title: Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA.
abstract: As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels.
  
  
  
  Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed.
  
  
  
  Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69∗), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69∗, and sG145R by co-expression of wild-type HBsAg.
  
  
  
  The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements.
  
  
  
  The hepatitis B surface antigen (HBsAg) is a major viral protein of the hepatitis B virus (HBV) secreted into patient blood serum and its quantification value serves as an important marker for the evaluation of chronic HBV infection and antiviral response. We found a few new amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These different substitutions might impair virion secretion, change the ability of HBsAg to bind to antibodies, or impact HBV replication. These could all result in decreased detectable levels of serum HBsAg. The factors affecting circulating HBsAg level and HBsAg detection are varied and caution is needed when interpreting clinical significance of serum HBsAg levels. Clinical trial number: NCT01088009.
authors.affiliation: Department of Microbiology and Center of Infectious Disease, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China; Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Kuan-Hui
authors.initials: KH
authors.lastname: Xiang
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Eleftherios
authors.initials: E
authors.lastname: Michailidis
authors.affiliation: Department of Microbiology and Center of Infectious Disease, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Hai
authors.initials: H
authors.lastname: Ding
authors.affiliation: Department of Microbiology and Center of Infectious Disease, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China; Department of Clinical Laboratory, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
authors.forename: Ya-Qin
authors.initials: YQ
authors.lastname: Peng
authors.affiliation: Department of Microbiology and Center of Infectious Disease, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Ming-Ze
authors.initials: MZ
authors.lastname: Su
authors.affiliation: Department of Microbiology and Center of Infectious Disease, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Microbiology and Center of Infectious Disease, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Xue-En
authors.initials: XE
authors.lastname: Liu
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Viet Loan
authors.initials: VL
authors.lastname: Dao Thi
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Xian-Fang
authors.initials: XF
authors.lastname: Wu
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, USA.
authors.forename: William M
authors.initials: WM
authors.lastname: Schneider
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rice
authors.affiliation: Department of Microbiology and Center of Infectious Disease, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China. Electronic address: zhuangbmu@126.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhuang
authors.affiliation: Department of Microbiology and Center of Infectious Disease, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China. Electronic address: tongli08@vip.sina.com.
authors.forename: Tong
authors.initials: T
authors.lastname: Li
mesh_terms: D000328:Adult; D019943:Amino Acid Substitution; D004279:DNA, Viral / Q000032:analysis / Q000097:blood; D005260:Female; D006514:Hepatitis B Surface Antigens / Q000097:blood / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification; D019694:Hepatitis B, Chronic / Q000175:diagnosis / Q000821:virology; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D014764:Viral Proteins / Q000235:genetics; D014771:Virion / Q000235:genetics / Q000302:isolation & purification; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D014764:Viral Proteins
keywords: Amino acid substitution; Antigenicity; DNA; HBV; HBsAg mutation; Hepatitis B surface antigens; Membrane proteins; Viral; Virion secretion; ‘a’ determinant