RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA.
Previously, a cDNA was constructed so that transcription by T7 RNA polymerase yielded a approximately 1-kb negative-sense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini. When transfected into RSV-infected cells, this minigenome was "rescued," as evidenced by high levels of CAT expression and the production of transmissible particles which propagated and expressed high levels of CAT expression during serial passage (P.L. Collins, M. A. Mink, and D. S. Stec, Proc. Natl. Acad. Sci. USA, 88:9663-9667, 1991). Here, this cDNA, together with a second one designed to yield an exact-copy positive-sense RSV-CAT RNA antigenome, were each modified to contain a self-cleaving hammerhead ribozyme for the generation of a nearly exact 3' end. Each cDNA was transfected into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase, together with plasmids encoding the RSV N, P, and L proteins, each under the control of a T7 promoter. When the plasmid-supplied template was the mini-antigenome, the minigenome was produced. When the plasmid-supplied template was the minigenome, the products were mini-antigenome, subgenomic polyadenylated mRNA and progeny minigenome. Identification of progeny minigenome made from the plasmid-supplied minigenome template indicates that the full RSV RNA replication cycle occurred. RNA synthesis required all three RSV proteins, N, P, and L, and was ablated completely by the substitution of Asn for Asp at position 989 in the L protein. Thus, the N, P, and L proteins were sufficient for the synthesis of correct minigenome and antigenome, but this was not the case for subgenomic mRNA, indicating that the requirements for RNA replication and transcription are not identical. Complementation with N, P, and L alone yielded an mRNA pattern containing a large fraction of molecules of incomplete, heterogeneous size. In contrast, complementation with RSV (supplying all of the RSV gene products) yielded a single discrete mRNA band. Superinfection with RSV of cells staging N/P/L-based RNA synthesis yielded the single discrete mRNA species. Some additional factor supplied by RSV superinfection appeared to be involved in transcription, the most obvious possibility being one or more additional RSV gene products.
Grosfeld
H
H
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Disease, Bethesda, Maryland 20892-0720, USA.
Hill
M G
MG
Collins
P L
PL
D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D002213:Capsid / Q000096:biosynthesis*; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis; D005822:Genetic Vectors; D016679:Genome, Viral; D015707:HN Protein; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection; D014616:Vaccinia virus; D014758:Viral Core Proteins / Q000096:biosynthesis*; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000096:biosynthesis*
D015707:HN Protein; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases