Abstract
The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env) that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER) and its trafficking to the trans-Golgi network (TGN) were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions.
Highlights
Gag (Pr55) is the predominant structural protein of HIV-1 and drives the assembly of virus-like particle in the absence of other viral proteins
To examine the possibility that in the absence of Ubc9 the decrease in intracellular gp120 could be due to an increase in gp160 degradation in the endoplasmic reticulum (ER), we examined two major ER degradation pathways (ERAD and unfolded protein response (UPR))
Even though our previous study analyzing transfected cell lysates at steady state, using monoclonal antibodies that were specific for gp120 and gp41, did not suggest that endoplasmic reticulum associated degradation (ERAD) was occurring [49], we could not rule out the possibility that a portion of the 120 kDa proteins observed could be gp160 undergoing ERAD in our pulse chase assays
Summary
Gag (Pr55) is the predominant structural protein of HIV-1 and drives the assembly of virus-like particle in the absence of other viral proteins. Gag multimerizes and initiates the budding process as the immature procapsid assembles. Gag is involved in the selective packaging of the viral genome, various cellular host factors, and incorporation of the viral protease and replication enzymes through co-assembly with Gag-Pol fusion proteins. The replication enzymes are cleaved into the mature forms along with Pr55 during virion maturation events (Gag function reviewed in [1,2,3]). The envelope glycoprotein is an essential viral structural protein that is incorporated into the assembling virions, and is responsible for virion binding and entry into a target cell. Envelope is expressed as an immature precursor protein (gp160) within the lumen of the endoplasmic reticulum (ER)
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