Study On The Mechanism Of Hepatitis B Virus S Protein Mutation And Modification In The Occurrence Of Occult Infection

Occult hepatitis B virus infection(OBI)is a special form of HBV infection,which is barely identified in the clinical practice due to its particular serological status of HBsAg-/HBV DNA+.However,emerging evidence showed that OBI was associated with mild continuous liver necro-inflammation that may favor progression to cirrhosis and eventually constitute an important risk factor for hepatocellular carcinoma development.But the occurrence mechanism of OBI is largely unknown.To explore this question from the virus aspects,this study verified the potential role of mutations and modification of S protein in the generation of OBI.From January 2010 to December 2013,1261 HBsAg-/HBV DNA+plasma samples were screened in 29 blood centers from 19 provinces and shipped to our lab,from which 970 were confirmed as OBI.Information of host and viral markers were analyzed in overall and stratified according to serological status for the 970 OBIs.S sequences were obtained from 116/275(42%)OBI samples randomly selected and 530 HBsAg+samples from the same population as a control group.Above sequences were analyzed from two aspects of N-glycosylation mutations and amino acid substitutions in S protein transmembrane domains(TMDs).Candidate mutations were identified and employed to functional analysis,which were introduced in wt plasmids using site-directed mutagenesis to construct mutated plasmids.Wt and mutated plasmids were transfected and expressed in huh-7 cell lines and supernatant and cell lysis were collected and tested HBsAg,which were confirmed by western blot(WB).The majority of OBI donors were male(70%),39.7%were repeat donors,median age was 40 years old(range:18-60 years),and all had a normal ALT level(<50 IU/mL).Sixty-nine percent of OBI donors were with HBV DNA levels less than 12 IU/mL.Additional serological testing identified 659(68%)samples as anti-HBc only reactive,242(25%)carrying both anti-HBc and anti-HBs,and 69(7%)anti-HBs only reactive.Overall,higher frequencies of N-glycosylation mutations(40.7%vs 11.8%;P<0.001)and hydrophilic amino acid substitutions in TMDs(65%vs 25%;P<0.001)were observed in the S sequences of OBIs compared to non-OBI controls.Nine additional and 3 abolishing N-glycosylation mutations at 7 sites in major hydrophilic region(MHR)were identified as candidate for functional analysis.WB results showed that all additional mutations except for GSS112-114NAT introduced a new glycan successfully.On plasmids M88 and p38.?,there was no significant effects for all the mutations except for TCT123-125NCT/NFT.When it comes to plasmid M86,all additional mutations decreased extracellular(EC)HBsAg to 1/10 or even 1/1000 of wt level while mutations abolishing glycan at site 146 increased EC HBsAg to 10 times of wt level.The secretion efficiency(EC/IC ratio of HBsAg)had the same change.In addition,R substitutions at 7 sites within TMD1-3 were identified for functional analysis.The results showed that 4 R substitutions within TMD2 decreased EC and IC HBsAg to undetectable level while there was no significant HBsAg decrease for mutations within TMD1 and TMD3.In conclusion,additional N-glycosylation mutations and hydrophilic substitutions within TMD2 impaired HBsAg production,secretion and stability and led to extremly low or undetectable HBsAg in circular,which may contribute to the multifactorial occurrence of OBI.The present study provided new view and evidence for OBI occurrence mechanism,laying the foundation of further study of OBI.