Study On The Antiapoptotic Function Of Herpes Simplex Virus Type 2 Latency Associated Transcript Gene ORF2 In Vero Cells

Genital herpes (GH),a common sexually transmitted disease,is primary caused by Herpes Simplex Virus type 2(HSV-2).As a potential carcinogen,GH is easy to cause abortion.The incident of GH has greatly increased in recent years,and GH has become critical sex transimitted disease(STD) in many countries and districts.Following primary infection at skin or mucosal surfaces,viral DNA is rapidly transported to the innervating ganglia by retrograde axonal transport.The viral genome circularizes and establishes a life-long latency in sensory neurons.There are no any anti-virus drugs,which can effectively control the occurrence and recrudescence of HSV-2.However ,the mechanism underlying the transition between the lytic and latent infection and virus reactivation is poorly understood,so it is very hard to clear the infectious virions clinically.The latency associated transcript (LAT),is the only abundantly transcribed viral gene during latency,and plays an important role in the HSV latency-reactivation cycle. The HSV-2 LAT gene codes for a primary transcript (9.0kb LAT), and the major LAT (2.2kb LAT). Studies on LAT functions have been mainly focused on the 2.2kb LAT intron. The major LAT has three ORFs(Open Reading Frames).At present there is main hypotheses regarding the role of the LATs in neuronal survival is that LATs promote cell survival by preventing infected neurons from undergoing apoptosis.In the research,the possible anti-apoptotic gene ORF2 of pVAX-LAT was cloned .The recombinant eucaryotic expression plasmid with EGFP tag, pEGFP-ORF2 ,was constructed.Expression and function of pEGFP-ORF2 in SH-SY5Y/Vero cells were investigated.PCR primers were designed according to DNA sequence of HSV-2 LAT ORF2 offered by Gene Bank.The full length ORF2 gene of pVAX-LAT was specially amplified and ligated into the eucaryotic expression vector pEGFP-C2.Identified by enzyme digestion and sequenced ,the recombinant plasmid pEGFP-ORF2 was obtained.The recombinant vector pEGFP-ORF2 was transfected into SH-SY5Y/Vero cells in vitro. The fusion green fluorescent protein was observed by fluorescence microscope, and the expression of the target fragment was detected in the lysate of the transfected Vero cells by RT-PCR. Vero cell line expressed EGFP-ORF2 had been established successfully with G418 for 20 days.Eukaryotic expressive vector pEGFP-ORF2 was transfected into Vero cells Morphogenesis were observed by fluorescence microscopy and cell activity was analyzed by MTT. HSV-2 LAT ORF2 had evident change in cytomorphology ,the location of fusion protein of EGFP and HSV-2 LAT ORF2 in cells has changed,MTT analysis revealed the cell activity decreased. HSV-2 LAT ORF2 has impairment on cells. Vero cells transfected with pEGFP-ORF2 induced by 5-FU have no changes in cytomorphology.MTT assay showed that the cells activity transfected with HSV-2 LAT ORF2 remarkably higer than the negative control,While there were no such remarkable changes between them and normal cells. DNA ladder showed that there was no DNA fragment. The activity of Caspase-3 remarkably lower than the negative control.Therefore we concluded that HSV-2 LAT ORF2 gene can protect cells from apoptosis induced by 5-FU.In conclusion ,the eukaryotic expression plasmid pEGFP-ORF2 was successfully constructed and could express in SH-SY5Y/Vero cells effectively.Meanwhile, the anti-apoptosis effect of HSV-2 LAT ORF2 on Vero cells was explored, these results may shed light on the further study of the function of HSV-2 LAT in the establishment and maintenance of latent infection and reactivation of HSV-2.