Rescue of synthetic analogs of genomic RNA and replicative-intermediate RNA of human parainfluenza virus type 3

Abstract

The genome of human parainfluenza virus type 3 (PIV3) is a single negative-sense RNA strand (vRNA) that is 15,463 nucleotides in length. A cDNA was constructed to encode an 898-nucleotide, internally deleted version of PIV3 vRNA, PIV3-CAT vRNA, in which the viral genes were replaced with the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. The CAT gene was flanked in turn by sequences representing (i) nontranslated sequences of the first and last genes in the PIV3 genome, (ii) PIV3 gene-start and gene-end sequences, which are presumed to be transcription signals, and (iii) 3' extracistronic (leader) and 5' extracistronic (trailer) terminal regions of PIV3 vRNA. A second cDNA was constructed to encode the exact complement of PIV3-CAT vRNA; this positive-sense RNA, PIV3-CAT vcRNA, would correspond to the predicted replicative intermediate of PIV3-CAT vRNA. When synthesized in vitro by runoff transcription with T7 RNA polymerase and transfected separately into PIV3-infected cells, both PIV3-CAT vRNA and vcRNA were rescued with similar efficiencies; that is, they were expressed to yield CAT and were packaged into particles that could be used to infect fresh cells. Rescue of PIV3-CAT vRNA was strictly dependent on complementation by PIV3; PIV3 could not be replaced by respiratory syncytial virus or, unexpectedly, by a bovine strain of PIV3. Passage was blocked by prior incubation with neutralizing monoclonal antibodies specific to the PIV3 attachment protein. Also, during nine serial passages, the expression of CAT by PIV3-CAT vRNA increased more than 3,000-fold. These results indicated that the 3'-terminal 111 nucleotides and the 5'-terminal 115 nucleotides of PIV3 vRNA, which are present in PIV3-CAT vRNA, contained all of the cis-acting RNA sequences required for replication, gene expression, and transmission.