title: Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription.
abstract: During the early stages of HIV-1 replication the conical capsid composed of p24(CA) protein dissociates from the rest of the cytoplasmic viral complex by a process called uncoating. Although proper uncoating is known to be required for HIV-1 infection, many questions remain about the timing and factors involved in the process. Here we have used two complementary assays to study the process of uncoating in HIV-1-infected cells, specifically looking at the timing of uncoating and its relationship to reverse transcription. We developed a fluorescent microscopy-based uncoating assay that detects the association of p24(CA) with HIV-1 viral complexes in cells. We also used an owl monkey kidney (OMK) cell assay that is based on timed TRIM-CypA-mediated restriction of HIV-1 replication. Results from both assays indicate that uncoating is initiated within 1 h of viral fusion. In addition, treatment with the reverse transcriptase inhibitor nevirapine delayed uncoating in both assays. Analysis of reverse transcription products in OMK cells revealed that the generation of early reverse transcription products coincides with the timing of uncoating in these assays. Collectively, these results suggest that some aspect of reverse transcription has the ability to influence the kinetics of uncoating.
authors.affiliation: Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Hulme
authors.forename: Omar
authors.initials: O
authors.lastname: Perez
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Hope
mesh_terms: D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D016655:HIV Core Protein p24 / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D054298:Human Immunodeficiency Virus Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D019829:Nevirapine / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D048348:Reverse Transcription; D013997:Time Factors; D014771:Virion / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics*; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; D016655:HIV Core Protein p24; D054298:Human Immunodeficiency Virus Proteins; D008164:Luminescent Proteins; D018942:Macrolides; D011993:Recombinant Fusion Proteins; C514799:p24 protein, Human Immunodeficiency Virus Type 1; D054325:vpr Gene Products, Human Immunodeficiency Virus; C514912:vpr protein, Human immunodeficiency virus 1; C057620:bafilomycin A; D049452:Green Fluorescent Proteins; D016572:Cyclosporine; D019829:Nevirapine