Study On The Molecular Mechanism Of Inhibting Interferons Responses By Human Rhinovirus C3C^pro

Human rhinoviruses (HRVs) belonging to the genus Enterovirus, family Picornaviridae, are the most common cause of viral infection, leading to considerable economic burdens in terms of medical visits and work absenteeism. HRVs are the major pathogen of upper and lower respiratory tract diseases, and responsible for greater than50%of all upper respiratory tract infections. Furthermore, rhinovirus is implicated in other diseases including bronchitis, pneumonia and causing severe exacerbation of chronic obstructive pulmonary, asthma and cystic fibrosis.Human rhinoviruses are classified into three species, HRV-A, HRV-B and HRV-C. With the use of molecular diagnostic techniques HRV-C was identified in2006.30%to50%of patients with rhinovirus infection are infected with HRV-Cs. There are some evidences that HRV-Cs may be more virulent than other species. Reports suggest that HRV-Cs may be overexpressed in children with pneumonia or acute wheezing and exacerbation of asthma. HRV-C strains do not grow in standard cell culture. The specific pathogenic mechanism of HRV-C remains unclear.The innate immune system is the first line of defense against pathogen infiltration. Host cells sense the viruses leading to a signaling cascade that stimulate the antiviral immune response, limiting viral replication. The innate immune response to rhinovirus infection may play an important role in rhinovirus-induced asthma. Interferon (IFN) responses affect the outcome of HRV illnesses. It has reported that HRV-induced epithelial cell production of IFN-β and IFN-λ is impaired in asthma. Many viruses have evolved strategies to attenuate the production of IFNs for evading the innate immune responses in order to promote their replication and spread. Reports suggest that MAVS (mitochondrial antiviral signaling) is cleaved during HRV1a infection. While the specific mechanism of evading innate immune responses for rhinovirus especially HRV-C has not been comprehensively investigated.To elucidate the relationships of HRV-C and innate immune responses and the mechanisms to evade the innate immunnity for HRV-C, we designed a series of experiments. Our results illustrated the molecular mechanisms that proteins of HRV-C disrupted the production of host type I IFN and type III IFN, which will lead to a better understanding of the pathogenesis of HRV-C.The main results of this study are as follows:1. With the use of dual-reporter system, we tested the effect of proteins of LZ651(VPO, VP4, VP2, VP3, VP1,2A,2BC,2B,2C,3AB,3ABC,3A,3B,3C,3CD and3D) on the activation of promoters of IFN-P and IFN-λ1. The results showed that3Cpro attenuated the activation of promoters of IFN-P and IFN-λ1obviously.2. To ascertain the inhibition on the expression of IFNs, we tested the effect of3Cpro on the IRF3(interferon regulatory factor3). The results showed that LZ651-3C pro disrupted the dimerization and nuclear translocation of IRF3. In addition,3Cpro inhibited RIG-I (retinoic acid-inducible gene I), MDA5(melanoma-associated differentiation gene5) and TLR3(toll-like receptors3) signalings.3. In order to find the active site of3Cpro for the inhibition of IFNs responses, we constructed mutants of3Cpro (H40D, C147A, C147S, N165D and E71A). Results suggested that40-His played important role for the inhibition of activation of IFNs.4. In order to explore the target proteins of3Cpro, we tested the influence of3Cpro on the expression of some key signaling transducers. The preliminary results revealed that3Cpro disrupt the expression of MDA5, RIG-I, TRIF (TIR domain-containing adaptor inducing IFN-β) and MAVS.5. With the co-immunoprecipitation, we found there was direct relationship between3C and TRIF protein.3Cpro cleaved the TRIF protein. But the cleavage site is different from the cleaved site of TRIF proterin due to entero virus713Cpro.6. In order to explore the distinct pathogenesis among HRV-A, HRV-B and HRV-C, we compared HRV1B-3C, HRV6-3C and LZ651-3C. The results showed that LZ651-3C had greater inhibition of the IFNs signaling pathway than HRV1B-3C and HRV6-3C.Taking together, our findings reveal the strategy employed by HRV-C to escape host anti-viral innate immunity that leads to a better understanding of HRV-C mediated immune-evasion mechanisms and contributes to reveal the pathogenic mechanisms of HRV-C.