HIV-1 Mutant Assembly, Processing and Infectivity Expresses Pol Independent of Gag

The pol retrovirus gene encodes required enzymes for virus replication and maturation. Unlike HIV-1 Pol (expressed as a Gag–Pol fusion protein), foamy virus (described as an ancient retrovirus) expresses Pol without forming Gag–Pol polyproteins. We placed a “self-cleaving” 2A peptide between HIV-1 Gag and Pol. This construct, designated G2AP, is capable of producing virions with the same density as a wild-type (wt) HIV-1 particle. The 2A peptide allows for Pol to be packaged into virions independently from Gag following co-translationally cleaved from Gag. We found that G2AP exhibited only one-third the virus infectivity of the wt, likely due, at least in part, to defects in Pol packaging. Attenuated protease (PR) activity, or a reduction in Pol expression due to the placement of 2A-mediated Pol in a normal Gag–Pol frameshift context, resulted in significant increases in virus yields and/or titers. This suggests that reduced G2AP virus yields were largely due to increased PR activity associated with overexpressed Pol. Our data suggest that HIV-1 adopts a gag/pol ribosomal frameshifting mechanism to support virus assembly via the efficient modulation of Gag–Pol/Gag expression, as well as to promote viral enzyme packaging. Our results help clarify the molecular basis of HIV-1 gene expression and assembly.