The 5' sequence of Sindbis viral RNA is m7G"pppApUpGp... Recent analyses have shown that the messenger polarity RNA plus strands of cytoplasmic polyhedrosis virus or human reovirus possess modified 5' nucleotide sequences (11, 12). The complementary minus strands of the doublestranded genomes of these viruses do not have modified nucleotides (11, 12). The 5' sequence of reovirus plus strand RNA (8) is believed to be m7Gr"pppGmpCp..., with a methyl residue present on the 7-position of the capping guanosine (m7G) and a second 2'-0-methyl group present on the ribose of the next guanosine nucleotide (Gmp). The presence of capping m 7G on the messenger or putative mRNA species of vesicular stomatitis or vaccinia viruses has also been demonstrated (13, 24). In this communication we present evidence indicating that the 5' terminal nucleotide of the alphavirus Sindbis is modified and that the sequence is m7G5'pppApUpGp ... MATERIALS AND METHODS Reagents. [3H ]nucleosides, ["4C ]guanosine, and ["2P ]phosphoric acid were obtained from International Chemical and Nuclear Corp., Irvine, Calif. [14C ]uridine and [methyl-'H Imethionine were obtained from New England Nuclear Corp., Boston, Mass. Nucleoside and nucleotide standards for chromatography were obtained from P. L. Biochemicals Inc., Milwaukee, Wis. Alkaline phosphatase and venom phosphodiesterase were obtained from Boehringer Mannheim Corp., New York, N.Y. Preparation and purification of 3H-, 14C-, or 32P-labeled virus. The procedures used to obtain labeled virus grown in the presence of [5-'Hluridine (25 uCi/ml, 8 Ci/mmol) and [5-3H]cytidine (25 MCi/ ml, 16.3 Ci/mmol), [8-_Hlguanosine (25 giCi/ml, 20 Ci/mmol), [8-"4C Iguanosine (0.5 XCi/ml, 55 mCi/ mmol), [8-_3H]adenosine (25 ACi/ml, 21 Ci/mmol), [32P]phosphate (200 ACi/ml), or [2-"4C]uridine (0.04 ACi/ml, 0.4 mCi/mmol) have been described (1, 15,