Show simple item record

dc.contributor.advisorEngelman, Alan N.
dc.contributor.authorMatreyek, Kenneth Anzai
dc.date.accessioned2014-02-25T22:04:50Z
dc.date.issued2014-02-25
dc.date.submitted2013
dc.identifier.citationMatreyek, Kenneth Anzai. 2013. HIV-1 capsid engages nucleoporin NUP153 to promote viral nuclear entry. Doctoral dissertation, Harvard University.en_US
dc.identifier.otherhttp://dissertations.umi.com/gsas.harvard:11210en
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:11745723
dc.description.abstractLentiviruses can infect non-dividing cells, and various cellular nuclear transport proteins provide crucial functions for lentiviral nuclear entry and integration. Genome-wide small interfering RNA screens previously identified nuclear pore complex component nucleoporin 153 (NUP153) as being important for infection by human immunodeficiency virus type 1 (HIV-1). We found that HIV-1 infection of NUP153 depleted cells resulted in normal levels of reverse transcription, a moderate reduction of 2-long terminal repeat circles, and a relatively large reduction in integrated proviruses, consistent with a role for NUP153 during nuclear entry of the HIV-1 pre-integration complex. We ascertained the capsid (CA) to be the major viral determinant for NUP153 dependence during infection, and accordingly observed a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153C). NUP153C fused to the effector domains of the rhesus Trim5alpha restriction factor (Trim-NUP153C) potently restricted HIV-1, providing an intracellular readout for the NUP153C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153C under these conditions, results that correlated with direct binding between purified recombinant proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 function during infection. Mutagenesis experiments identified NUP153C and CA residues important for binding, and different FG motifs within NUP153C mediated binding to HIV-1 versus EIAV CA proteins. HIV-1 CA binding mapped to residues that line a common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 each competed with NUP153C for binding to HIV-1 CA, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicated that the NUP153C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. We propose that HIV-1 CA binds NUP153 FG motifs to affect viral nuclear import, serving as a novel example of viral hijacking of a fundamental cellular process.en_US
dc.language.isoen_USen_US
dash.licenseLAA
dc.subjectVirologyen_US
dc.subjectMolecular biologyen_US
dc.subjectCellular biologyen_US
dc.subjectCapsiden_US
dc.subjectFG motifen_US
dc.subjectHIVen_US
dc.subjectLentivirusen_US
dc.subjectNuclear Importen_US
dc.subjectNuclear Pore Complexen_US
dc.titleHIV-1 capsid engages nucleoporin NUP153 to promote viral nuclear entryen_US
dc.typeThesis or Dissertationen_US
dash.depositing.authorMatreyek, Kenneth Anzai
dc.date.available2014-02-25T22:04:50Z
thesis.degree.date2013en_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.committeeMemberSodroski, Josephen_US
dc.contributor.committeeMemberLuban, Jeremyen_US
dc.contributor.committeeMemberJohnson, Welkinen_US
dc.contributor.committeeMemberAllen, Todden_US
dash.contributor.affiliatedMatreyek, Kenneth Anzai


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record