Characterization of heteroclite subgenomic RNAs associated with PRRSV infection

In this study, porcine reproductive and respiratory syndrome virus (PRRSV) heteroclite (uncommon forms) RNAs were characterized. Nucleotide sequencing of 11 additional defective RNA species verified that heteroclites are formed between the 5' and 3' termini of PRRSV at short stretches of identity, with variability seen between the junction sites utilized. Northern blot and RT-PCR analyses indicated that heteroclite RNA species were likely to be packaged into purified virions. To study whether heteroclite RNAs and viral genomic RNAs could be packaged into the same virions, PRRSV strain VR-2332 was purified by sucrose density gradient centrifugation. RT-PCR amplification of the viral RNAs isolated from three distinct gradient bands, using genomic- and heteroclite-specific primer pairs, demonstrated that heteroclite RNAs could not be readily dissociated from genomic RNA. Partial segregation of full-length and larger heteroclite genomes to the upper two gradient bands was seen, but smaller species could be found in all three fractions. These results strongly suggest that heteroclite RNAs retain the PRRSV RNA packaging signal. In vitro transcription and translation of one heteroclite cDNA clone verified that the RNA could express a predicted 32.6 kDa protein, indicating that these RNA species have the potential to produce abnormal proteins in infected cells.