The Biological Mechanism Of HSV2 RL1,UL41 And US12 Proteins In Viral Pathogenicity Was Studied By Mutation Technique

Herpes simplex virus type 2 is one of the main pathogens causing genital herpes and it can cause herpes encephalitis and more serious sexually transmitted diseases in immunodeficiency patients.The virus can be transmitted sexually and from mother to child.After HSV-2 infection,the latent infection can be established retrograde through the sensory nerve axis to the sacral dorsal root ganglion,this latent infection can present as the symptoms of dominant infection when exposed to external stimulation.Recurrent infection has caused physical and psychological effects on patients,seriously affecting the quality of lif.e.Due to this latent and repeated infection,the development of therapeutic drugs and vaccines for HSV-2 is extremely difficult.In addition,infection with HSV-2 can increase the chance of infection with HIV.Therefore,the study of the pathogenesis of HSV-2 infection,the host immune response and the interactions between viral proteins and the host,will contribute to the development of therapeutic drugs and vaccines for HSV-2 and our deeper understanding of the pathogenesis of HSV-2.Based on the above facts,the first part of this study focused on the gene RL1 UL41 US 12,which is related to the immune escape of HSV-2.First,HSV-2 mutant was constructed using CRISPR/Cas9 system:M2(HSV-2 RL1-1UL41-)M3(HSV-2 RL1-US12-)M4(HSV-2 RL1-UL41’US12-).Then,the biological characteristics of the three mutant strains were analyzed in cell and animal experiments,including proliferation kinetics detection for plaque detection,acute infection and latent infection,and immunogenicity analysis.The results indicate that M2,M3,and M4 exhibit decreased proliferation ability in both acute infection and latent infection at the animal level and cellular level,and cause milder clinical symptoms,of which M4 has the most significant decline in proliferation ability.In the immunogenicity analysis test,it was found that although the mutant strain group can reduce the replication ability of the virus,it cannot be 100%protected,the survival rate of the M4 group was 90%,and the survival rate of the M2/M3 group was 80%.In view of this discovery,the second part of the study performed transcriptome sequencing analysis on the vagina,uterus and ovaries of mice infected with wild strains and mutant strains M1(HSV-2 RL-),M2,M3.The results showed that compared with the wild strains,the most interesting is that mutant strain M1,M2,M3 can activate metabolic signaling pathways in vagina,uterus and ovarian tissues,of which steroid hormone metabolism signaling pathway and cytochrome metabolism signaling pathway are the most obvious.It was found that M1 had a significant effect on the signaling pathways of steroid hormone metabolism and cytochrome metabolism,the possible mechanism is related to RL1’s ability to inhibit translational arrest in response to viral infection,including the process of steroid hormones and cytochrome metabolism.In summary,the mutant strain M2,M3,M4 was compared and analyzed in cell and animal experiments,and M4 strain was with low replication ability,low pathogenicity,low latent infection ability,and a certain immune protective effect,which can be used as a candidate strain for HSV-2 attenuated live vaccine.Analysis of transcriptome data showed that the M1 mutant had a significant effect on steroid hormone metabolism and cytochrome metabolism,but this effect was limited when UL41/US12 was continued to be absent,indicating that there was a state of equilibrium between viral proteins.However,it is undeniable that the loss of RL1 has an important impact on steroid hormone metabolism and cytochrome metabolism.This discovery provides a new research direction for the interaction between the viral protein and the host during HSV-2 infection.