Abstract
BackgroundAfter viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. Previously we have utilized the CsA washout assay, in which TRIM-CypA mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of HIV-1 uncoating in owl monkey kidney (OMK) and HeLa cells. Here we have extended this analysis to the human microglial cell lines CHME3 and C20 to characterize uncoating in a cell type that is a natural target of HIV infection.MethodsThe CsA washout was used to characterize uncoating of wildtype and capsid mutant viruses in CHME3 and C20 cells. Viral fusion assays and nevirapine addition assays were performed to relate the kinetics of viral fusion and reverse transcription to uncoating.ResultsWe found that uncoating initiated within the first hour after viral fusion and was facilitated by reverse transcription in CHME3 and C20 cells. The capsid mutation A92E did not significantly alter uncoating kinetics. Viruses with capsid mutations N74D and E45A decreased the rate of uncoating in CHME3 cells, but did not alter reverse transcription. Interestingly, the second site suppressor capsid mutation R132T was able to rescue the uncoating kinetics of the E45A mutation, despite having a hyperstable capsid.ConclusionsThese results are most similar to previously observed characteristics of uncoating in HeLa cells and support the model in which uncoating is initiated by early steps of reverse transcription in the cytoplasm. A comparison of the uncoating kinetics of CA mutant viruses in OMK and CHME3 cells reveals the importance of cellular factors in the process of uncoating. The E45A/R132T mutant virus specifically suggests that disrupted interactions with cellular factors, rather than capsid stability, is responsible for the delayed uncoating kinetics seen in E45A mutant virus. Future studies aimed at identifying these factors will be important for understanding the process of uncoating and the development of interventions to disrupt this process.
Highlights
After viral fusion with the cell membrane, the conical capsid of Human immunodeficiency virus (HIV)-1 disassembles by a process called uncoating
Similar to our previous results in cell lines that are not natural targets of HIV infection (OMK and HeLa cells), uncoating initiated within the first hour of infection and was facilitated by reverse transcription
These results support the model in which uncoating is initiated by early steps of reverse transcription in the cytoplasm and proceeds during transport of the viral complex to the nucleus
Summary
After viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. In order for infection to progress this conical capsid structure disassembles by a process called uncoating. During this time, the viral RNA is reverse transcribed into a double stranded DNA. The viral complex of nucleic acid and associated proteins must traffic through the cytoplasm on microtubules [4,5,6] This viral complex gains access to the nucleus through a nuclear pore and the viral DNA is integrated into the chromosomal DNA to establish infection of a cell. It is necessary to characterize the process of uncoating in order to fully understand the early events of HIV replication
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