title: Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus.
abstract: BACKGROUND
  HIV-1 infects non-dividing cells. This implies that the virus traverses the nuclear pore before it integrates into chromosomal DNA. Recent studies demonstrated that TNPO3 is required for full infectivity of HIV-1. The fact that TNPO3 is a karyopherin suggests that it acts by directly promoting nuclear entry of HIV-1. Some studies support this hypothesis, while others have failed to do so. Additionally, some studies suggest that TNPO3 acts via HIV-1 Integrase (IN), and others indicate that it acts via capsid (CA).
  
  
  RESULTS
  To shed light on the mechanism by which TNPO3 contributes to HIV-1 infection we engineered a panel of twenty-seven single-cycle HIV-1 vectors each bearing a different CA mutation and characterized them for the ability to transduce cells in which TNPO3 had been knocked down (KD). Fourteen CA mutants were relatively TNPO3-independent, as compared to wild-type (WT) HIV-1. Two mutants were more TNPO3-dependent than the WT, and eleven mutants were actually inhibited by TNPO3. The efficiency of the synthesis of viral cDNA, 2-LTR circles, and proviral DNA was then assessed for WT HIV-1 and three select CA mutants. Controls included rescue of TNPO3 KD with non-targetable coding sequence, RT- and IN- mutant viruses, and pharmacologic inhibitors of RT and IN. TNPO3 KD blocked transduction and establishment of proviral DNA by wild-type HIV-1 with no significant effect on the level of 2-LTR circles. PCR results were confirmed by achieving TNPO3 KD using two different methodologies (lentiviral vector and siRNA oligonucleotide transfection); by challenging three different cell types; by using two different challenge viruses, each necessitating different sets of PCR primers; and by pseudotyping virus with VSV G or using HIV-1 Env.
  
  
  CONCLUSIONS
  TNPO3 promotes HIV-1 infectivity at a step in the virus life cycle that is detectable after the preintegration complex arrives in the nucleus and CA is the viral determinant for TNPO3 dependence.
authors.affiliation: Department of Microbiology and Molecular Medicine, University of Geneva, 1 Rue Michel Servet, CH-1211 Geneva, Switzerland.
authors.forename: Alberto
authors.initials: A
authors.lastname: De Iaco
authors.forename: Jeremy
authors.initials: J
authors.lastname: Luban
mesh_terms: D002213:Capsid; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000821:virology*; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D055785:Gene Knockdown Techniques; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs; D009154:Mutation; D009685:Nuclear Envelope / Q000821:virology; D034622:RNA Interference; D034741:RNA, Small Interfering; D018894:Reverse Transcriptase Inhibitors; D016662:Virus Integration; D014779:Virus Replication; D028961:beta Karyopherins / Q000235:genetics* / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; D035683:MicroRNAs; D034741:RNA, Small Interfering; D018894:Reverse Transcriptase Inhibitors; C546476:TNPO3 protein, human; D028961:beta Karyopherins; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1