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dc.contributor.advisorEngelman, Alan N.
dc.contributor.authorOztop, Ilker
dc.date.accessioned2014-10-21T17:51:46Z
dc.date.issued2014-10-21
dc.date.submitted2014
dc.identifier.citationOztop, Ilker. 2014. The functional interplay between TNPO3, CPSF6 and HIV-1 CA. Doctoral dissertation, Harvard University.en_US
dc.identifier.otherhttp://dissertations.umi.com/gsas.harvard.inactive:11725en
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:13067468
dc.description.abstractLentiviruses can infect postmitotic cells, indicative of a role for the nucleocytoplasmic transport machinery. Genome-wide RNA interference screens identified transportin 3 (TNPO3) that may regulate human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) nuclear import but plays no role during murine leukemia virus (MLV) infection. Independently, TNPO3 was shown to bind HIV-1 integrase (IN), a PIC component, suggesting a potential mechanism for nuclear import. We demonstrated direct binding between TNPO3 and several retroviral INs, which did not correlate with TNPO3 dependency profiles of the respective retroviruses. Infectivity assays employing HIV-1/MLV chimeric viruses ascertained that the capsid (CA) domain, but not IN, was the functional determinant of TNPO3 dependence. A carboxy-terminal truncation mutant of the serine-arginine rich (SR) protein family member, cleavage and polyadenylation specific factor 6 (CPSF6), CPSF6-358, which lacks its RS domain, was shown to restrict HIV-1 PIC nuclear import. We demonstrated that CPSF6 interacts with HIV-1 CA, and a single point mutation in CA, Asn74Asp (N74D), abolished this interaction. N74D also rendered HIV-1 TNPO3-independent and impaired cyclophilin A (CypA) binding to CA. The CA:CPSF6 binding interface, as described in a partial co-crystal structure, defined a surface pocket on CA that faces the CA hexamer:hexamer interspace. Infectivities and CA binding profiles of CA mutants within this pocket or with aberrant CypA-related phenotypes were assessed to compare their CPSF6-358 sensitivity and TNPO3 dependence, which largely correlated. We showed an overall correlation between the CPSF6/CPSF6-358 binding profiles of these HIV-1 CA mutants and their CPSF6-358 sensitivity, whereas TNPO3 binding and TNPO3 dependence did not correlate. Based on similar infectivity profiles of CA mutants and the loss of the RS domain from CPSF6-358 we tested for a direct interaction between CPSF6 and TNPO3. We demonstrated specific binding between recombinant TNPO3 and the CPSF6.RS domain. Mutagenesis experiments suggested a multicontact binding interface. The interaction was downmodulated by Ras-related nuclear protein (Ran)-GTP, indicating that CPSF6 is a bona fide import substrate of TNPO3. Our results support a model where TNPO3 regulates nuclear CPSF6 localization and that in its absence CPSF6 may restrict infection by directly interacting with HIV-1 CA at the hexamer:hexamer interface.en_US
dc.language.isoen_USen_US
dash.licenseLAA
dc.subjectVirologyen_US
dc.subjectBiochemistryen_US
dc.subjectMolecular biologyen_US
dc.subjectcapsiden_US
dc.subjectCPSF6en_US
dc.subjectCypAen_US
dc.subjectHIVen_US
dc.subjectNuclear Importen_US
dc.subjectTNPO3en_US
dc.titleThe functional interplay between TNPO3, CPSF6 and HIV-1 CAen_US
dc.typeThesis or Dissertationen_US
dash.depositing.authorOztop, Ilker
dc.date.available2014-10-21T17:51:46Z
thesis.degree.date2014en_US
thesis.degree.disciplineBiology: Medical Sciences, Division ofen_US
thesis.degree.grantorHarvard Universityen_US
thesis.degree.leveldoctoralen_US
thesis.degree.namePh.D.en_US
dc.contributor.committeeMemberKnipe, Daviden_US
dc.contributor.committeeMemberMünger, Karlen_US
dc.contributor.committeeMemberSheehy, Annen_US
dash.contributor.affiliatedOztop, Ilker


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