Up until this point, we had not co-expressed CIITA with HLA-DR in producer cells to determine if CIITA could alleviate retention of Gag and/or restore infectious virus release as would be expected. Interestingly, when CIITA was transiently expressed with the HLA-DR heterodimers in producer cells, Gag still accumulated intracellularly and infectious virus production remained reduced ( Figure 3B and C, respectively), suggesting that expression of CIITA is not sufficient to compensate for HLA-DRmediated Gag retention. However, when HLA-DR is endogenously expressed under the control of CIITA, retention is alleviated. [8] . Ubiquitination of a conserved lysine residue on the HLA-DR b chain induces intracellular accumulation of class II heterodimers in MHC class II compartments (MIICs) [35, 36] . Thus, the ubiquitination state of HLA-DR, might influence Gag retention. To address this idea, Lys225Arg (pcDRabK225R) and Lys222/ 225Arg (pcDRabK222/225R) point mutations were made in the HLA-DR beta chain. Despite these mutations, HLA-DR expression on the surface of A293T cells was not significantly altered (flow cytometric data not shown), nor was intracellular retention of Gag alleviated or infectious virus release restored ( Figure 4A and B, respectively). Therefore, we attempted to alleviate Gag retention and restore infectious virus release, as demonstrated by Finzi et al. (2006) [8] , by complete truncation of the cytoplasmic tails of both HLA-DR a and b (pcDRaDcyto and pcDRbDcyto, respectively). However, loss of either cytoplasmic tail (pcDRaDcytob and pcDRabDcyto), or both cytoplasmic tails (pcDRaDcy-tobDcyto), from the heterodimer did not alleviate Gag retention and even further reduced infectious virus release from these cells ( Figure 4A and B, respectively). Our results may differ from Finzi's because of the differences in cell type, or HLA-DR gene alleles, nevertheless they strongly suggest that transient HLA-DR expression in producer cells induces Gag retention. Further, flow cytometry to monitor surface HLA-DR expression demonstrates that transient transfection of HLA-DR induces an approximate half-log to log-fold increase in HLA-DR as compared to cells stably or transiently expressing CIITA, respectively ( Figure S1 ). Therefore, it is possible that HLA-DR induced Gag retention is a consequence of HLA-DR overexpression in this transient system To test this possibility, virus producer cells were transfected with increasing amounts of HLA-DR in the presence of HIV-1 NL4-3 DNA, and infectious virus release was monitored. As HLA-DR expression increased, the level of infectious virus release decreased ( Figure 4C ). Further, while not statistically significant, there was a positive correlation (r = 0.9954) between Gag accumulation (Gag hi ) and increasing HLA-DR expression ( Figure 4D ), suggesting Gag retention is likely related to HLA-DR overexpression. Similarly, when HLA-DM, which is structurally similar to HLA-DR, was overexpressed in producer cells, Gag retention was also induced ( Figure 4D ) and infectious virus release reduced (data not shown). As endogenous expression of the class II antigen presentation pathway by CIITA does not induce Gag retention and limiting expression of HLA-DR or -DM likewise has a limited effect on Gag retention in producer cells, these data collectively suggest that overexpression of the class II pathway alters Gag trafficking in producer cells leading to reduced infectious virus release. However, these data do not provide an explanation for the increased infectivity of virions released from CIITA-expressing cells.