Conditions are described for the synthesis of long cDNA transcripts of Sindbis virus 26S and 49S RNA in high yield. This single-stranded cDNA could be cut with Type II restriction endonucleases including Hae III, Hha I, Rsa I, or Taq I to give reproducible patterns of discrete, virus-specific fragments which were suitable for subsequent end-labeling and direct sequence analysis. Using these methods, the strategy used for obtaining nearly the entire 26S RNA sequence from cDNA synthesized in vitro is presented. The 26S RNA is approximately 4.2 kb in length, and from the AUG codon initiating synthesis of the capsid protein, contains an open reading frame for 3735 nucleotides. From this sequence, the amino acid sequences of the encoded virus structural proteins, which include a basic capsid protein and two integral membrane glycoproteins (El and E2), as well as the sequences of two nonstructural polypeptides have been deduced. Features of the primary structure of these proteins and the proteolytic cleavage sites involved in their processing are discussed.
The orientation of the virion glycoproteins with respect to the lipid bilayer was studied by digesting intact Sindbis virus with α-chymotripsin. A single membrane-associated peptide is produced from each of the two virion glycoproteins. These peptides contain covalently attached palmitic acid, are rich in hydrophobic amino acids and are located at the extreme COOH-terminal end of each glycoprotein. Both peptides contain uninterrupted sequences of uncharged amino acids of sufficient length to span the lipid bilayer, and it is suggested that they serve to anchor the viral glycoproteins in the membrane. The properties of these and other well-characterized transmembrane segments are discussed.
Specific antisera to each of the virus structural proteins was produced and used to study the association of the virion glycoproteins and their precursors. E1 and E2 could be cross-linked into heterodimers using bifunctional amino-reactive imidates. This association is present both in intact virions and infected cells and is stable after solubilization of the virion envelope by Triton X-100. Cross-linking data of pulse-labeled monolayers and cells infected with ts mutants are summarized. These data suggest that PE2 (the precursor to E2) and E2 are in different conformations with respect to E1, and that the glycoprotein precursors synthesized at elevated temperatures have an increased tendency to undergo intracellular aggregation.
