The Role Of Interferon-Stimulated Gene15in HIV-1Infection

ISG15, the product of interferon (IFN)-stimulated gene15, is the first identified ubiquitin-like protein, consisting of two ubiquitin-like domains.The covalent conjugation of ISG15to target proteins is defined as ISGylation. Similar to Ubiquitination, ISGylation is catalyzed by a set of three-step cascade enzymes, an El activating enzyme (UBE1L), an E2conjugating enzyme (UBCH8), an E3ligase (HERC5) and ISG15specific protease USP18. The ISGylation is regarded as one of the most important antiviral pathways in IFN mediated antiviral effect as ISG15and related enzymes are induced by type I IFNs or other stimuli, such as exposure to viruses and lipopolysaccharide.As we know, Ubiquitination plays a critical role in HIV-1budding, assembly, maturation and the formation of infectious virions. It has been reported that ISG15was up-regulated through the IFN pathway when virus invaded and HIV-1replication was disturbed by ISGylation of Gag protein. The competitive inhibition of ubiquitination mediated by ISG15may be an effective antiretroviral procedure. Some reports showed that ISG15blocked Gag assembly so as to inhibit HIV-1budding. One of the speculations is through the ISGylation of Endosomal Sorting Complex Required for Transport system. However, no direct interaction between ISG15and HIV-1protein was reported. Our Longitudinal observation of a HFV-1chronic infection cohort indicated that ISG15was correlated with HIV/AIDS disease progression. HIV-1regulates cellular restriction factors while the latter interfere with HIV-1replication which determins the infection results (infected or not, typical progress or slow progress) and a balance may be reached in the combat. Nevertheless, little is known about ISG15functional pathway and related mechanism involved in HIV-1infection. Fortunately, no antagonistic effect against ISG15was reported which renders the application of ISG15in anti-HIV-1infection very promising. Accordingly further understanding of ISG15expression and function in different cells subsets is very necessary.To investigate the influence ofISG15expression with HIV-1infection and its antiviral effect on HIV-1with in vitro cell line mode. HIV-1molecular clone pNL4-3was used to transfect293T, TZM-bl and Hela cells while HIV-1pseudo-typed virus was used to infect Jurkat, MT-1and THP-1cells. Cells were harvested for extraction of total RNAs and subsequently used in real time PCR for quantification of ISG15transcriptional expression. Forty-eight hours post infection or transfection, cells were harvested for extraction of total proteins to detect ISG15protein expression. A significant up-regulation of ISG15at transcription level was observed in HIV-1infected or transfected cell lines especially in THP-1and TZM-bl cells. But the up-regulation of ISG15protein was observed only in TZM-bl cell. Two ISG15recombinant eukaryotic expression plasmids were constructed to assess the inhibition effect of ISG15on HIV-1replication. Cotransfection results suggested that ISG15inhibited HIV-1producing and assembly as a dose and time depend manner in293T cell but not TZM-bl cell which exhibited no dose dependent effect. These results suggested that HIV-1can escape from innate immune system by a new mechanism remains to be addressed.To comfirm the ISG15expression pattern in HIV-1infection and further investigate the correlation between ISG15expression and HIV-1disease progression we recruited71HIV-1chronic infected patients in Shanxi province and25healthy donors in Shanghai. Peripheral blood mononuclear cells were separated from blood samples and ISG15expression was detected both at transcription level and protein level. The patients were divided into ART+, ART-but slow progression, ART-but typical progression groups.Systematic analysis revealed that ISG15was up-regulated in HIV-1chronic infected patients compared to healthy donors. And Patiens’ PBMCs had a much lower response when stimulated with IFN-α2b with regard to the ISG15expression. Different cell subsets exerted different ISG15level and were conferred with some anti-HIV-1effect. Interestingly, we found that monocyte had the highest ISG15expression level not only in healthy donors but also in HIV-1chronic infected patients which indicated that the antiviral function of ISG15may happen mostly in monocyte and through related cellular factors. Additionally, ISG15expression was correlated with HIV-1RNA copies and CD4count, indicating that ISG15may be regared as auxiliary HIV-1progression monitor in the future. Collectively we discribled ISG15expression profile in HIV-1chronic infected patients and provided a new insight on HIV-1prevention and curation.