Encapsidation studies of poliovirus subgenomic replicons

The inclusion of a foreign marker gene, chloramphenicol acetyltransferase (CAT) gene, into the poliovirus genome allows its replication and encapsidation to be easily monitored using a simple enzyme assay. Such poliovirus replicons require the presence of helper virus for their successful propagation and thus are similar to defective interfering (DI) viruses. In genomes containing the CAT gene, the majority of the P1 virus capsid region of the poliovirus genome could be removed without destroying viability. The smallest replicon was significantly smaller than any naturally occurring DI particle so far reported, yet it retained the ability to replicate and be encapsidated by structural proteins provided by helper virus in trans. The efficiency with which the replicons were encapsidated was investigated using a direct immunostaining technique that allows individual cells infected with either a replicon or helper virus to be quantified. These results were compared to the frequencies of trans-encapsidation of polioviruses and coxsackievirus B4 using a two-stage neutralization assay. Poliovirus types 1, 2 and 3 but not coxsackievirus B4, coxsackievirus A21 or rhinovirus 14 provided efficient trans-encapsidation of poliovirus type 3 or type 3-derived replicons. These results suggest that a specific encapsidation process operates and that it does not involve RNA sequences within the region of the genome encoding the capsid proteins.