Characterization of recombinant hepatitis A virus genomes containing exogenous sequences at the 2A/2B junction

Abstract

Hepatitis A virus (HAV) differs from other members of the family Picornaviridae in that the cleavage of the polyprotein at the 2A/2B junction, commonly considered to be the primary polyprotein cleavage by analogy with other picornaviruses, is mediated by 3C(pro), the only proteinase encoded by the virus. However, it has never been formally demonstrated that the 2A/2B junction is the site of primary cleavage, and the actual function of the 2A sequence, which lacks homology with sequence of other picornaviruses, remains unknown. To determine whether 2A functions in cis as a precursor with the nonstructural proteins, we constructed dicistronic HAV genomes in which a heterologous picornaviral internal ribosome entry site was inserted at the 2A/2B junction. Transfection of permissive FRhK-4 cells with these dicistronic RNAs failed to result in the rescue of infectious virus, indicating a possible cis replication function spanning the 2A/2B junction. However, infectious virus was recovered from recombinant HAV genomes containing exogenous protein-coding sequences inserted in-frame at the 2A/2B junction and flanked by consensus 3C(pro) cleavage sites. The replication of these recombinants was less efficient than that of the parent virus but was variable and not dependent upon the length of the inserted sequence. An HAV recombinant containing a 420-nt insertion encoding the bleomycin resistance protein Zeo was stable for up to five passages in cell culture. Inserted sequences were deleted from replicating viruses, but this did not result from homologous recombination at the flanking 3C(pro) cleavage sites, since the 5' and 3' segments of the inserted sequence were retained in the deletion mutants. These results indicate that the HAV polyprotein can tolerate an insertion at the 2A/2B junction and that the 2A polypeptide does not function in cis as a 2AB precursor. Recombinant HAV genomes containing foreign protein-coding sequences inserted at the 2A/2B junction are novel and potentially useful protein expression vectors.

FIG. 1

Schematic representation of the genetic organization of HAV dicistronic genomes. Inserted foreign nucleotide sequences from either EMCV (p18f2A-E-2B) or Rhinovirus (p18f2A-Rh-2B) IRESs are shown below the scheme of dicistronic HAV genomes interrupted at the 2A/2B junction. Positions of HAV and EMCV or rhinovirus nucleotides within respective genomes are indicated above the sequences, and encoded amino acids, when appropriate, are shown below the nucleotide sequences. Stop and initiation codons are indicated in an open font.

FIG. 2

In vitro translation and processing of polyproteins synthesized from dicistronic transcript p18f2A-E-2B. (A). Coupled TNT transcription-translation reactions (Promega) were programmed with 1 μg of HaeII-linearized plasmids containing either full-length parental HAV cDNA (p 18f) or full-length HAV dicistronic cDNA (p18f2A-E-2B). [³⁵S]methionine-labeled HAV proteins were separated by SDS–10% PAGE either directly (lanes 0) or following immunoprecipitation with anti-HAV capsid (anti-caps), anti-HAV 2C (anti-2C), or anti-HAV 3C (anti-3C) antibodies. Positions of molecular mass standards and of precursors and mature HAV polypeptides are indicated. (B) Rabbit reticulocyte lysate reactions were programmed with 100 ng of either full-length parental HAV transcript (p18f) or full-length HAV dicistronic transcript (p18f2A-E-2B). [³⁵S]methionine-labeled HAV proteins were separated by SDS–10% PAGE. Positions of molecular mass standards are indicated on the left of the panel, and positions of precursors and mature HAV polypeptides, as determined after immunoprecipitation with appropriate antibodies (not shown), are indicated on each side of the panel.

