Sindbis virus mutant ts20 of complementation group E contains a lesion in glycoprotein E2.
A technique has been devised to readily obtain the entire structural protein region of Sindbis virus cloned into a plasmid vector. This method uses the fact that the nearest site for restriction enzyme HindIII to the 3' terminal poly(A) occurs at nucleotides 6266-6271 in the genomic RNA. Inserts extending from the poly(A) tract to this HindIII site are 5438 nucleotides long (excluding the poly A tract) and contain the entire 4106-nucleotide structural protein region. Using an oligo(dT)-tailed vector as a primer for first strand cDNA synthesis such clones could be obtained in high yield. We were interested in a precise determination of the mutation responsible for the temperature-sensitive phenotype of ts20, a mutant belonging to complementation group E which has a defect in the function of glycoprotein E2 at the nonpermissive temperature. Using this technique we have cloned and sequenced the structural protein region of ts20 and of several revertants and concluded that the mutation was a change from histidine to leucine at amino acid 291 of E2. Reversion to temperature insensitivity occurred by same site reversion to the parental nucleotide, restoring the original histidine as amino acid 291. Thus, complementation group E of Sindbis virus results from changes in glycoprotein E2 and together with previous results from our laboratory (Arias et al., 1983; Hahn et al., 1985) demonstrates that the three RNA+ complementation groups of Sindbis virus, C, D, and E, result from changes in the three structural proteins of the virus, capsid, glycoprotein E1, and glycoprotein E2, respectively.
D003001:Cloning, Molecular; D004247:DNA; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII; D005814:Genes, Viral; D005816:Genetic Complementation Test; D006023:Glycoproteins / Q000032:analysis / Q000235:genetics*; D009154:Mutation; D012845:Sindbis Virus / Q000032:analysis / Q000235:genetics*; D013696:Temperature; D014759:Viral Envelope Proteins / Q000032:analysis / Q000235:genetics*
D006023:Glycoproteins; D014759:Viral Envelope Proteins; D004247:DNA; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII