Mutations in the nsP1 coding sequence of Sindbis virus which restrict viral replication in secondary cultures of chick embryo fibroblasts prepared from aged primary cultures.
SVMPA, a mutant of Sindbis virus (SV), which is able to replicate in Aedes albopictus cells treated with mycophenolic acid (MPA) or ribavirin, also has a host range phenotype. This phenotype is most clearly demonstrated by means of efficiency of plaquing (EOP) assays on secondary chick embryo fibroblasts (CEF) prepared from aged primary CEF. For example, in one experiment in which standard SV (SVSTD) had a relative EOP (EOP on primary CEF divided by EOP on secondary CEF) of 1.6 the corresponding value for SVMPA was 2340. The host restriction of SVMPA was also seen with similarly prepared secondary cultures of duck embryo fibroblasts, but not with established lines of quail cells. The finding that the accumulation of viral RNA was much lower in SVMPA-infected secondary CEF than in SVSTD-infected CEF indicated that the replication of SVMPA in these cultures was blocked at an early step. Revertants of SVMPA were isolated which were no longer host-restricted but had retained their resistance to MPA. Of the three mutations [nucleotide (nt) 120, 127, and 963] in the nsP1 coding sequence of SVMPA, which lead to amino acid changes, these revertants had retained the nt 127 and nt 963 mutations but had lost the nt 120 mutation. This, along with earlier findings, indicated that only the nt 127 and nt 963 mutations are required for resistance to MPA. This result also associated the nt 120 mutation with the host restriction phenotype. In other experiments derivatives of pToto1101 (a plasmid from which infectious Sindbis virus RNA can be transcribed) were constructed by site-directed mutagenesis and used to test the effect of specific mutations on the viral phenotype. Although we were unable to obtain viable virus with the nt 120 mutation alone, virus with the nt 120 and nt 127 mutations was viable and host-restricted. We suggest that the nt 120 mutation by itself is lethal and that the nt 127 mutation suppresses the lethal effect of the nt 120 mutation. The SVMPA mutation at nt 120, which is associated with the host range phenotype, changes Gln21 of nsP1 to Lys. When a more conservative change was engineered, i.e., to Asn, the virus was not host-restricted. Although the reason for the restriction of SVMPA replication in secondary CEF is not known, some possible explanations are discussed.
Rosenblum
C I
CI
Department of Molecular Genetics and Microbiology, UMDNJ-Robert Wood Johnson Medical School, Piscataway 08854-5636.
Scheidel
L M
LM
Stollar
V
V
D000330:Aedes / Q000166:cytology; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D006224:Cricetinae; D005347:Fibroblasts / Q000166:cytology / Q000382:microbiology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D012367:RNA, Viral / Q000032:analysis; D012845:Sindbis Virus / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D013045:Species Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication
D012367:RNA, Viral; D017361:Viral Nonstructural Proteins