Anti-apoptotic Function Of HSV-2Latency-associated Transcript RL1Sequence And The Screening Of Its Encoded MicroRAN

Herpes simplex virus type2(HSV-2) is a common pathogen of human diseases, latent inthe body lead to continued infection, when Immunocompromised it can be activated again andit is difficult to cure.Latent infection is the main cause for genital herpes recurrence.Thelatency-associated transcript (LAT) is the only genes expressed effectively during HSVlatency and it plays an important role in HSV life cycle. Currently,there are many hypothesesabout the roLe of LAT,but the specific mechanism is not clear.LAT can not encodeproteins,but can encode a plurality of functional miRNA.microRNA can target mRNAthrough specific base pairing, causing degradation of the target mRNA or inhibiting itstranslation, so that regulate gene expression in post-transcriptional level.miRNA may play animportant role in HSV latent infection,during Latency.During viral latency, LAT-encodedmiRNA interfere with its target genes expression,to inhibit cells apoptosis and to cause viruslatency.Therefore, in this study,we use HSV-2333genome as a template,PCR amplified HSV-2LAT RL1sequence and then constructed eukaryotic expression vector pEGFP-RL1.Liposometransfection, and electroporation transfection were used for the transfection of recombinantsinto Vero cells and PC12cells respectively, G418were used for the screenning of therecombinants.To study the Expression of recombinant in Vero cells and PC12, and itsanti-apoptotic effects, we use real-time PCR method to determine RL1encoded microRNA,and then make clear that RL1perform its anti-apoptotic function by encoding microRNA.First, according to the GenBank, LAT RL1primers were designed, HSV-2333genomewere used as a template,PCR amplified HSV-2LAT RL1fragments, and then constructrecombinant plasmid pEGFP-RL1.Recombinant plasmids were transfected into Vero cells andPC12cells, G418resistance screening for recombinants,and LAT RL1mRNA successfullytranscribed in Vero cells and PC12cells were confirmed by RT-PCR.RL1sequence having anti-actinomycin D function in Vero cells and PC12,G418resistance screening for recombinants,were determined by Hochest33342stained,thefluorescent observation, and JC-1staining.Both in Vero cells and in PC12cells.MTT results showed that after transfection of cellsand apoptosis by actinomycin D,cells activities of recombinant plasmid pEGFP-RL1groupwere no statistically difference compared with no treatment control group (P>0.05), buthigher than cells transfected with with the empty vector pEGFP-C2and actinomycinD-induced apoptosis group, the difference was statistically significant (P <0.05). Flow cytometry results showed that both in Vero cells and in PC12cells,the apoptosisrate of recombinant plasmid pEGFP-RL1group were no statistically difference comparedwith control group (P>0.05), but lower than cells transfected with with the empty vectorpEGFP-C2and actinomycin D-induced apoptosis group, the difference was statisticallysignificant (P<0.05).Caspase-3activity assay showed that both in Vero cells and in PC12cells,the activity ofcaspase-3of recombinant plasmid pEGFP-RL1group were no statistically differencecompared with control group (P>0.05), but lower than cells transfected with with the emptyvector pEGFP-C2and actinomycin D-induced apoptosis group, the difference was statisticallysignificant (P <0.05).DNA Ladder indicated that there was not DNA fragmentation in either cells transfectedwith pEGFP-RL1induced by ActD,or normal cells, but transfected with the empty vectorpEGFP-C2and actinomycin D-induced apoptosis group appeared DNA fragmentation.Real time-PCR displayed RL1sequence can encode five kinds of microRNA,(miR-H3,miR-H4-3p, miR-H4-5p, miR-H24and miR-H19) both in Vero cells and in PC12cells, butmicroRNA expression levels is a difference in the two cells, indicating that the expression ofmicroRNA is tissue-specific.In conclusion, the above results show that, HSV-2LAT RL1fragment has antiactinomycin D function in both PC12cells and Vero cells.Further more, this function wasperformd mainly by its encoded microRNA. This study has layed a foundation for the explorethe of the mechanisms relating to HSV-2LAT-mediated viral latency and recurrence.