Determinants in 3Dpol modulate the rate of growth of hepatitis A virus

Abstract

Hepatitis A virus (HAV), an atypical member of the Picornaviridae, grows poorly in cell culture. To define determinants of HAV growth, we introduced a blasticidin (Bsd) resistance gene into the virus genome and selected variants that grew at high concentrations of Bsd. The mutants grew fast and had increased rates of RNA replication and translation but did not produce significantly higher virus yields. Nucleotide sequence analysis and reverse genetic studies revealed that a T6069G change resulting in a F42L amino acid substitution in the viral polymerase (3D(pol)) was required for growth at high Bsd concentrations whereas a silent C7027T mutation enhanced the growth rate. Here, we identified a novel determinant(s) in 3D(pol) that controls the kinetics of HAV growth.

FIG. 1.

Construction and rescue of cell culture-adapted HAV containing a Bsd selectable marker. (A) Schematic representation of HAV constructs. Synthetic oligonucleotides containing SalI, SnaBI, and KpnI restriction sites flanked by three G codons (Gly hinges) and HAV 3C^pro cleavage sites were cloned into the 2A-2B junction of the full-length infectious cDNA of cell culture-adapted strain HM-175 of HAV in pT7HAV (29). This construct, pHAVvec9, was cleaved with SalI and KpnI to insert a gene coding for Bsd-deaminase lacking translation initiation and termination codons (16) to generate pHAVvec9-Bsd. (B) Immunofluorescence (IF) analysis of HAV-infected cells. HAV/7, HAVvec9, and HAVvec9-Bsd were rescued from FRhK4 cells transfected with in vitro-synthesized HAV transcripts (16) from pT7HAV, pHAVvec9, and pHAVvec9-Bsd, respectively. FRhK4 cells were infected with virus stocks for 1 week, fixed, and stained with neutralizing anti-HAV MAb K2-4F2 as described previously (16). (C) Titration of HAV stocks. Tenfold serial dilutions of virus stocks were titrated in 96-well plates containing monolayers of African green monkey kidney cells. Plates were incubated for 2 weeks at 35°C, and viral titers were determined by an endpoint dilution enzyme-linked immunosorbent assay (ELISA) using rabbit anti-HAV antibodies as described previously (16). Viral titers and standard deviations, shown as bars, were calculated using the ID50 program developed by John L. Spouge (National Center for Biotechnology Information, NIH).

FIG. 2.

Selection of HAVvec9-Bsd variants under increasing concentrations of Bsd. (A) FRhK4 cells infected with HAVvec9-Bsd survive selection with 1 μg/ml Bsd. Subconfluent monolayers of FRhK4 cells were infected with HAV/7, HAVvec9, or HAVvec9-Bsd at an MOI of 1 TCID₅₀/cell or mock infected and grown in a selection medium containing 1 μg/ml Bsd (16). Phase-contrast micrographs were taken in a Zeiss Axiscope microscope at ×200 magnification. (B) Schematic representation of the selection of HAVvec9-Bsd variants under increasing concentrations of Bsd. Serial passages of HAVvec9-Bsd in FRhK4 cells were performed under increasing concentrations of Bsd. To do so, FRhK4 cells were infected with HAVvec9-Bsd at an MOI of 1 TCID₅₀/cell and incubated for 6 weeks in the presence of Bsd. Viral stocks were prepared and used to infect naive FRhK4 cells at the following higher concentration of Bsd. Passages were performed at 1, 5, and 20 μg/ml Bsd, and the viral variants that grew at each concentration of the antibiotic were termed HAVvec9-Bsd, -5, and -20, respectively. (C) Growth of FRhK4 cells infected with fr HAV variants in the presence of different concentrations of Bsd. Subconfluent monolayers of FRhK4 cells in six-well plates were infected with HAVvec9-Bsd, -5, or -20 at an MOI of 1 TCID₅₀/cell or mock infected and selected with 1, 5, and 20 μg/ml Bsd. After 1 week of incubation at 35°C, cells were fixed with 5% trichloroacetic acid, stained with crystal violet, and photographed. Each image represents a well of the six-well plate. Cells that survived antibiotic selection and formed monolayers or grew in colonies were stained with the dye (dark areas). (D) One-step growth curve analysis of cells infected with HAVvec9-Bsd variants. FRhK4 cells were infected with an MOI of 2 to 5 TCID₅₀/cell of HAVvec9-Bsd, -5, or -20 in the presence of 2 μg/ml Bsd. At different times postinfection, virus stocks were prepared and titrated in 96-well plates containing FRhK4 cell monolayers in the presence of 2 μg/ml Bsd as described previously (17). Plates were observed under the microscope 8 days postinfection, and wells containing surviving cells were counted as positive. Viral titers and standard deviations, shown as error bars, were calculated using the ID50 program. (E) IF analysis of cells infected with HAV variants. IF analysis of FRhK4 cells mock infected or infected with HAVvec9-Bsd or HAVvec9-Bsd-20 for 4 days in the presence of 2 μg/ml Bsd was performed as described for Fig. 1B.

