Characterization of Replication-competent Hepatitis A Virus Constructs Containing Insertions at the N Terminus of the Polyprotein

To determine whether hepatitis A virus (HAV) could tolerate the insertion of exogenous sequences, we constructed full-length HAV cDNAs containing in-frame insertions at the N terminus of the polyprotein and transfected the derived T7 RNA polymerase in vitro transcripts into FRhK-4 cells. Replication of HAVvec1, a construct containing an insertion of 60 nucleotides coding for a polylinker, a 2B/2C cleavage site for HAV protease 3Cpro, and two initiation codons that restored the sequence of the N terminus of the polyprotein, was detected 2 weeks after transfection by indirect immunofluorescence analysis using anti-HAV monoclonal antibodies. Western blot analysis of HAVvec1-infected cells using anti-VP2 and anti-VP4 antibodies failed to detect the expression of the inserted sequences. Insertion of a 24-mer oligonucleotide coding for a FLAG epitope into HAVvec1 resulted in its HAV-mediated expression which was retained upon deletion of a Gln residue from the inserted 2B/2C cleavage site. Western blot analysis using anti-FLAG and anti-VP2 antibodies showed that the FLAG epitope accumulated in infected cells fused to VP0. Replacement of the FLAG epitope with an epitope of the circumsporozoite protein (CSP) of Plasmodium falciparum resulted in its stable HAV-mediated expression for at least six serial passages in FRhK-4 cells. Sedimentation analysis in sucrose density gradients showed that the CSP epitope accumulated in infected cells fused to VP0, forming 80S empty capsids which also contained native VP0. Our data suggest that the HAV internal ribosome entry site can efficiently direct dual initiation of translation of the polyprotein from AUG codons separated by 66 to 78 nucleotides and show that HAV can tolerate insertions at the N terminus of the polyprotein.