Importance of amino acid 216 in nonstructural protein 2B for replication of hepatitis A virus in cell culture and in vivo

Abstract

Clinical isolates of hepatitis A virus (HAV) replicate inefficiently in cell culture unless mutations are acquired throughout the genome. An Ala-to-Val substitution in the nonstructural protein 2B (2B-216) was known to have a major impact on replication in cell culture. Analysis of chimeric viruses confirmed that the 2B-A[216]V change was critical for efficient replication and that Leu or Ile could substitute for Val. Viruses containing Val, Ile, or Leu at 2B-216 all replicated with similar kinetics in cell culture, whereas the virus containing Ala at this position grew 10- to 20-fold less efficiently. In contrast, in vivo, virus with either Ala or Val at 2B-216 replicated equally efficiently when tested in a chimpanzee and in tamarins, and each amino acid was stably maintained. Attempts to complement wild-type 2B in trans with adapted 2B provided by co-infection with a second viable HAV mutant failed to enhance replication of the virus containing the wild-type 2B sequence.

A simple and rapid Hepatitis A Virus (HAV) titration assay based on antibiotic resistance of infected cells: evaluation of the HAV neutralization potency of human immune globulin preparations

“…To develop a simple and rapid titration method, we used a wild type HAV construct containing a Bsd resistance gene inserted into the 2A-2B junction [ 24 ]. In addition to this selectable marker, this HAV-Bsd construct contained an Ala-to-Val substitution at amino acid 216 of the 2B protein (Figure 1 ) that enhanced its growth in cell culture but did not attenuate the virus [ 25 ]. HAV-Bsd grew efficiently in Huh7-A-I cells, a clone of human hepatoma Huh7 cells that supports the stable growth of wt HAV [ 24 ].…”

Stable Growth of Wild-Type Hepatitis A Virus in Cell Culture

“…A mutant of the wt HM-175 strain of HAV containing only an Ala-to-Val change at position 216 of protein 2B (2B/A216V), termed HAV8Y, was rescued from Huh7 cells transfected with in vitro SP6 polymerase transcripts from pHAV8Y (11,13,22). Attenuated and cell cultureadapted HAV/7 was rescued from FRhK-4 cells transfected with in vitro SP6 polymerase transcripts from pHAV/7 (5-7).…”

The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

“…Since HAV is a slow growing virus, the suppression of innate immune responses through non-structural proteins like 2B is probably instrumental in allowing the virus to maintain replication 11 12 13 . Also, mutations in 2B appear to be essential in allowing cell culture adaptation of HAV 14 15 . A single mutation at alanine 216 in 2B, converting it into any hydrophobic amino acid, can increase virus yield by 10–20 fold 15 .…”

“…Also, mutations in 2B appear to be essential in allowing cell culture adaptation of HAV 14 15 . A single mutation at alanine 216 in 2B, converting it into any hydrophobic amino acid, can increase virus yield by 10–20 fold 15 . There is no mechanistic information about how this point mutation engineers such a large increase in virus production.…”