HIV-1 Protease Has a Genetic T-Cell Adjuvant Effect Which Is Negatively Regulated by Proteolytic Activity

HIV protease (PR) mediates the processing of human immunodeficiency virus (HIV) polyproteins and is necessary for the viral production. Recently, HIV PR was shown to possess both cytotoxic and chaperonelike activity. We demonstrate here that HIV PR can serve as a genetic adjuvant that enhances the HIV Env and human papillomavirus (HPV) DNA vaccine-induced T-cell response in a dose-dependent manner, only when codelivered with DNA vaccine. Interestingly, the T-cell adjuvant effects of HIV PR were increased by introducing several mutations that inhibited its proteolytic activity, indicating that the adjuvant properties were inversely correlated with its proteolytic activity. Conversely, the introduction of a mutation in the flap region of HIV PR limiting the access to the core domain of HIV PR inhibited the T-cell adjuvant effect, suggesting that the HIV PR chaperonelike activity may play a role in mediating T-cell adjuvant properties. A similar adjuvant effect was also observed in adenovirus vaccine, indicating vaccine type independency. These findings suggest that HIV PR can modulate T-cell responses elicited by a gene-based vaccine positively by inherent chaperonelike activity and negatively by its proteolytic activity.Human immunodeficiency virus protease (HIV PR) is a typical aspartic protease required for processing HIV polyproteins such as Gag and Pol and is essential for HIV production (5). HIV PR consists of three domains: terminal, core, and flap domains, and each has a unique role in the proteolytic process (21). The substrate-binding site is found in the core domain that contains the catalytic triad (DTG). The terminal domain is required for dimerization, which is also important for its proteolytic activity, and the flap domain regulates substrate access (2).In addition to the above-described processes, HIV PR also cleaves cellular proteins important to cell survival including the antiapoptotic protein, Bcl-2 (26), and procaspase-8 (20), which mediates the apoptosis of HIV-infected cells or of cells transfected with DNA encoding HIV PR. The importance of HIV PR proteolytic activity in mediating cell death was highlighted in studies that demonstrated the inhibition of cell death after mutations to the DTG catalytic site (26) or after the treatment with protease inhibitors such as ritonavir or saquinavir (19).HIV PR was also shown to have inherent chaperonelike activity mediated by the core domain. The introduction of a mutation that removed the aspartic acid residue of the catalytic site or inhibition of dimerization necessary for proteolytic activity did not affect its chaperonelike activity in vitro, suggesting that the chaperonelike activity was independent of its proteolytic properties (8).Typical chaperones, such as heat shock protein 70 (Hsp70), Hsp90, and gp96, were reported to chaperone antigenic peptides and mediate cross-priming of cognate antigen-specific CD8 T cells in vivo (1). In addition, the minimal 136-amino-acid peptide binding domain of the mycobacterial Hsp70 efficiently generated CD8 T-cell responses against the complexed peptide. Hsps can also protect processed peptides from further proteosomal degradation and enhance targeting to dendritic cells via the interactions of Hsps with surface molecules, including CD91, TLR4, or CCR5, resulting in CD8 T-cell cross-priming (10).In the present study, we demonstrated that the codelivery of HIV PR could enhance the T-cell response, but not the humoral response, elicited by DNA and adenoviral vaccines and that the T-cell response was further augmented by HIV PR mutations that inhibited proteolytic activity. Interestingly, the T-cell adjuvant effect of the catalytic mutant was reduced by the introduction of a point mutation that stabilized the flap domain into a closed position and not by a mutation that inhibited dimerization. These data suggested that HIV PR has a T-cell adjuvant effect presumably due to the intrinsic chaperonelike activity which is veiled by its proteolytic activity.