In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. II. Characterization of the leader RNA

Abstract

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...

Transcription and replication of rhabdoviruses.

“…The RNA-synthetic activity of purified RNP of VSV has also been demonstrated in various serotypes in addition to the widely used Indiana serotype (43,54,78). Similar activity has also been shown for rabies virus (76,115), although this activity is significantly lower than that of VSV.…”

Nucleotide sequence of the leader RNA of the New Jersey serotype of vesicular stomatitis virus

“…Reaction mixtures were incubated with 0.1 mg/pl of purified VSV for 6 hr at 300. The reaction was terminated by the addition of SDS to 0.5% and the product RNA was directly extracted with phenol and purified by Sephadex G-50 chromatography, oligo(dT) cellulose chromatography, and electrophoresis on a 20% polyacrylamide slab gel as previously described (8,9). The leader RNA was recovered from the gel as described previously (10).…”

“…The reaction mixture was incubated at 30°for 6 hr with 0.1 mg purified virions of clonally purified Ogden strain of New Jersey serotype of VSV. RNA synthesis was terminated by addition of SDS to 0.5% and the product RNA purified by phenol extraction and Sephadex G-50 chromatography (9). Poly(A) containing mRNA was removed by oligo(dT)-cellulose chromatography and the leader RNA isolated from nonpoly(A)-containing product RNA by electrophoresis on a 20% polyacrylamide slab gel (140 V for 16 hr) as described previously (6,9).…”

“…RNA synthesis was terminated by addition of SDS to 0.5% and the product RNA purified by phenol extraction and Sephadex G-50 chromatography (9). Poly(A) containing mRNA was removed by oligo(dT)-cellulose chromatography and the leader RNA isolated from nonpoly(A)-containing product RNA by electrophoresis on a 20% polyacrylamide slab gel (140 V for 16 hr) as described previously (6,9). The band migrating in the center of the gel was located by autoradiography and recovered as previously described (6).…”

“…Keller, Maxam and Gilbert (11). New Jersey leader RNA was synthesized in the presence of [3H] UTP and purified as described previously (9). The 5'terminal phosphates were removed with alkaline phosphatase and the terminus was labeled with [y32p] ATP using polynucleotide kinase (11).…”