FIG. 3.

Characterization of HAVvec9-Bsd variants. (A) Analysis of HAV RNA replication by quantitative RT-PCR. Total RNA was extracted from FRhK4 cells infected with an MOI of 2 to 5 TCID₅₀/cell of HAVvec9-Bsd or -20 at different times postinfection. HAV RNA was quantitated by real-time RT-PCR as described previously (24). The HAV RNA copy numbers were determined in triplicate and expressed as mean values with standard deviations shown as error bars. Statistical significance at 2 days postinfection was determined by t test. (B) Western blot analysis of HAV-infected cells. FRhK4 cells were infected with an MOI of 2 to 5 TCID₅₀/cell of HAVvec9-Bsd or -20, cell extracts were prepared at different dpi, and equal amounts of extracts were resolved by 4 to 12% SDS-PAGE under denaturing conditions, transferred to polyvinylidene difluoride (PVDF) membranes, and probed with rabbit anti-HAV VP2 antibodies as described previously (29). Bands corresponding to VP0 and VP2 specifically detected by the anti-VP2 antibodies are marked with arrows (upper panel). Bands corresponding to a constitutive FRhK4 cellular protein that cross-reacted with the anti-VP2 antibodies are shown as loading controls (lower panel). Molecular size markers are indicated in kilodaltons. (C) Mutations in 3D^pol that increased the growth kinetics of the HAVvec9-Bsd variants. The T6069G, mutation, the C7027T mutation, or both mutations were introduced into the infectious cDNA of HAVvec9-Bsd to generate HAVvec9-Bsd-6069, HAVvec9-Bsd-7027, and HAVvec9-Bsd-6069 + 7027, respectively. One-step growth curve analysis of constructs and variants in FRhK4 cells was done as described for Fig. 2D. (D) Alignment of 3D^pol N-terminal 69-amino-acid sequences of HAV and other members of the Picornaviridae. Single-letter amino acid symbols of 3D^pol from coxsackievirus (CV) B3, poliovirus (PV), human rhinovirus (HRV), foot-and-mouth virus (FMDV), and hepatitis A virus (HAV), corresponding to accession numbers AAW83322, NP_740478, 1XR7B, CAA59731, and AAA45466, respectively, were aligned using the Clustal W program, which introduced gaps in the sequence shown as dashes. Conserved KT and PAA/V motifs are shown in blue, and the HAV F42, which changed to L due to the T6069G mutation, is shown in magenta. (E) Space-filling model of the poliovirus 3D^pol (1RA6) (26) showing amino acids F30 (red), F34 (green), K44 (magenta), and G64 (yellow) and the remaining N-terminal 69 amino acids that from the “index finger” in blue.