Currentanti-HIV-1therapiespreventprogressiontoAIDSbutdonotcureinfection.HIV-1persistsinlong-lived memoryCD4+Tcellsasatranscriptionallysilentprovirus,whereitisundetectablebytheimmunesystem,and therefore resistant to extirpation. Recently, we reported that primate immunodeficiency virus Vpx and Vpr proteins activate HIV-1 provirus transcription by degrading the three proteins of the human silencing hub (HUSH) complex. Disruption of the HUSH complex in bulk CD4+ T cells increased transcription from HIV-1 provirusesandkineticsofHIV-1spreadinginfections,indicatingthattheHUSHcomplexplaysadominantrole inHIV-1provirussilencing.Nonetheless,examinationofindividualclonesshowedheterogeneityinresponseto HUSHdisruption,andinconsistentcorrelationwithknownsilencingfactorssuchasSETDB1.
Aim1 willbeto identify requirements for HUSH complex silencing of the HIV-1 provirus. Sets of CD4+ T cell clones bearing HIV-1 proviruses that exhibit a range of HUSH responsiveness will be subjected to loss-of-function screenstoidentifyhostsilencingfactorsthatdistinguishcloneswithdifferentHUSHphenotypes.Suchfactors willbecharacterizedindependentlyforeffectsonprovirustranscriptionandproviruschromatinfeatures.From these experiments we expect to better understand how HUSH is recruited to, and maintains transcriptional silencing of, HIV-1 proviruses.
Aim 2 will be to examine the role of the HUSH complex in CD4+ T cell transcriptionanddevelopment.DisruptionoftheHUSHcomplexactivatesLINE-1expressionincertaincell lines raising questions about possible consequences of HUSH complex disruption. Global transcription and chromatinprofilingwillbeperformedonprimaryhumanCD4+TcellsinwhichHUSHcomplexcomponentsare disrupted. Increased expression of particular retrotransposons is expected, but also immune-related genes of relevance to HIV-1, and markers that may be used to monitor HUSH complex activity in cells. Examination of transcriptionfactormotifswithinlostATAC-SeqpeakswillaididentificationofDNA-bindingproteinsthatrecruit theHUSHcomplex.TheHUSHcomplexwillalsobedisruptedincordbloodhumanCD34+hematopoieticstem cells used to reconstitute an immune system in mice. These experiments will tell us whether the HUSH complex is essential for human hematopoietic development generally or for CD4+ T cells specifically.
Aim 3 will assess the contribution of the HUSH complex to HIV-1 latency in vivo. The effect of HUSH complex inactivation on HIV-1 provirus reactivation will be examined with CD4+ T cells harvested from HIV-1+ individuals on anti-HIV-1 suppressive therapy and from humanized mice. These studies are expected to improve mechanistic understanding of HIV-1 transcriptional regulation, help prognosticate the transcriptional status of a given provirus, develop new approaches for disrupting the HIV-1 provirus in the clinical context, and,moregenerally,increasefundamentalunderstandingofgeneregulation.

Public Health Relevance

Anti-HIV-1 drugs prevent HIV-1-infected people from developing AIDS but the drugs do not cure infection because HIV-1 becomes a permanent genetic element in human chromosomes that is called a provirus. We discovered that hostcellproteinscalledtheHUSHcomplexsilencetheHIV-1provirus,makingit difficult for drugs, or the immune system, to detect and eliminate the virus. The experiments proposed here, which are intended to better understand how the HUSHcomplexsilencestheHIV-1provirus,arehopedtoprovideinformationthat willfacilitateeffortstocureHIV-1infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
1R37AI147868-01
Application #
9846851
Study Section
HIV Molecular Virology, Cell Biology, and Drug Development Study Section (HVCD)
Program Officer
Mcdonald, David Joseph
Project Start
2019-07-01
Project End
2024-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655