Construction Of HSV-2 Mutant Strains And Analysis Of Their Biological Characteristics

To date,we have identified eight types of herpes viruses related to human diseases,of which herpes simplex virus type 2(HSV-2)is a member of the alpha herpes virus family and the main pathogen causing genital herpes.HSV-2 is a neurotropic virus that can extend axons retrograde to the dorsal root ganglion to establish latent infection and cause virus reactivation when stimulated specifically.HSV-2 encodes a large number of proteins,and these proteins have important biological significance in the primary infection,latent infection and recurrent infection of the virus.Among them,ICP34.5 is an important functional molecule encoded by the RL1 gene of the human herpes simplex virus(HSV)and involved in the viral pathological process probably via its neurotropic feature and the inhibition of cellular stress-induced translational arrest.As a latent-associated transcription factor,LAT gene can inhibit the apoptosis of neural cells and promote the survival of neural cells,it plays a key role in the formation,maintenance,and reactivation of latent infection of HSV-2 virus in neural tissues.In addition,the miRNA of LAT gene map antisense to the 5' untranslated region of RL1 gene and the LAT gene can regulate the expression of the RL1 gene.In addition,there are no reports about the interaction between the two neurotropic genes.In order to further explore the ability of HSV-2 virus to infect nerve cells and related regulatory mechanisms,our work first deleted the "neurovirulence factor" RL1 gene,then we constructed RL1-HSV-2 mutant strain,and analyzed its clinical and pathological manifestations,proliferation characteristics,induced cellular stress response and involvement in the regulation of virus-related genes of RL1 molecular.Based on the deletion of RL1,we also constructed RL1-LAT-HSV-2 which mutated both the RL1 and LAT genes simultaneously,and compared LAT-HSV-2 which only deleted the LAT gene,the level of proliferative capacity in cells and animals of RL1-LAT-HSV-2 was analyzed,we also explored acute infection,latent infection,and the immune response by RL1-LAT-HSV-2,compared with the biological characteristics of LAT-HSV-2.In our first part of the work,infection of mice by an HSV-2 strain with a mutated RL1 gene leads to more serious clinical pathological manifestations,which present as a cachexia-like syndrome,than infection by the wild-type HSV-2 strain.However,this pathological characteristic is not due to viral proliferation in tissues,the viral loads of RL1-HSV-2 in mouse tissues is much lower than that of wild strain infection group.Our experimental observation indicate that the defective ICP34.5 protein was found to be responsible for an excessive host stress response that upregulated the expression of various chemokine and the inflammatory reaction in some tissues,leading to systemic failure in infected animals.In addition,the fact that defective ICP34.5 enables the downregulation of the viral ?-gene followed by some ?-and y-genes suggests that ICP34.5 plays a role in the transcriptional regulation necessary for stable viral proliferation in infected tissues.These experimental results provide a new understanding of the function of ICP34.5.In the second part of the work,the HSV-2 mutant strains LAT-HSV-2 and RL1-LAT-HSV-2 present different biological properties.The proliferation of RL1-LAT-HSV-2 in nerve cells was decreased significantly,and the plaques induced by RL1-LAT-HSV-2 in Vero cells were smaller than those induced by LAT-HSV-2 mutant and wild-type strains.The observation of mice infected with these two mutants compared to mice infected with the wild-type strain indicated that the mutant RL1-LAT-HSV-2 has an attenuated phenotype with reduced pathogenicity during both acute and latent infections and induces a stronger specific immune response than the wild-type strain,whereas the attenuation effect was not found in mice infected with the LAT-HSV-2 mutant containing the LAT gene deletion.However,the simultaneous mutation of both the RL1 and LAT genes did not completely restrict viral proliferation in nerve cells,indicating that other HSV genes are involved in viral replication in the neural system.The above results indicate that both RL1 gene and the LAT gene of HSV-2 have biological characteristics related to the pathogenic mechanism of the virus during the process of viral infection and proliferation,there may be some mutual checks and balances between the two genes,the two genes in the course of mutual regulation also showed specific pathological significance in the process of virus-cell interaction,so that the virus and the host have a co-balanced survival state.This is also the first discovery and exploration of the interaction relationship between RL1 and LAT genes in the study of the neurotropic characteristic infection mechanism of HSV-2.