Hepatitis A Virus Adaptation to Cellular Shutoff Is Driven by Dynamic Adjustments of Codon Usage and Results in the Selection of Populations with Altered Capsids

Abstract

Hepatitis A virus (HAV) has a highly biased and deoptimized codon usage compared to the host cell and fails to inhibit host protein synthesis. It has been proposed that an optimal combination of abundant and rare codons controls the translation speed required for the correct capsid folding. The artificial shutoff host protein synthesis results in the selection of variants containing mutations in the HAV capsid coding region critical for folding, stability, and function. Here, we show that these capsid mutations resulted in changes in their antigenicity; in a reduced stability to high temperature, low pH, and biliary salts; and in an increased efficacy of cell entry. In conclusion, the adaptation to cellular shutoff resulted in the selection of large-plaque-producing virus populations. IMPORTANCEHAV has a naturally deoptimized codon usage with respect to that of its cell host and is unable to shut down the cellular translation. This fact contributes to the low replication rate of the virus, in addition to other factors such as the highly inefficient internal ribosome entry site (IRES), and explains the outstanding physical stability of this pathogen in the environment mediated by a folding-dependent highly cohesive capsid. Adaptation to artificially induced cellular transcription shutoff resulted in a redeoptimization of its capsid codon usage, instead of an optimization. These genomic changes are related to an overall change of capsid folding, which in turn induces changes in the cell entry process. Remarkably, the adaptation to cellular shutoff allowed the virus to significantly increase its RNA uncoating efficiency, resulting in the selection of large-plaque-producing populations. However, these populations produced much-debilitated virions.

Molecular Basis of the Behavior of Hepatitis A Virus Exposed to High Hydrostatic Pressure

“…Five HAV populations derived from the cell-adapted pHM175 43c strain were used throughout the present study (15): L0 (parental type), F0.05A (population adapted to low levels of cellular shutoff), F0.05LA (population long adapted to low levels of cellular shutoff), F0.2A (population adapted to high levels of cellular shutoff), and F0.2LA (population long adapted to high levels of cellular shutoff). The physical, antigenic, and biological properties of these virus populations have been previously described (15). Virus stocks of these populations were titrated [50% tissue culture infective dose(s) (TCID 50 )] in the FRhK-4 cell line as previously described (15).…”

“…The physical, antigenic, and biological properties of these virus populations have been previously described (15). Virus stocks of these populations were titrated [50% tissue culture infective dose(s) (TCID 50 )] in the FRhK-4 cell line as previously described (15).…”

“…Suspensions with high virus titers were used in the enzyme-linked immunosorbent assays (ELISAs) and in the sucrose gradients. Concentrated viral stocks were obtained as previously described (15). At 5 to 6 days postinfection, FRhK-4-infected cells (multiplicity of infection of 1) from a T-175 flask were trypsin treated, collected by low-speed centrifugation at 1,000 ϫ g, resuspended in 500 l of NT buffer (0.1 M NaCl, 10 mM Tris-HCl, 1% NP-40 [pH 7.4]), and incubated for 30 min at room temperature.…”

“…Inhibition of cellular mRNA synthesis may be achieved by treating cells with actinomycin D, which specifically inhibits the cellular DNA-dependent RNA polymerases with no effect on viral RNA-dependent RNA polymerases. Under these conditions, tRNA pools available for HAV translation are modified (14,15). Adaptation of HAV to actinomycin D-induced cellular shutoff resulted in the selection of mutant virus populations with subtle capsid-folding changes, induced by adjustments of their codon usage, which have profound influences in capsid biological functions such as cell binding and genome uncoating (15).…”

“…Under these conditions, tRNA pools available for HAV translation are modified (14,15). Adaptation of HAV to actinomycin D-induced cellular shutoff resulted in the selection of mutant virus populations with subtle capsid-folding changes, induced by adjustments of their codon usage, which have profound influences in capsid biological functions such as cell binding and genome uncoating (15). These newly generated capsids showed a completely different phenotype and became highly sensitive to high temperature, low pH and bile salts (15).…”