Mechanism Of HCMV-miR-US33as-5p In Immune Evasion

Human Cytomegalovirus(HCMV), a prevalent human pathogen, is a member of the subfamily of ?-herpesviruses, which is an enveloped, double-stranded DNA virus that maintain a persistent latent infection during host’s lifetime, transmitted through blood, sex, mother to child, organ transplantation, and so on. Healthy individuals remain asymptomatic when infected with HCMV, but turn to series complications once activated, especially in immunocompromised individuals. Congenital HCMV infection leads to neonatal deafness, mental retardation and even death, one of the key factors affecting the quality of births. However, lacking of effective cure or vaccine, prevention and treatment of HCMV infection has been troubling clinicians and appealed much attention and research. Proportion of HCMV infected cells is extremely low, with the fact that HCMV is highly species-specific, significantly limiting the research process of HCMV infection.Due to its ability of escaping the host immune surveillance, HCMV could be latent in the host for quite a long period. HCMV encodes various proteins, for example, early gene products IE1 and IE2 encoded by HCMV could disturb the IFN signal pathway. IE1 integrates into STAT2, inhibiting ISGF3 to combine with ISG promoter. IE2 prevents transcriptional factor NF?B combining to IFN-?, down-regulating expression of IFN-?, impeding immune killing of IFN. On the other hand, HCMV encodes kinds of mi RNAs. Studies show that HCMV encoded mi RNAs play important roles in immune evasion, imparing host immune elimination by targeting corresponding m RNAs. Nevertheless, HCMV encoded mi RNAs reported by now has been found during lytic infection. Whether HCMV encoding mi RNAs during latent infection and the mechanism they exerted for immune evasion need to be further explored and studied.The purpose of this study is to construct a cell model of HCMV latent infection, to explore HCMV encoded mi RNAs in latent infection and their molecular mechanisms and biological function on the basis of the cell model. The results of the study are as follows:1. Construction of the HCMV latent infection cell model. We infected THP-1 cells with HCMV, detected HCMV DNA copies, changes in HCMV genome and trascriptome genome, observed if THP-1 cells turned to lytic state. Then we infected THP-1 cells with HCMV for 30 days, divided into direct infected group and induced infected group, the distinguishing changes in the HCMV genome and trascriptome genome of the two groups state that we have constructed the HCMV latent infection model. Expression of pp65 protein of THP-1 cells in lytic infection, undifferentiated infection, differentiated infection by immunofluorescence confirm that this model conforms to the property of HCMV latentency and activation without cell damage.2. HCMV can also encode multiple mi RNAs during latent infection. Deep-sequencing of HCMV latent infected THP-1 cells by high-throughput Solexa sequencing platform, we found that HCMV also encoded mi RNAs during latent infection, including mi R-US25-2-3p, mi R-US25-2-5p, mi R-UL112, mi R-US25-1, mi R-UL22 A and PC-5p-148467 with a predicted length of 25 bp. Conductive analyses of PC-5p-148467 with bioinformatics methods displayed that the sequence was conservative and contain a hairpin structure which was a necessity to form mi RNA. Then according to the gene location and naming rules of mi RNA, we named PC-5p-148467 as HCMV-mi R-US33as-5p.3. IFNAR1 is one of the potential targets of HCMV-mi R-US33as-5p. We predicted the possible targets of mi R-US33as-5p with bioinformatics software, constructed dual luciferase reporter system, detected that mi R-US33as-5p could down-regulated the relative luciferase activity of IFNAR1. Then we constructed mutation and reverse mutation expression plasmid of binding site between IFNAR1 and mi R-US33as-5p seed region, screening stable expressed 293 cell line tranfected with the vectors above. Dual luciferase reporter assay verified that IFNAR1 was one of the targets of HCMV-mi R-US33as-5p, and revealed the main binding site between mi R-US33as-5p and its target IFNAR1.4. Once IFN binds to IFNAR1, Jak-STAT1 signal transduction pathway was activated, ISGs were released, the anti-virus effect was exerted by host cells. We constructed stable expressed 293 cell line tranfected with IFNAR1-RNAi plasmid, compared the expressions of p-STAT1 protein, nuclear translocation of STAT1, and relative m RNA levels of ISGs in all stable transfected 293 cells. The expressions or levels were up-regulated and down-regulated accordingly. Oppositely, we constructed recombine lentivirus containing anti-US33 as sequence(HCMV-mi R-US33as-5p inhibition sequence), and stably transfected HFF cells. Results showed that, HCMV encoded mi R-US33as-5p could target IFNAR1, block Jak-STAT1 signal transduction pathway, reducing release of ISGs, finally resist the immune impairment of the host.In summary, this study successfully constructed the HCMV latent infected cell model, expecting to offer basis for research on HCMV latent infection; demonstrated that HCMV could also encode mi RNAs during latent infection, including the newly discovered HCMV-mi R-US33as-5p; illustrated that IFNAR1 was one of the targets of HCMV combining bioinformatics methods with molecular biological skills. Because of the binding of mi R-US33as-5p and IFNAR1, the immune function by IFN was beyond effectiveness, HCMV then could keep lifelong lantency in host. This study stated the target of HCMV-mi R-US33as-5p discovered in HCMV latent infection, helping to enhance the understanding of mechanism of HCMV inmmune evasion, find out new target for prevention and treatment, develop new antiviral drugs against HCMV, and open up promising prospects.