Background:  The TRIM family of proteins has recently been identified as being responsible for the cross-species restriction of a number of retroviruses, including restriction of HIV-1 by the TRIM5α protein from rhesus monkeys. While this restriction is known to occur following the specific recognition of a capsid determinant by TRIM proteins, subsequent steps in the TRIM5α-mediated restriction of HIV-1 remain poorly defined, although restriction occurs early during infection, prior to the generation of late reverse transcription (RT) products.

Methods:  HeLa cells and HeLa cells stably expressing TRIM5α were infected, and the infection examined microscopically and the degree of infection and reverse transcription examined using flow cytometry and quantitative polymerase chain reaction (PCR) in the presence or absence of proteasome inhibitors.

Results:  We find that proteasome inhibition relieves this TRIM5α-mediated block to RT accumulation, although 2 LTR circle formation and infection remains impaired. This is true in the case of HIV-1 restriction by rhesus (rh) TRIM5α and owl monkey TRIM-Cyp and in the case of murine leukemia virus (MLV) restriction by human (hu) TRIM5α. Integration assays with extracted preintegration complexes revealed that the reverse transcribed HIV-1 DNA rescued from rhTRIM5a restriction by proteasome inhibition was integration competent, although viral infection was blocked. By examining the fate of GFP-Vpr-labeled virions in cells expressing HA-tagged rhTRIM5α, we observe that proteasome inhibition results in the accumulation of HIV-1 virions associated with rhTRIM5α cytoplasmic bodies. This accumulation is specific to virions that have entered the host cell cytoplasm, as indicated by the loss of their fluorescent membrane label, and requires elements in the TRIM5α SPRY domain known to be required for restriction specificity. Extended treatment with proteasome inhibitor dramatically extends the half-life of GFP-Vpr-labeled virions in the cytoplasm of TRIM5α-expressing cells. Arrest in this state results in TRIM5α cytoplasmic bodies associating with ubiquitin and proteosomal subunits.

Conclusions:  We propose that the TRIM5a proteins restrict by encapsidating the incoming viral core, altering trafficking of the reverse transcribing viral cDNA to the nucleus and targeting the reverse transcription complex for disruption involving the proteasome.