Abstract
The RNA-dependent RNA polymerase of human respiratory syncytial (RS) virus was expressed in a functional form from a cDNA clone. Coexpression of the viral polymerase (L) protein, phosphoprotein (P), and nucleocapsid (N) protein allowed us to develop a system for expression and recovery of replicable RS virus RNA entirely from cDNA clones. cDNA clones of the N, P, and L genes were constructed in pGEM-based expression plasmids and shown to direct expression of the appropriate polypeptides. Two types of RS virus genomic RNA analogs were expressed from an intracellular transcription plasmid that directed the synthesis of RNAs with defined 5' and 3' ends. One analog included the authentic 5' and 3' termini of the genome, and the second contained the authentic 5' terminus and its complement at the 3' terminus as found in copyback defective interfering RNAs of other negative-strand RNA viruses. Both types of genomic analogs were encapsidated and replicated in cells expressing the RS virus N, P, and L proteins. Omission of any of the three viral proteins abrogated replication, thereby defining the N, P, and L proteins as the minimal trans-acting proteins required for RNA replication. This system has the advantages that expression occurs at a level sufficient to allow direct biochemical analysis of the products of RNA replication and that neither the use of reporter genes nor wild-type RS helper virus is required. These features allow analysis of both cis- and trans-acting factors involved in the control of replication of RS virus RNA.