The Molecular Mechanism Of The Deubiquitinating Activity And The Inhibition Of NF-KB Of PRRSV Nsp11

Porcine reproductive and respiratory syndrome (PRRS), caused by Porcine reproductive and respiratory syndrome virus (PRRSV), is a relatively new viral diseases. The disease was characterized by severe reproductive failure in sows and high mortality and respiratory symptoms in the newborn piglets. PRRSV infection only induces tardy and week neutralizing antibodies, leading persistent existence and secondary infection. This was difficult for the prevention and control of the great PRRSV.Virus invading organism can initiate the immune system of host, which mainly including non-specific natural immune defensive system and specific immune responsive defensive system. Natural immunity was the first defensive line to pathogens, which has very crucial effects in anti-virus infection for organism. The production and anti-virus effects of interferon are the majority for organism to against infection in the early stage of natural immune defensive. Many pathogens encode proteins that can modify immune system of host, thus escape from anti-virus effects of host. The interaction between pathogen and host, provided more space for the immune system development of host. Many articles reported the ubiquitin plays an important role in viral replication proliferation process, we had found that the PRRSV infection had obvious influence on the ubiquitin level of the cellular. Interferon, as an important anti-virus factor, was the dundamental role for anti virus invadd. Many reports said that PRRSV had the ability of escaping form the natural immune of host, also caused the body’s immune suppression. The ubiquitin plays an important role in the natural immune signal transduction, many viruses encode proteins that can modify the host’s ubiquitin and ubiquitin-like machinery, blocking natural immune response, and interferon produce, thus escaped from the host of anti-viral infection reponse.In our research, we found PRRSV infection caused the polyubiquitin reduced and Nsp11had the deubiquitylation activity, modifying the ubiquitin of I-IFN signal transduction. To clarify the precise molecular mechanism between the deubiquitylation activity of Nsp11and the production of I-IFN, the main research works were as following:1. PRRSV infection regulates polyubiquitination in celler substratesTo investigate the levels of ubiquitinated cellular proteins in the context of PRRSV infection, Marc-145cells were infected with PRRSV strain WUH3. Compared to the mock-uninfected cells, the polyubiquitination was varied dynamically in the time-course of PRRSV infection:a gradually decrease in the early phase (0-12h) and reaching a plateau phase for24h (12-36h), while a slightly increase at48h compared to that observed at12h after PRRSV infection. Notably, infection with UV-irradiated inactivated PRRSV, did not alter the level of protein-ubiquitin conjugates. These results indicated that the decreased level of ubiquitinated cellular proteins depends on viral replication.2. PRRSV Nsp11possesses DUB activity.Because the decreased cellular protein ubiquitination depended on PRRSV replication, we tried to understand whether there are Nsp(s) with DUB activity. All of the PRRSV Nsps derived from PRRSV strain WUH3were cloned and then co-transfected with a plasmid expressing Flag-tagged ubiquitin into HEK293cells. We found expression of Nsp2and Nsp11resulted in decreased expression of ubiquitin-conjugated proteins. Nsp11appeared to have stronger DUB activity than Nsp2. Further experiments demonstrated the degree of deubiquitination depends directly on the amount of transfected Nsp11expression plasmid.3. PRRSV Nspll processes K48-linked, but not K63-linked polyubiquitin.The the main forms of ubiquitin modification, including:K63-links and K48-links.To further identify which Ub linkage type is targeted by Nsp11, HEK293cells were transfected with HA-K48-Ub or HA-K63-Ub. We saw that K48-linked Ub chains were processed by Nsp11in a dose-dependent manner, but K63-linked Ub chains had no change, indicating that PRRSV Nspll processes K48-linked, but not K63-linked polyubiquitin in vivo.4. Sites D144, K173, D180and Y219were essential for the DUB activity of Nsp11.PRRSV Nsp11encodes an endoribonuclease (NendoU), which is unique and conserved throughout the Nidovirales order but have not been identified in other RNA viruses. To identify whether these NendoU sites are associated with the DUB activity of Nsp11, various mutants(H129A, H144A, K173A, D180A, D204A, Y219A) were constructed by replacing these conserved sites with Ala. The result of Western Blot revealed that mutants H144A, K173A, D180A, and Y219A, decreased the DUB activity to some degrees, compared to the wt Nsp11. 5. The DUB activity of Nspll is essential for its ability to inhibit NF-κB activationThe ubiquitin plays an important role in the natural immune signal transduction, using luciferase reporter system, we demonstrated that Nsp11down-regulated SEV/poly(I:C) induced promoter activation of IFN-(3, NF-κB and IRF3in a dose-dependent manner. To determine whether the DUB activity of Nsp11is involved in the inhibition of type Ⅰ IFN induction, various Nspll mutants with differing DUB activities were analyzed for their abilities to affect signaling to SeV/poly(I:C)-induced IFN-β, NF-κB and IRF3in HEK293cells. To our surprise, nearly all of Nsp11mutants lacking the ability to block SeV/poly(I;C)-induced IFN-β and IRF3. Rather, only the mutants losing the DUB activity could abolish the inhibition to ploy(I:C)-induced NF-κB. These results suggested that the DUB activity of Nspll was key to inhibit NF-κB activation, but not for IRF3and IFN-β.6. Nspll inhibits NF-κB through degradation of K48-linked polyubiquitination of IκBα.A key step that leads to NF-κB activation in response to many extracellular stimuli is degradation of IκB proteins. To test whether the PRRSV Nspll inhibits the NF-κB activation by interfering with the ubiquitination of IκBα and subsequently preventing IκBα degradation, using TNF-a stimulated HEK293cell, we observed that the degradation of TNF-α-induced IκBα was prevented to some degrees in cells transfected with Nspll, compared to cells transfected with empty vector, indicating that Nspll inhibits NF-κB activation through preventing IκBα degradation. Further test shown IκBα had interaction with K48-Ub, and Nsp11could obviously remove the K48-Ub of IκBα. Using COIP experiment, we further investigated the Nspll mutants (H129A, K173A, D180, and Y21lacking DUB activity were incapable of degrading K48-Ub of IκBα, demonstrating that Nsp11degraded K48-Ub of IκBα and inhibitd the degradation of IκBα, then inhibit NF-κB activation...