To determine the mechanism by which CIITA mediates protease activity in these cells, we analyzed the expression of the Gag and Gag-Pol in CIITA-expressing cells. Expression of the HIV protease is extremely toxic to cells, and is thus very tightly regulated during virus replication [60, 61, 62] . The viral protease arises from the alternative Gag translation product ( Figure 5A ), Gag-Pol which, accounts for only 2-10% of Gag polyproteins and results from ribosomal frameshifting at a conserved heptamer ''slippery sequence'' and a secondary stem-loop structure on gag-pol transcripts [51, 63] . Therefore, increased viral protease-mediated processing of the Gag polyproteins from CIITA-expressing cells may be due to an increase in overall viral protease levels, due to increased Gag-Pol synthesis. To establish the ability of CIITA to mediate the enhanced production of HIV protease, cells were transiently co-transfected with either CIITA or pcDNA and HIV-1 NL4-3 DNA, and were treated with 80 nM of Lopinavir to inhibit the majority of Gag polyprotein processing. Gag-Pol levels relative to Gag were then determined by western blotting. While 1-2% of all Gag polyprotein in cell lysates of vector-only cells was in the form of Gag-Pol, expression of CIITA increased this level to almost 5% ( Figure 7C and D) , indicating that CIITA increases Gag-Pol levels in virus producer cells. We also measured the potential of the cytoplasmic CIITA mutants to increase Gag-Pol levels relative to Gag and, interestingly, Gag-Pol made up approximately 13, 20 and 15% of all Gag polyproteins in GTP2, GTP3 and L1035P -expressing cells, respectively. This result further demonstrates a novel transcription-independent role for cytoplasmic CIITA during HIV infection, where CIITA enhances Gag-Pol protease expression, which subsequently enhances virus maturation and infectivity. [16] . We speculated that complete expression of the antigen presentation pathway, through expression of CIITA, would alleviate this phenotype in virus producer cells. Interestingly, we noticed two phenomena: i) CIITA did not induce intracellular retention of Gag or impair virus release in producer cells despite expression of HLA-DR and ii) the virus released from cells expressing CIITA was significantly more infectious. Upon investigation of CIITA-mediated alleviation of HLA-DR-induced Gag retention, we found that this effect was not due to the lack of Ii or HLA-DM (key components of antigen presentation), as had been expected, nor was it a consequence of Rab4B expression in these cells. Further, when lysine residues in the HLA-DR chain were mutated or both of the HLA-DR chain cytoplasmic tails were truncated, Gag was still retained and virus release inhibited. In addition, when CIITA was expressed in conjunction with HLA-DR this effect could not be alleviated, suggesting that Gag retention is a consequence of HLA-DR overexpression. Indeed, retention of Gag is directly correlated to levels of HLA-DR expression. Further, we did not observe Gag retention when class II antigen presentation pathway genes were expressed at endogenous levels via CIITA expression. Overexpression of HLA-DM, which is structurally similar to HLA-DR also induced Gag retention, suggesting that retention of Gag is a likely consequence of altered trafficking of overexpressed class II antigen presentation pathway components rather than a physiologically relevant phenomenon.