FUNCTIONAL CDNA CLONES OF THE HUMAN RESPIRATORY SYNCYTIAL (RS) VIRUS N-PROTEINS, P-PROTEINS, AND L-PROTEINS SUPPORT REPLICATION OF RS VIRUSGENOMIC RNA ANALOGS AND DEFINE MINIMAL TRANS-ACTING REQUIREMENTS FOR RNA REPLICATION

The RNA-dependent RNA polymerase of human respiratory syncytial (RS) v irus was expressed in a functional form from a cDNA clone. Coexpressio n of the viral polymerase (L) protein, phosphoprotein (P), and nucleoc apsid (N) protein allowed us to develop a system for expression and re covery of replicable RS virus RNA entirely from cDNA clones. cDNA clon es of the N, P, and L genes were constructed in pGEM-based expression plasmids and shown to direct expression of the appropriate polypeptide s. Two types of RS virus genomic RNA analogs were expressed from an in tracellular 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' te rminus and its complement at the 3' terminus as found in copyback defe ctive interfering RNAs of other negative-strand RNA viruses. Both type s of genomic analogs were encapsidated and replicated in cells express ing the RS virus N, P, and L proteins. Omission of any of the three vi ral proteins abrogated replication, thereby defining the N, P, and L p roteins as the minimal trans-acting proteins required for RNA replicat ion. 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 R S virus RNA.