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Cytopmasic CPSF6 and Cyclophilin A Modulate HIV-1 Trafficking

Zhong, Zhou (2020) Cytopmasic CPSF6 and Cyclophilin A Modulate HIV-1 Trafficking. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Human immunodeficiency virus type 1 (HIV-1) capsid binds to multiple host cell proteins after entry into a cell, including cyclophilin A (CypA) and cleavage and polyadenylation specificity factor 6 (CPSF6), which is expressed predominantly in the nucleus. As CPSF6 expression was observed in the cytoplasm, we examined the effect of CPSF6 on HIV-1 capsid and nucleic acid trafficking in the cytoplasm of HeLa cells and primary macrophages. High-speed live-cell microscopy was performed with fluorescently labeled wild-type (WT) HIV-1 and capsid (CA) mutants and cells expressing labeled CPSF6. Imaging data were complimented with infectivity assays in HeLa cells, primary CD4+ T lymphocytes, and primary macrophages and in vitro binding assays with CA tubular assemblies and purified full-length CPSF6 protein and CypA. In cells, CPSF6 forms higher order complexes in the cytoplasm upon infection with WT HIV-1 but not N74D HIV-1, which does not bind to CPSF6. CPSF6 complexes associate with WT HIV-1 capsid and traffic on microtubules. Full-length CPSF6 protein and CPSF6 lacking the R/S domain (CPSF6-358) form higher order complexes that bind to and disrupt WT CA assemblies but not N74D CA assemblies in vitro. CPSF6-capsid complex trafficking can be altered by mutations in HIV-1 CA (e.g. N74D) or mutations in or truncation of the C-terminus of CPSF6 (e.g. CPSF6-358), which is associated with decreased HIV-1 infection. In addition, disruption of HIV-1 capsid binding to CypA (e.g. cyclosporine A treatment or G89V mutation in CA) leads to increased CPSF6 binding to capsid in vivo and in vitro and altered capsid trafficking in HeLa cells and macrophages, resulting in reduced infectivity. Altered trafficking and reduced infectivity due to capsid-CypA perturbation can be partially restored by depletion of CPSF6 in HeLa cells but not in macrophages. Our data suggest that both CPSF6 and CypA are important for proper HIV-1 cytoplasmic capsid trafficking and infection. We propose that CypA prevents HIV-1 capsid from premature binding to cytoplasmic CPSF6. Differences in CypA cellular localization and type I interferon responses may explain cell-specific variations in HIV-1 capsid trafficking and uncoating as well as subsequent infectivity.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Zhong, Zhouzhz70@pitt.eduzhz700000-0001-5418-4990
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorAmbrose, Zandreazaa4@pitt.eduZAA40000-0002-8610-2766
Committee MemberEngelman, AlanAlan_Engelman@dfci.harvard.edu0000-0002-9709-2591
Committee MemberKinchington, Paulkinch@pitt.eduKINCH0000-0002-1901-9970
Committee MemberSluis-Cremer, Nicolasnps2@pitt.eduNPS20000-0003-3897-7450
Committee MemberWatkins, Simonsimon.watkins@pitt.eduSWATKINS0000-0003-4092-1552
Date: 21 December 2020
Date Type: Publication
Defense Date: 7 October 2020
Approval Date: 21 December 2020
Submission Date: 1 December 2020
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 169
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Microbiology and Immunology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: HIV-1 Trafficking Imaging Microtubules CPSF6 CypA
Date Deposited: 21 Dec 2020 16:42
Last Modified: 21 Dec 2020 16:42


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