FIG. 3

Genetic organization and RIFA of HAV recombinants containing insertions at the 2A/2B junction. The genetic organization of the genome of parental p18f and of HAV recombinants bearing insertions at the 2A/2B junction of foreign sequences coding for either the Zeo sequence (p18f2A-Zeo-2B), EGFP (p18f2A-EGFP-2B), or Rluc (p18f2A-Rluc-2B) is schematically shown. The 3C^pro cleavage sites and Gly-Gly-Gly hinges, as well as the position of unique restriction sites XbaI and BamHI, are indicated. Petri dish cultures of BS-C-1 cells were infected with 2-week-old lysates of FRhK-4 cells transfected with either parental or recombinant synthetic genome-length RNA and maintained for 1 week at 37°C before being processed for detection of HAV radioimmunofoci as described in Materials and Methods.

FIG. 4

Retention of the Zeo gene in v18f2A-Zeo-2B after two passages in BS-C-1 cells. (A) The genome of either the parent (v18f) or v18f-Zeo-2B (18f-Zeo) recombinant virus, after two successive passages in BS-C-1 cells, was analyzed by RT-PCR using two different pairs of primers as schematically depicted. The left panel represents RT-PCR products obtained using oligonucleotide 1, which is derived from the Zeo sequence, and HAV-specific oligonucleotide 3. The right panel shows RT-PCR products obtained using two HAV-specific oligonucleotides, 2 and 3, spanning the Zeo insertion. Fragments corresponding to v18f and 18f-Zeo sequences are indicated by an arrowhead. (B) Viral RNA isolated after two passages from BS-C-1 cells infected with either v18f or v18f2A-Zeo-2B (18f-Zeo) was bound to nitrocellulose and probed with a ³²P-labeled probe specific either for the Zeo sequence or for HAV RNA.

FIG. 5

The recombinant HAV v18f2A-Zeo-2B shows no defect in HAV polyprotein processing. Cytoplasmic extracts at 72 h p.i. from uninfected, v18f-infected, or v18f2A-Zeo-2B (18f-Zeo)-infected FRhK-4 cells were prepared and were separated by SDS–12% PAGE. HAV polypeptides were identified by immunoblotting using anti-VP1 (A), anti-VP2 (B), or anti-2B (C) antibodies. Positions of molecular weight markers (M) and relative positions of HAV polypeptides are shown on the left and the right of each panel, respectively.

FIG. 6

Immunofluorescence analysis of v18f- and v18f2A-Zeo-2B-infected FRh-K4 cells. Monolayers of FRhK-4 cells grown in four well chamber-slides were either infected with v18f (A) or v18f2A-Zeo-2B (B and D) or mock infected (C and E), fixed in acetone at 4 days p.i., and incubated with the anti-HAV monoclonal antibody K3.2F2 (A, B, and C) or a rabbit polyclonal anti-Zeo antibody (D and E). Specific antigens were visualized using fluorescein isothiocyanate-conjugated rabbit anti-mouse or goat anti-rabbit antisera.

FIG. 7

Analysis of the stability of recombinant HAV genomes. BS-C-1 cells were mock infected or infected with either HAV parent v18f or v18f2A-Zeo-2B (18f-Zeo) (A) or v18f2A-EGFP-2B (18f-EGFP) or v18f2A-Rluc-2B (18f-Rluc) (B). The presence of foreign sequence insertions in the genomes of recombinant viruses obtained after six passages (A) or only one passage (B) in BS-C-1 cells was determined by RT-PCR using primers 2 and 3, as depicted in Fig. 4A. Molecular size markers (M) indicate relative mobilities. Fragments corresponding to the parent genome (v18f) are shown on the left, and fragments representing complete and partially deleted recombinant genomes are indicated on the right of each panel. Fragments noted with an asterisk were purified for sequence analysis (see Fig. 8).

FIG. 8

Nucleotide sequence analysis of revertant HAV recombinant genomes. RT-PCR products representing deletion events in the genomes of v18f2A-Zeo-2B (A) and v18f2A-Rluc-2B (B) (noted by an asterisk in Fig. 7) were gel purified and subjected directly to automated sequencing. The resulting sequences are shown in comparison to the input recombinant sequences. Sizes of the respective foreign sequences, as well as the size of the resulting deletion, are indicated.