An amino acid change in the exodomain of the E2 protein of Sindbis virus, which impairs the release of virus from chicken cells but not from mosquito cells.
In order to obtain a mutant of Sindbis virus (SV) with a low methionine-resistant (LMR) phenotype, i.e., able to replicate in methionine-deprived Aedes albopictus mosquito cells, standard SV (SV(STD)) was passaged 17 times in mosquito cells maintained in a low methionine medium and then plaque-purified, also in mosquito cells. Although the virus obtained by this procedure, SV(LM17), did have the desired LMR phenotype, it also appeared to have acquired a host-range phenotype. We have now characterized the host-range phenotype of SV(LM17) in greater detail. In yield assays, the titer of SV(LM17) produced by chick embryo fibroblasts (CEF) was 100- to 1000-fold lower than that from mosquito cells. SV(STD), in contrast, produced a similar titer of virus from the two cell types. On the other hand, when SV(LM17) was assayed directly by plaque formation on CEF and on mosquito cell monolayers, no host restriction in CEF was observed. When CEF were infected with SV(LM17), viral proteins were synthesized normally, pE2 was processed to E2, and E2 was demonstrated by the fluorescent antibody method to reach the cell surface. However, electron microscopy of SV(LM17)-infected cells revealed an absence of extracellular virions and of budding particles; also, nucleocapsids were not aligned beneath the plasma membrane. By sequence determination and by site-directed mutagenesis, it was determined that the host restriction of SV(LM17) was due to a change from Ala to Val at position 251 of the E2 protein. Substitution of Gly or Leu at this position also resulted in the same host range phenotype.
Li
M L
ML
Department of Molecular Genetics and Microbiology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854-5635, USA.
Liao
H J
HJ
Simon
L D
LD
Stollar
V
V
D000330:Aedes / Q000821:virology; D019943:Amino Acid Substitution; D000818:Animals; D002642:Chick Embryo; D002999:Clone Cells; D004279:DNA, Viral / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000821:virology; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011994:Recombinant Proteins / Q000378:metabolism; D012845:Sindbis Virus / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics / Q000502:physiology*
D004279:DNA, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C064950:glycoprotein E2, Sindbis virus