Journal Article

Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription

Hulme, Amy E, Hope, Thomas J, Perez, Omar

2011

Overview

During the early stages of HIV-1 replication the conical capsid composed of p24CA 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 p24CA 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.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Animals / Anti-HIV Agents - pharmacology / Aotus (Cebidae) / Biological Sciences / Capsid / Capsids / Cell Line / Cells / Cyclosporine - pharmacology / Fluorescence / Green Fluorescent Proteins - genetics / Green Fluorescent Proteins - metabolism / Half lives / HEK293 Cells / HeLa Cells / HIV / HIV 1 / HIV Core Protein p24 - genetics / HIV Core Protein p24 - metabolism / HIV infections / HIV-1 - genetics / HIV-1 - metabolism / Human immunodeficiency virus / Human immunodeficiency virus 1 / Human Immunodeficiency Virus Proteins - genetics / Human Immunodeficiency Virus Proteins - metabolism / Humans / Infection / Infections / Kidney / kidneys / Kinetics / Luminescent Proteins - genetics / Luminescent Proteins - metabolism / Macrolides - pharmacology / Memory interference / Microscopy / Microscopy, Fluorescence / Monkeys & apes / Nevirapine / Nevirapine - pharmacology / Proteins / Recombinant Fusion Proteins - genetics / Recombinant Fusion Proteins - metabolism / Replication / Reverse Transcription / RNA-directed DNA polymerase / Time Factors / Uncoating / Virion - genetics / Virion - metabolism / Virions / virus replication / Virus Replication - drug effects / Virus Replication - genetics / Viruses / vpr Gene Products, Human Immunodeficiency Virus - genetics / vpr Gene Products, Human Immunodeficiency Virus - metabolism