Abstract
Retroviral capsid (CA) cores undergo uncoating during their retrograde transport (toward the nucleus), and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC) using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable) CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating.
Highlights
The mature capsid (CA) core of human immunodeficiency virus 1 (HIV-1) is a ~60 nm × 120 nm [1] cone-shaped protein lattice, encasing viral genomic RNA, enzymes and other viral proteins
dynein heavy chain (DHC) depletion was efficient as assessed by Western blotting, since transfection of DHC siRNA led to a reduction of ~85.4% ±6.7% (n = 10) in protein levels (Figure 1A), which is consistent with previous observations that a large proportion of cells showed altered dynein-dependent transport in these conditions as seen by LAMP-1 staining [33]
Previous articles showed that HIV-1 uncoating could be modulated by CA interactions with cellular factors such as cleavage and polyadenylation specific factor 6 (CPSF6) [26] and cyclophilin A [24]
Summary
The mature capsid (CA) core of human immunodeficiency virus 1 (HIV-1) is a ~60 nm × 120 nm [1] cone-shaped protein lattice, encasing viral genomic RNA, enzymes (integrase and reverse transcriptase) and other viral proteins (reviewed in [2]). It is composed of ~1000 CA monomers [3] arranged in ~170 hexamers and 12 pentamers through interactions involving their. It is possible that a small fraction of CA proteins remain associated with pre-integration complexes and are transported to the nucleus [12] These hypotheses are not mutually exclusive, considering that uncoating could take place through several sequential steps rather than through a rapid single-step process [13]. Uncoating could be triggered by an as yet unknown cellular factor [18]
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