Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. LAY SUMMARY: 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.

A resonance Raman spectroscopic and CASSCF investigation of the Franck-Condon region structural dynamics and conical intersections of thiophene.

Resonance Raman spectra were acquired for thiophene in cyclohexane solution with 239.5 and 266 nm excitation wavelengths that were in resonance with ∼240 nm first intense absorption band. The spectra indicate that the Franck-Condon region photodissociation dynamics have multidimensional character with motion mostly along the reaction coordinates of six totally symmetry modes and three nontotally symmetry modes. The appearance of the nontotally symmetry modes, the C-S antisymmetry stretch +C-C=C bend mode ν(21)(B(2)) at 754 cm(-1) and the H(7)C(3)-C(4)H(8) twist ν(9)(A(2)) at 906 cm(-1), suggests the existence of two different types of vibronic-couplings or curve-crossings among the excited states in the Franck-Condon region. The electronic transition energies, the excited state structures, and the conical intersection points (1)B(1)/(1)A(1) and (1)B(2)/(1)A(1) between 2 (1)A(1) and 1 (1)B(2) or 1 (1)B(1) potential energy surfaces of thiophene were determined by using complete active space self-consistent field theory computations. These computational results were correlated with the Franck-Condon region structural dynamics of thiophene. The ring opening photodissociation reaction pathway through cleavage of one of the C-S bonds and via the conical intersection point (1)B(1)/(1)A(1) was revealed to be the predominant ultrafast reaction channel for thiophene in the lowest singlet excited state potential energy hypersurface, while the internal conversion pathway via the conical intersection point (1)B(2)/(1)A(1) was found to be the minor decay channel in the lowest singlet excited state potential energy hypersurface.