Recombinant Cell Lines Expressing ShRNA Targeting HSV2 VP16 Inhibit Virus Replication

Background:Herpes simplex virus 2 (HSV2) is highly prevalent world wide, especially in developing countries. It is not only a main cause of genital infection, but also can cause several atypical diseases and neurological diseases, such as fatal chorioretinitis, meningoencephalitis in neonates and meningitis in adults. Meanwhile, it is an important cofactor for HIV-1 infection. VP16 is a tegument protein encoded by UL48 gene which belongs to late gene. It is expressed in the late phase of the viral replication cycle, and initiates the cascade of HSV gene transcription. Research shows that as a multifunctional virion protein, VP16 is not only a key factor in the lytic mode infection, but also critical for the virus reactivation from latency. RNA interference (RNAi) refers to a regulatory mechanism of most eukaryotic cells that small double stranded RNA (dsRNA) molecules was used to direct sequence specific control of gene activity. There are two basic strategies in using RNAi including RNA-based strategy which uses chemical synthesized siRNA, and DNA-based strategy in which vector based shRNA is used to established a stable siRNA express cell line. Comparing with the chemical synthesized siRNA, the latter one can produce longer gene silencing effect.Aims:To establish HSV2 VP16 targeting shRNA expressing cell line;To investigate the antiviral effect of shRNA targeting HSV2 VP16.Methods:Ligated annealed DNA templates into the linearized plko-mcs-GFP vector using T4 ligase;Transfected the ligation products into DH-5acompetent cells. Correct clones were identified by PCR and sequencing; plko-shRNA, envelope plasmid VSVG, and packaging plasmid A8.2 were cotransfeccted into 293T cells. The supernates were collected 48 hours later. The viral titer of recombined lentivirus was mearured by endpoint dilution assay.24 hours before infection, vero cells were plated into 6 well plates. Each plate was infected by different recombinant lentivirus.8μg/ml puromycin was added to each plate 24 hours later for screening. One screening period lasted 24 hours. After three selecting periods, cell clones expressing GFP and could not be killed by puromycin survived. Picked monoclone and enriched the cells in 10% DMEM culture medium. Three cell lines were established using the same method. They were named vero-shRNA642 (expressing the shRNA 642), vero-shRNA24 (expressing the shRNA 24), and vero-shCON (expressing the control shRNA);24 hrs,72hrs and 96hrs after infection of HSV2, their inhibition effects on VP16 mRNA expression were tested by real-time fluorescent quantitative PCR;The 5 groups of cell lines (A:Vero, B:Vero-shCON, C:Vero-shRNA24,D:Vero-shRNA642 and E:Vero-shRNA24+642) were infected by HSV2 with the MOI of 5,1 and 0.05. Cytopathic Effect (CPE) of HSV2 on each cell line was observed 24,48,72,84 and 96 hours latter to identify the virus replication speed.And The progency virus was tittered were evaluated by yield reduction assay to evaluate their antiviral effects The virus titer described as 50% tissure culture infectious dose (TCID50)/ml was cauculated by Reed-Muench formula The influence of passage numbers on the inhibition ability of cell lines was researched.Results:According to the sequencing results, plko-shRNA24 and plko-shRNA642 plasmids' genomes contained the exact VP16 targeting shRNA genes.24 hrs after three plasmids cotransfection, green fluorescent could be detected.After 48hrs, almost 90% 293T cells express GFP in both groups.Recombinant virus of each group was harvested and concentrated in ultracentrifuge.The viral titer of recombinant virus expressing shRNA24 was 106 TCID50/ml, and the viral titer of shRNA 642 expressing virus was 105 TCID50/ml;Vero cells were infected by the recombinant virus, after three cycle screening by puromycin. The survival and GFP positive clones were picked, and expanded in incubator. Vero-shRNA24 targeting the upper stream, vero-shRNA642 targeting the lower stream, and vero-shCON were established;24hrs, 72hrs and 96hrs after HSV2 infection (MOI=1), VP16 mRNA expression in each vero-shRNA cell line was measured by real time fluorescent quantitative PCR. Comparing to Vero group, vero-shCON could slightly reduce the VP16 expression level by 7% after 24hrs and 6.8% after 72hrs, both of which were of no statistical significance. However, vero-shRNA24, vero-shRNA642, and vero-shRNA24+642 could all reduce the VP16 mRNA significantly comparing with the vero-shCON group either 24hrs or 72hrs later(P<0.05). Vero-shRNA24 was the most efficient with the inhibiting rate of 90.55% after 24hrs and 93.92% after 72hrs, whereas, Vero-shRNA642 resulted in 69.36% reduction of HSV2 VP16 mRNA after 24hrs and 76% reduction after 72hrs; The combination of the two cell lines expressing two different shRNAs didn't show enhancement effect,82.77% HSV2 VP16 mRNA was decreased 24hrs later, and 89.2% decreased 72hrs later.96hrs after HSV2 infection, the inhibiting rate of all the cell lines generally decreased.Vero-shCON showed no obvious reduction effect. Vero-shRNA24 caused 61.37% reduction;Vero-shRNA64 caused 49.28% reduction;Vero-shRNA24+642 reduced the VP16mRNA by 58.36%. The 5 groups of cells:A:Vero, B:Vero-shCON, C:Vero-shRNA24, D:Vero-shRNA642and E:Vero-shRNA24+642 were infected with HSV-2 of three titers MOI= 5,1,0.05.The rate of CPE of infected cells was evaluated 24hrs after infection. MOI= 5,nearly 80% cells in group A and group B got CPE.For group C, the CPE rate was 40%.For group D and E, the CPE rates were 50% and less than 50% respectively;MOI= 1, about 40% cells in group A and group B got CPE.15% cells got CPE in group C,20% cells got CPE in group D, and 18% cells got CPE in group E;MOI= 0.05,30% cells in group A and group B got CPE.For group C, the CPE rate is 5%. For group D and E, the CPE rates were nearly 10%; The yield reduction assay was performed to investigate the antiviral effects of each cell line after infected by HSV2 with MOI=0.05.Compared with the wild type vero cells, the control vero-shCON cell lines had no effect on the virual titer;vero-shRNA 24, vero-shRNA 642, and vero-shRNA 24+642 cell lines could decrease the HSV2 yield by 2 log10,1 log10,1.4 log10, respectively 24hrs after infection. The inhibition kept increasing until 72hrs, with the titer decrease by 3 log10,21og10,2.4 log10. The HSV2 titer of vero and vero- shCON was the highest at 72hr, and started decreasing afterwards. But the virual titers of the shRNAs groups reached peak after 84hr and the highest titer were lower than in the Vero group. The influence of passage numbers on the inhibition ability of cell lines was researched. Virus tittering was also done on passage 10,20 and 50 of Vero-shRNA24 cell lines 24hrs after infecting by HSV2 with MOI=1.And they could decrease the HSV2 yield by 21og10,1.91og10 and 1.51og10, respectively;The inhibiting effect on VP16 mRNA expression and viral replication of vero-shRNA 24 cell lines of passage 10 and 20 had no significant difference with the primary cell line.Although of no statistical significance, the passage 50 cell line showed decreased inhibiting ability.Conclusions:Recombinant cell lines expressing shRNA targeting HSV2 VP16 were established. They can stably inhibit HSV2 VP16mRNA expression and viral replication within passage 50.