
Yoon, John.
2019
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Despite important
therapeutic breakthroughs, HIV infection remains one of the most challenging problems
the global health community faces today. Patients on highly active antiretroviral
therapy maintain viral suppression for years, unless therapy is interrupted, at which
point viral replication quickly rebounds from a small, but stable reservoir.
Interestingly, recent in vivo HIV integration ... read moresite analysis (ISA) suggest that
integration into genes related to growth and survival, such as BACH2 and MKL2, can
impart certain long-term proliferative or survival benefits to the infected host cell.
Given the need for clarity surrounding these findings, we designed two in vitro
approaches to help us study the effects of HIV insertional mutagenesis on T cells.
CRISPR-mediated insertion of an HIV reporter sequence upstream of the first BACH2 coding
exon and subsequent T cell activation resulted in the formation of HIV-BACH2 chimeric
transcripts and elevated BACH2 protein expression. Chimeric transcripts were even
detected during baseline conditions, consistent with recent in vivo findings.
Furthermore, we show BACH2 protein expression was sustained over a prolonged period.
Promoter insertion-mediated expression of BACH2 was also seen in cells treated with
various histone deacetylase inhibitors (HDACi), many of which are currently being tested
in ongoing clinical trials for the treatment of HIV infection. Curiously, HIV
integration into MKL2 resulted in reduced full-length protein expression, and possibly
the reduced expression of an alternate short protein isoform. Interestingly, this
resulted in reduced CD25 and CD69 expression upon TCR stimulation using
phytohemagglutinin (PHA) or CD3/CD28, discovering a previously unknown
connection between MKL2 and T cell signaling. The identification of additional genes
that can participate in this mode of HIV insertional mutagenesis may help us in
understanding the events leading to these types of occurrences. To understand these
processes in detail, we designed an in vitro primary T cell infection assay analyzing
the integration site patterns found in clonally expanded T cells over long periods of
time in culture to look for signs of selective outgrowth. Through these experiments, we
were able to demonstrate the selective clonal expansion of infected cells containing
integration events in the STAT3 gene, but only upon repeat T cell activation. The
cluster of highly expanded clones in STAT3 is very reminiscent of the most recent
notable cases of selective clonal outgrowth due to an HIV integration event. Our data
suggest that these types of proliferative events are readily inducible and warrant
further investigation.
Thesis (Ph.D.)--Tufts University, 2019.
Submitted to the Dept. of Immunology.
Advisor: John Coffin.
Committee: Peter Brodeur, Karl Munger, Naomi Rosenberg, and Jose Caro.
Keywords: Virology, and Immunology.read less - ID:
- 41687w96b
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