In addition to the five mRNA species and 47 nucleotide long leader RNA synthesized by purified virions of vesicular stomatitis virus, at least three discrete low molecular weight RNA species having approximate chain lengths of 28, 42 and 70 nucleotides can be detected in vitro. Each of these RNA species displays a unique and characteristic T1 fingerprint profile and contains (p)ppAA as its 5' terminus. By partial sequence analyses, two of the small RNA products, 42 and 28 bases long, were found to contain 5' terminal sequences identical to those in the N and NS mRNAs, respectively. Ultraviolet inactivation studies demonstrate that each of these RNA species has a target size in agreement with its molecular weight indicating independent initiation. Kinetic studies show that the small RNA species are synthesized within 1 min, while mRNA chain completion occurs later in the sequential order N-NS-M-G. These results indicate that viral mRNA synthesis occurs in vitro by multiple initiations at different promoter sites on the genome RNA, and that the elongation and completion of the individual mRNAs depend on prior transcription of 3' proximal genes. We present a model for viral mRNA synthesis in vitro.

The in vitro RNA synthesis by the virion-associated RNA polymerase of vesicular stomatitis virus (VSV), New Jersey serotype, was compared with that of the serologically distinct Indiana serotype of VSV. The New Jersey serotype of VSV synthesized five distinct mRNA species in vitro, three of which were smaller than the corresponding species synthesized by the Indiana serotype of VSV. These included the mRNA's coding for the G, M, and NS proteins. By hybridization experiments, virtually no sequence homology was detected between the mRNA's of the two serotypes. Despite this lack of overall homology, the 12 to 18S mRNA species of both serotype contained a common 5'-terminal hexanucleotide sequence, G(5')ppp(5')A-A-C-A-G. The signicance of this finding in light of specific interactions between the two serotypes of VSV in vivo is discussed.

The oligonucleotides synthesized by purified vesicular stomatitis virus in vitro in the absence of one or more ribonucleoside triphosphate precursors have been studied. The oligonucleotides contained the 5'-terminal sequences of the leader RNA and one or more mnRNA's. The promoter-proximal oligonucleotides lacked 5'-terminal cap structure and contained triphosphate A. These results suggest that the RNA polymerase is located at multiple promoter sites on the genome RNA from where it initiates transcription. The capping reaction appears to occur subsequently during RNA chain elongation. We have also demonstrated that a unique dinucleotide, pppGpC, of presently unknown function is synthesized in vitro in large amounts during RNA synthesis or in the presence of GTP and CTP only.

The New Jersey serotype of vesicular stomatitis virus (VSV) was able to synthesize a small RNA (leader RNA) approximately 70 bases in length similar to the leader RNA synthesized in vitro by the genetically distinct Indiana serotype of VSV. Also, the New Jersey leader RNA contained the same 5'-terminal sequence, ppA-C-G, as the Indiana leader RNA and had a very similar base composition, with 42% AMP, 16% CMP, 18.6% GMP, and 23.4% UMP. The 3'terminal sequence of the VSV New Jersey genome RNA was determined and found to contain the sequence-Py-G-UoH, again the same as that of the Indiana serotype of VSV. Evidence that the New Jersey leader RNA is transcribed from the 3' end of the genome RNA was obtained from the fact that it can protect the 3'-terminal base of [3H]borohydride-labeled New Jersey genome RNA from RNase digestion. Although the New Jersey and Indiana leader RNAs were similar in many respects, they were unable to form RNase-resistant hybrids when annealed to heterologous genome RNA. Vesicular stomatitis virus (VSV), a rhabdoviminus, similar base composition, and is also tranrus, contains five structural proteins and a sin-scribed from the 3' end of genome RNA, but its gle-stranded genome RNA of negative polarity base sequence is specific for the VSVNJ genome which sediments at 42S (21). Studies on the RNA. Indiana serotype (VSVInd) have demonstrated that the virion contains an RNA polymerase MATERIALS AND METHODS which sequentially transcribes the genome RNA MATEIALaAN M S into a leader RNA followed by five monocis-Purification of VSV. VSVInd and VSVNJ were tronic mRNA's (1, 2). In the preceding commugrown in baby hamster kidney cells (BHK-21, clone tronic mRNA's (1, 2). In the preceding commu-13), adapted to suspension culture, and purified as nication (16), it was shown that the serologically described previously (5). distinct New Jersey serotype of VSV (VSVNJ) Synthesis and purification of RNA in vitro. can also synthesize five mRNA species in vitro Standard in vitro RNA polymerase reactions (1 ml) which contain very little, if any, sequence hocontained 50 mM Tris-hydrochloride (pH 8.0), 0.1 M mology with the corresponding mRNA species NaCl, 5 mM MgCl2, 4 mM dithiothreitol, 0.05% Triton synthesized by VSVI.d. Surprisingly, it was N101, 1 mM ATP, CTP, and GTP, and 0.1 mM UTP. found that a common 5'-terminal sequence, In vitro labeling conditions were as follows: ATP, 250 G(5')ppp(5')A-A-C-A-G, identical to that of MCi of [a-32P]ATP (16.4 Ci/mmol) with or without 500 VSVIDd mRNA's, was present in VSVNJ mRNA's. MCi of [3H]ATP (25.8 Ci/mmol) at a final ATP con-ThVisd obsRvAtionwa lredeusto VSpeclat thNAt'th centration of 0.4 mM; CTP, 250 fLCi Of [a_32p]CTP This observation led us to speculate that the (37.4 Ci/mmol) with or without 500 MCi of [3H]CTP conservation of this sequence was important (23.1 Ci/mmol) at a final CTP concentration of 50 ,uM; during evolution of various rhabdoviruses, since GTP, 250 liCi of [a-32P]GTP (19.5 Ci/mmol) with or this sequence may be an integral part of the without 500 ,i...