Restriction of a Sindbis virus mutant in BHK cells and relief of the restriction by the addition of adenosine.
SV(PZF) is a mutant of Sindbis virus (SV) which we selected on the basis of its ability to replicate in mosquito cells treated with pyrazofurin (PZF), a drug which inhibits pyrimidine nucleotide biosynthesis (Lin et al., 2000, Virology 272, 61-71). Three mutations, A6627U, A7543U, and C7593A, were identified in the nsP4 (the viral RNA polymerase) coding region, which were required for the PZF-resistant phenotype. We report here that SV(PZF) has a second phenotype. Its replication in BHK cells is severely restricted; yields of SV(PZF) from BHK cells are 100- to 1000-fold lower than the yields of standard SV (SV(STD)). However, addition of adenosine to the SV(PZF)-infected cultures completely relieves this restriction and results in yields comparable to those observed with SV(STD). Adenosine has no effect on the yield of SV(STD) from BHK cells. Synthesis of the viral structural proteins is markedly depressed in SV(PZF)-infected BHK cells, as is synthesis of the viral subgenomic (SG) RNA from which these proteins are translated. In contrast, normal amounts of genomic RNA are made. Experiments with mutagenized viruses indicated that the SV(PZF) mutation, C7593A, by itself, was sufficient to produce the restriction phenotype. However, this mutation not only changes Pro 609 of nsP4 to Thr, it also changes the nucleotide at the minus sign5 position of the SG promoter. To evaluate the relative contributions of the change in nsP4 and the change in the SG promoter to the restriction phenotype, we made use of double SG viruses, in which nsP4 and the promoter for the SG RNA which encodes the structural proteins can be changed independent of each other. Our results indicated that both the change in nsP4 and the change in the SG promoter were required to produce the full restriction phenotype. We suggest that the changes in nsP4 and the SG promoter destabilize the RNA initiation complex assembled at the SG promoter and that since ATP is the initiating nucleotide in the SG RNA transcript, the increased level of ATP resulting from the addition of adenosine is able to compensate for this destabilization and restore the synthesis of SG RNA to normal levels.
Lin
Yen-Huei
YH
Department of Molecular Genetics and Microbiology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854-5635, USA.
Simmonds
H Anne
HA
Stollar
Victor
V
D000241:Adenosine / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D012367:RNA, Viral / Q000378:metabolism; D012845:Sindbis Virus / Q000235:genetics* / Q000502:physiology*; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D012367:RNA, Viral; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases; D000241:Adenosine