The Influence Of LncRNA4.9 On HCMV Latent Infection And Its Mechanism

Objectives:Human cytomegalovirus(HCMV),belonging to the betaherpesvirus family,mainly causes latently,mild,and subclinical infection.Whereas,as an important pathogen for newnatals and immunocompromised individuals,HCMV may lead to deafness,neurodevelopmental abnormalities,or pneumonia.The mechanism of HCMV latent infection and reactivation remains unclear,neither the relationship between the long non-conding RNAs(lncRNAs)encoded by HCMV and latent infection.Our study aims to clarify the influence of lncRNA4.9 encoded by HCMV on its latent infecton,reveal the mechanism,and then help us to understanding the mechanisms of HCMV latent infection,provide new ideas for clinical prevention and treatment of HCMV infection.Methods:1.Constructed interference lentiviral vector of lncRNA4.9 and NAB2:designed and synthesized four shRNAs including NAB2-shRNA,lncRNA4.9-shRNA-1,lncRNA4.9-sh:RNA-25 and lncRNA4.9-shRNA-3,linked those shRNAs to the digested linearization vectors,and then transformed them into germ competent cells.Next,sequenced and blasted the positive clones,choose the right one for later amplifying and packaging of lentivirus.Lastly,transformed them into THP-1 cells which infected with HCMV,evaluating the interference effect by performing real-time RT-PCR and Western-Blot.2.Establishment of HCMV latent infection:infected THP-1 and MRC-5 cells with HCMV towne strain for 1,2,3,5,and 7 days,collected the cells for extraction of DNA,RNA,and protein,in order to detect the titer of HCMV within THP-1 cells and MRC-5 cells,protein levels of UL122 and UL138 by performing Western-Blot,as well as mRNA levels of UL122 and UL138,together with lncRNA4.9 levels by performing real-time PCR respectively.3.Regulated the levels of lncRNA4.9 and NAB2:divided into two part.Firstly,transfected the HCMV infected THP-1 cells with negative control vector and lncRNA4.9 interference lentiviral vector respectively for 3 and 7 days,collected the cells,extracted DNA,RNA,and protein for detection of HCMV titers within THP-1 cells,protein levels of IE2 by performing Western-blot,mRNA and protein levels of NAB2;Secondly,transfected the HCMV infected THP-1 cells with negative control vector and NAB2 interference lentiviral vector respectively for 3 and 7 days,collected the cells,extracted DNA,RNA,and protein.Then detected the HCMV titers within THP-1 cells,protein levels of IE2 by performing Western-blot,mRNA and protein levels of EGR1;Results:1.Successfully constructed the interference lentiviral vectors carrying NAB2-shRNA or lncRNA4.9-shRNA:blast results showed the sequences of shRNAs are in accordance with expectation.The results of real-time RT-PCR and Western-Blot showed the LV-NAB2-RNAi group significantly downregulated the expression of NAB2,and the LV-lncRNA4.9-RNAi-2,LV-lncRNA4.9-RNAi-3 group downregulated the expression of lncRNA4.9 significantly.2.HCMV latent infection were established at 5 days after infected THP-1 cells:After infected by HCMV towne strain,THP-1 cells clustered and expanded gradually.The intracellular titers of HCMV decreased significantly on the fifth day post infection,and the mRNA level of UL122 began to decline on the fifth day and disappeared on the seventh day,in accordance with the protein level of IE2.The lncRNA4.9 level,mRNA and protein levels of UL138 persist consistently.In contrast,the intracellular titers of HCMV in MRC-5 cells increased consistently,and the lncRNA4.9 level,the mRNA level of UL122 also climbed during the study period,mRNA and protein levels of UL138,protein level of IE1/IE2 persist consistently.3.(1)Comparing with negative control group,the LV-lncRNA4.9-RNAi-2 group showed a higher intracellular HCMV titer level and an increased expression level of NAB2 protein on the seventh day.7 days post HCMV infection,IE2 protein level cannot be detected in negative control group,while exist in the LV-lncRNA4.9-RNAi-2 group.(2)The LV-NAB2-RNAi group showed a lower intracellular HCMV titer level than negative control group both on 3 and 7 days post infection;the mRNA level increased also,while the protein level increased on the seventh day.7 days post HCMV infection,IE2 protein level cannot be detected in both groups.And the immunofluences showed that EGR1 may linked with IE1/IE2,which we should confirm further by performing co-immunoprecipitation.Conclusions:1.Constructed lncRNA4.9 and NAB2 interference lentiviral vectors successfully,which provide a solid foundation for exploration of their physiological function.2.lncRNA4.9 can inhibit the expression of NAB2.3.We validate primarily that the lncRNA4.9 can directly inhibit the expression of IE2,and can also inhibit the expression of IE2 by inhibiting the expression of NAB2,thus increase the level of EGR1 and the binding of EGR1 to IE2,thereby prompoting the occurrence of HCMV latent infection.