Fates of Retroviral Core Components during Unrestricted and TRIM5-Restricted Infection

TRIM5 proteins can restrict retroviral infection soon after delivery of the viral core into the cytoplasm. However, the molecular mechanisms by which TRIM5 alpha inhibits infection have been elusive, in part due to the difficulty of developing and executing biochemical assays that examine this stage of the retroviral life cycle. Prevailing models suggest that TRIM5 alpha causes premature disassembly of retroviral capsids and/or degradation of capsids by proteasomes, but whether one of these events leads to the other is unclear. Furthermore, how TRIM5 alpha affects the essential components of the viral core, other than capsid, is unknown. To address these questions, we devised a biochemical assay in which the fate of multiple components of retroviral cores during infection can be determined. We utilized cells that can be efficiently infected by VSV-G-pseudotyped retroviruses, and fractionated the cytosolic proteins on linear gradients following synchronized infection. The fates of capsid and integrase proteins, as well as viral genomic RNA and reverse transcription products were then monitored. We found that components of MLV and HIV-1 cores formed a large complex under non-restrictive conditions. In contrast, when MLV infection was restricted by human TRIM5 alpha, the integrase protein and reverse transcription products were lost from infected cells, while capsid and viral RNA were both solubilized. Similarly, when HIV-1 infection was restricted by rhesus TRIM5 alpha or owl monkey TRIMCyp, the integrase protein and reverse transcription products were lost. However, viral RNA was also lost, and high levels of preexisting soluble CA prevented the determination of whether CA was solubilized. Notably, proteasome inhibition blocked all of the aforementioned biochemical consequences of TRIM5 alpha-mediated restriction but had no effect on its antiviral potency. Together, our results show how TRIM5 alpha affects various retroviral core components and indicate that proteasomes are required for TRIM5 alpha-induced core disruption but not for TRIM5 alpha-induced restriction.