Replication of a hepatitis A virus replicon detected by genetic recombination in vivo

Unlike other picornaviruses, hepatitis A virus (HAV) replicates so inefficiently in cell culture that the study of its RNA biosynthesis presents a major experimental challenge. To assess viral RNA replication independent of particle formation, a subgenomic replicon representing a self-replicating RNA was constructed by replacing the P1 domain encoding the capsid proteins with the firefly luciferase sequence. Although translation of the HAV replicon was as efficient as a similar poliovirus replicon, the luciferase activity derived from replication of the HAV construct was more than 100-fold lower than that of poliovirus. The replication capacity of the HAV replicon was clearly demonstrated by its ability to recombine genetically with a non-viable, full-length HAV genome that served as capsid donor and thus to rescue a fully infectious virus. In contrast to a replication-deficient replicon, co-expression of the genetically marked and replication-competent HAV replicon with several lethally mutated HAV genomes resulted in the successful rescue of infectious HAV with a unique genetic marker. Our data suggest: (i) that autonomous HAV RNA replication does not require sequences for the HAV structural proteins; and (ii) that low-level genome replication can unequivocally be demonstrated by the rescue of infectious virus after co-expression with nonviable genomes.