Rescue of rubella virus replication-defective mutants using vaccinia virus recombinant expressing rubella virus nonstructural proteins.
The genome of rubella virus (RV) is translated into a polyprotein precusor, p200, of the nonstructural proteins (NSPs). This is proteolytically processed by a viral-encoded protease into two mature products, p150 and p90. p150 contains sequence corresponding to the predicted methyltransferase and protease activities, while p90 has sequence for the proposed helicase and RNA-dependent RNA polymerase activities. Processing of p200 is essential for RV viral replication. RV NSPs are responsible for viral RNA replication, in which a full-length negative-strand RNA serves as the intermediate for the replication of positive-strand genomic RNA and the transcription of subgenomic RNA. Previously we demonstrated that p200 synthesizes negative- but not positive-strand RNA, and that cleavage products p150/p90 are required for efficient production of positive-strand RNA. To determine whether p150 or p90 alone or together is involved in positive-strand RNA synthesis, vaccinia virus recombinants expressing individual NSPs were constructed and characterized. These were used in in vivo rescue experiments to complement replication-defective mutants in virus replication. A protease-inactive mutant was rescued by p200 or p150 provided in trans by using vaccinia virus recombinants. Thus this protease can function in trans. Rescue of cleavage-defective mutant by either p200 alone, or p150 plus p90 but not by p150 or p90 alone suggests that p150 and p90 function together as a replication complex in positive-strand RNA synthesis.
Wang
Xiaojie
X
Department of Pathology and Laboratory Medicine, University of British Columbia, BC Research Institute for Children's and Women's Health, 950 West 28th Avenue, Vancouver, BC, Canada.
Liang
Yuying
Y
Gillam
Shirley
S
D002460:Cell Line; D003673:Defective Viruses / Q000502:physiology*; D016679:Genome, Viral; D009154:Mutation; D010957:Plasmids; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D012412:Rubella virus / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
D012367:RNA, Viral; D017361:Viral Nonstructural Proteins