Study On The Molecular Mechanism Of Latent Hepatitis B Virus Infection Based On High-frequency Mutations In The Pre-S Region Of Hepatitis B Viru

Hepatitis B virus(HBV)infection remains a major public health problem worldwide,posing a serious threat to human health and life.Occult HBV infection(OBI)is a special type of HBV infection that refers to the presence of replication-competent HBV DNA in the liver and/or HBV DNA in the blood of people who tested negative for HBV surface antigen(HBsAg)based on current available assays.The mechanism of OBI is not fully understood and mainly includes viral factors,host factors,and other factors such as coinfection.Among them,the viral factors are the focus of research,but most of the current studies have concentrated on the S region in the HBV genome,while research on other regions is lacking.Located upstream of the S region,the HBV pre-S region is not only involved in encoding HBsAg,but also plays a vital role in HBV infection,assembly and secretion of viral particles,and induction of host immune response.Some studies have shown that pre-S mutations can significantly reduce HBsAg expression,which may be associated with the occurrence of OBI.However,most of these studies have focused on pre-S deletion mutations,and fewer studies have been conducted on common mutations such as amino acid substitutions,and there are generally problems such as small sample size,insufficient mechanistic studies,and lack of systematicity.To solve these problems,this study performed sequencing and sequence analysis of the pre-S region in 370 OBI blood donor samples and 370 HBsAg-positive donor samples from 32 blood centers in 14 provinces across China to identify pre-S mutations that occur naturally in OBI donors at a higher frequency than in HBsAg-positive donors,and investigated the effect of these pre-S mutations on HBsAg and the specific molecular mechanisms involved in the occurrence of OBI by series of cellular and animal experiments.There were 307(82.97%)and 293(79.19%)samples sequenced successfully in the OBI group and HBsAg-positive control group,respectively;the sequence analysis results showed that the pre-S mutation frequencies of OBI samples were higher than those of HBsAg-positive samples in both genotypes B and C.By transfecting the constructed preS high-frequency mutation plasmids into Huh-7 cells,it was found that F25L,T68I,S78N,N98T and D133N mutations in genotype B and T97A,Q104K/R,Q121K and R135K mutations in genptype C could significantly reduce HBsAg production and might be associated with the occurrence of OBI.Mechanistically,the T68I,S78N and N98T mutations(genotype B)located in the pre-S2/S promoter region were found to significantly reduce pre-S2/S promoter activity;the qPCR results also confirmed that these three mutations significantly decreased the level of 2.1 kb RNA in transfected cells.Chromatin immunoprecipitation assays showed that the T68I and S78N mutations hindered the binding of Spl transcription factor to the pre-S2/S promoter,and the N98T mutation inhibited the binding of NFY transcription factor to the promoter,and Western blot results showed that Sp1 and NFY were able to promote HBsAg expression.Therefore,these above results suggest that the T68I,S78N and N98T mutations lead to reduced promoter activity and reduced 2.1kb RNA levels by inhibiting the binding of the corresponding transcription factors to the pre-S2/S promoter,resulting in decreased HBsAg synthesis and the occurrence of OBI.The effects of these three mutations on HBsAg were also confirmed in animal experiments.In genotype C,only the Q121K mutation mildly reduced pre-S2/S promoter activity,but did not significantly affect the 2.1 kb RNA levels.In addition,pre-S mutations in both genotype B and C,which caused reduced HBsAg levels,did not significantly affect HBsAg degradation,suggesting that pre-S mutations promoting the occurrence of OBI may not be related to this pathway.Finally,this study found that OBIassociated S78N and N98T mutations(genotype B)and Q121K mutation(genotype C)could lead to elevated intracellular large surface protein ratios,activate Akt and promote cell proliferation,which may be associated with hepatocellular carcinogenesis and of potential clinical significance.In conclusion,this study systematically analyzed the role of pre-S mutations in the occurrence of OBI and its molecular mechanism,which provides a new perspective to clarify the occurrence and development of OBI and also provides some research basis for an in-depth understanding of HBV infection.