title: Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid.
abstract: The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules.
authors.affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Ke
authors.initials: K
authors.lastname: Peng
authors.affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Walter
authors.initials: W
authors.lastname: Muranyi
authors.affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Bärbel
authors.initials: B
authors.lastname: Glass
authors.affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Vibor
authors.initials: V
authors.lastname: Laketa
authors.affiliation: Gilead Sciences Inc., Foster City, United States.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Yant
authors.affiliation: Gilead Sciences Inc., Foster City, United States.
authors.forename: Luong
authors.initials: L
authors.lastname: Tsai
authors.affiliation: Gilead Sciences Inc., Foster City, United States.
authors.forename: Tomas
authors.initials: T
authors.lastname: Cihlar
authors.affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Müller
authors.affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Kräusslich
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects*; D036022:Capsid Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism / Q000821:virology; D003600:Cytosol / Q000378:metabolism / Q000821:virology; D004279:DNA, Viral / Q000096:biosynthesis*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism / Q000821:virology; D018613:Microscopy, Confocal / Q000379:methods; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D061251:Primary Cell Culture; D048348:Reverse Transcription / Q000187:drug effects; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism / Q000821:virology; D016662:Virus Integration / Q000187:drug effects / Q000235:genetics; D053586:Virus Internalization / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics*; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; D007211:Indoles; C000591221:PF-3450074; C546476:TNPO3 protein, human; D028961:beta Karyopherins; C450562:cleavage factor Im, human; D039221:mRNA Cleavage and Polyadenylation Factors; D010649:Phenylalanine; D019427:HIV Integrase
keywords: CPSF6; HIV; infectious disease; macrophages; microbiology; reverse transcription; viral entry; viruses