Thursday, November 21, 2024
11/21/2024
ABSTRACT Chronic infection with hepatitis B virus (HBV) is a leading cause of liver cancer. HBV produces co-terminal large (L), middle (M), and small (S) envelope proteins containing preS1+preS2+S, preS2+S, and S domain alone, respectively. Central S domain and N-terminal preS1 domain respectively mediate virion attachment to HSPG and NTCP, the low- and high-affinity HBV receptors. Corresponding antibodies can neutralize HBV infectivity, although how anti-preS2 antibodies neutralize HBV infectivity remains enigmatic. Intriguingly, preventing M protein expression or artificially deleting most part of the preS2 domain does not impair HBV virion production or viral infectivity in cell culture, and late stage of chronic HBV infection often selects for point mutation of preS2 ATG codon to abolish M protein expression, or in-frame deletions to remove N-terminal preS2 domain. Such deletions are much more prevalent in liver cancer patients than those with mild liver diseases, but the driving force remains unclear. This R21 exploratory grant application aims to characterize the biological properties of such naturally occurring and clinically important HBV mutants. We hypothesize that 1) M-minus mutants and preS2 deletion mutants remain infectious; 2) preS2 deletions escape neutralization by anti-preS2 antibodies, which can work through binding to preS2 domain on either L or M protein; 3) consequently M-minus mutants partially escape anti-preS2 antibodies. Two specific aims are proposed to critically test our working hypotheses. Aim 1 will characterize clinically important M-minus and preS2 deletion mutants and establish their infectivity. The naturally occurring mutations will be introduced to an infectious clone of genotype C and genotype D, respectively, and viral particles generated from transfected HepG2 cells will be inoculated to HepG2/NTCP cells and differentiated HepaRG cells to test their infectivity. Aim 2 will determine susceptibility of such M-minus and preS2 deletion mutants to neutralizing antibodies and establish whether anti-preS2 antibodies work through L, M, or either protein. We will abolish M protein expression by mutation of preS2 ATG codon and enhance its expression via optimized Kozak sequence. In addition, viral particles containing neutralization escaping preS2 deletion in L, M, or both proteins will be generated by co-transfection experiments and their susceptibility to anti-preS2 antibodies determined. If the proposed studies reveal that anti-preS2 antibodies can neutralize HBV infectivity through M protein, then an additional host factor might be involved in infection by wild-type HBV. In this regard polymerized human serum albumin can bind to the preS2 domain on M protein, which was proposed over 30 years ago as an HBV entry mechanism.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
