Plasmids pM2PR8_EGFP or pM2PR8DsRed were transiently transfected into MDCK cells by lipofection. The cells were infected with influenza A/PR/8 virus 1 day after transfection. Virus titres were determined from supernatants after additional 24 h of incubation at 37°C. Class II Transactivator (CIITA) Enhances Cytoplasmic Processing of HIV-1 Pr55Gag BACKGROUND: The Pr55(gag) (Gag) polyprotein of HIV serves as a scaffold for virion assembly and is thus essential for progeny virion budding and maturation. Gag localizes to the plasma membrane (PM) and membranes of late endosomes, allowing for release of infectious virus directly from the cell membrane and/or upon exocytosis. The host factors involved in Gag trafficking to these sites are largely unknown. Upon activation, CD4+ T cells, the primary target of HIV infection, express the class II transcriptional activator (CIITA) and therefore the MHC class II isotype, HLA-DR. Similar to Gag, HLA-DR localizes to the PM and at the membranes of endosomes and specialized vesicular MHC class II compartments (MIICs). In HIV producer cells, transient HLA-DR expression induces intracellular Gag accumulation and impairs virus release. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that both stable and transient expression of CIITA in HIV producer cells does not induce HLA-DR-associated intracellular retention of Gag, but does increase the infectivity of virions. However, neither of these phenomena is due to recapitulation of the class II antigen presentation pathway or CIITA-mediated transcriptional activation of virus genes. Interestingly, we demonstrate that CIITA, apart from its transcriptional effects, acts cytoplasmically to enhance Pr160(gag-pol) (Gag-Pol) levels and thereby the viral protease and Gag processing, accounting for the increased infectivity of virions from CIITA-expressing cells. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that CIITA enhances HIV Gag processing, and provides the first evidence of a novel, post-transcriptional, cytoplasmic function for a well-known transactivator. HIV polyprotein Gag serves as a scaffold to promote assembly of progeny virions at cellular membranes [1] and recruits components of the vesicular protein sorting pathway to facilitate virus budding [2, 3, 4] . Concomitantly, the virally encoded protease begins to cleave Gag, which is required for complete virion maturation and infectivity [5, 6, 7] . Gag proteins can be detected at both the PM and the membranes of endosomes among different cell types, suggesting that budding is not limited to one cell-type specific locale [8, 9, 10, 11, 12, 13, 14, 15, 16] . Further, host factors which participate in targeting Gag trafficking to particular membranes are largely unknown. As Gag and infectious virus can originate from two cellular locations, two models for Gag trafficking have emerged. The first model proposes that following synthesis, Gag traffics to endosomal membranes, and upon exocytosis is deposited on the PM, where it serves as the site for productive virus assembly [14, 17] . The second model proposes that Gag is first trafficked to the PM, where virus assembly occurs, and then excess Gag is internalized to intracellular compartments [14, 18, 19, 20] , that serve as sites of productive virus assembly [15, 21] .