HIV is a complex retrovirus containing a number of genes not commonly found in other retroviruses. One of these genes, vpu, which is encoded only by HIV-1 and does not have any known homologues in HIV-2 or SIV, encodes for a small integral membrane protein. We have in the past identified two biological activities for Vpu: (i) enhancement of particle release; (ii) degradation of CD4. To study the effect of Vpu on particle release, we introduced a series of mutations into the infectious molecular clone, pNL43, eliminating the Vpu gene and/or the Env gene. Mutant constructs were analyzed in transient expression assays in HeLa cells by pulse/chase analyses for their ability to produce and secrete virus particles. We found that the presence of Env is not required for HIV-1 particles to be secreted into the culture medium. In fact, particle release was similarly Vpu dependent in the presence or absence of Env. This indicates that Vpu enhances particle release through a mechanism that does not involve the Env product. To study the mechanism of Vpu-induced CD4 degradation, we developed an in vitro assay which involves the expression of Vpu and CD4 in rabbit reticulocyte lysates in the presence of canine microsomal membranes. The advantage of this in vitro system over a whole cell system is its simplicity and accessability for experimental variations. We constructed a series of in vitro expression plasmids that allowed us to synthesize wild-type CD4 or Vpu or specific mutants of both proteins in vitro. We found that Vpu can indeed induce degradation of CD4 in vitro. Successful CD4 decay required the presence of Vpu and CD4 in the same membrane compartment. Co-expression of CD4 and Vpu in rabbit reticulocyte lysate in the absence of microsomal membranes did not result in any appreciable destabilization of CD4. Analyses involving specific mutants of CD4 and Vpu indicate that (i) glycosylation of CD4 is not a prerequisite for degradation; (ii) CD4 decay occurs post-translational; (iii) Vpu targets a short sequence in the cytoplasmic tail of CD4; (iv) degradation in vitro is significantly slower than in vivo, yet there are no degradation intermediates detectable; therefore, (v) CD4 decay is likely to be a multi-step process; (vi) short deletions from the C-terminus of Vpu or an internal highly conserved domain result in a loss of biological activity.
Showing the most recent 10 out of 54 publications
