Abstract
Uncoating of Human Immunodeficiency Virus type 1 (HIV-1) and type 2 (HIV-2) conical cores is an important early step for establishment of infection. In Old World Monkey (OWM) cells, the TRIM5α cellular factor potently suppresses an early step of infection by HIV-1. Previously, biochemical studies using whole cell lysates of infected cells revealed that OWM TRIM5α accelerates the uncoating of HIV-1, leading to premature reverse transcription. In the present study, we re-evaluated uncoating kinetics of HIV-1 in the presence of OWM TRIM5α by using an in situ uncoating assay, which allowed us to differentiate productive HIV-1 entry from simple (non-productive) endocytosis. Results showed that the uncoating kinetics of HIV-1 was indeed accelerated in the presence of OWM TRIM5α. Furthermore, we adapted an in situ uncoating assay to HIV-2, which showed wide variations in TRIM5α sensitivity among different isolates. HIV-2 isolate GH123, whose infectivity was suppressed by cynomolgus monkey (CM) TRIM5α, showed accelerated uncoating in the presence of CM TRIM5α. In contrast, mutant HIV-2 ASA, whose infectivity was unaltered by CM TRIM5α, showed no change in uncoating kinetics in the presence of CM TRIM5α. These results confirmed and further extended the previous notion that accelerated uncoating is associated with restriction activity of TRIM5α against lentiviruses.
Highlights
Uncoating of the lentivirus core, which is composed of *1,000 capsid proteins (CA), is an important process for establishment of viral infection
The use of anti-HA antibody followed by FITC-labeled secondary antibody confirmed the presence of cynomolgus monkey (CM) TRIM5α protein in virtually all transfectants (Fig. 1B, lower panels); green fluorescence was barely detectable in Vc transfectants (Fig. 1B, upper panel)
In order to determine the restriction activity against Human Immunodeficiency Virus type 1 (HIV-1) and Human Immunodeficiency Virus (HIV)-2 in these cell lines, we performed a single-round infection assay, in which viral infection was detected as fluorescence generated by production of the green fluorescent protein (GFP) encoded by the viral RNA (Fig. 1C)
Summary
Uncoating of the lentivirus core, which is composed of *1,000 capsid proteins (CA), is an important process for establishment of viral infection. Human Immunodeficiency Virus (HIV) infection begins with the binding of viral glycoprotein to the cellular receptor and co-receptors, a step that is followed by fusion of the viral and cellular membranes. A conical core that contains two viral genomic RNAs and several viral proteins is released into the cytoplasm of the target cell. CAs eventually dissociate from the viral complex in a PLOS ONE | DOI:10.1371/journal.pone.0121199. Uncoating Kinetics of HIV in the Presence of TRIM5α CAs eventually dissociate from the viral complex in a PLOS ONE | DOI:10.1371/journal.pone.0121199 March 24, 2015
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