In vitro synthesis of the full-length complement of the negative-strand genome RNA of vesicular stomatitis virus.

Abstract

Under the normal conditions of in vitro RNA synthesis, the virion-associated RNA polymerase of vesicular stomatitis virus synthesizes five monocristronic mRNAs and a 48-nucleotide-long leader RNA that represents the exact 3'-terminal region of the genome RNA [Colpnno, R. J. & Banerjee, A. K. (1978) Cell, 15, 93-101]. When the transcribing core was preincubated with ATP and CTP, reisolated, and then incubated in the presence of the fty imido analoguq of ATP (AdoPFIINMI) and the three normal ribonucleoside. triphosphates, the fulllength complementary strand of the getlotne RNA was synthesized in vitro. The results suggest that specific phosphorylated states of regulatory proteins may control transcription in vitro to generate the full-length plus strands.The virion-associated RNA polymerase of vesicular stomatitis virus (VSV) transcribes the singlestranded genome RNA of negative polarity in vitro into five"mbnocistronic mRNA species(1, 2). The transcription process appears to be sequential, with the synthesis of a "leader RNA" representing the 3'-terminal 48-nucleotide region of the genome RNA (3) followed by the synthesis of the mRNA species coding for the structural proteins in the order N, NS, M, G, and L (4,5). The sum of the molecular weights of the mRNA species approximately corresponds to the total coding potential of the genome RNA, indicating that the entire genome is transcribed in vitro (2). However, the complete complement of the negative-strand genome RNA (the 42S plus strand), which is the required intermediate of replication, has not been detected in vitro although it has been identified in the infected cell (6, 7). Thus, it appears that transcription of the full-length VSV genome RNA in vitro possibly requires modified experimental conditions or mediation by host or intracellular viral proteins (8,9).We have recently shown that ATP plays a significant role during initiation of VSV mRNA synthesis in vitro (10). The apparent Km for ATP during the initiation step was significantly higher (0.5 mM) than that observed for the same nucleotide during the period of chain elongation (25MuM). These results coupled with the observation that the first RNA'product, the leader RNA, terminates with a 5'-terminal diphosphate ppA-C-G sequence (3) suggested that the removal of the y-phosphate of ATP may be directly involved in the initiation step of RNA synthesis. This MATERIALS AND METHODS Purification of VSV. VSV (Indiana serotype) was grown in baby hamster kidney cells (BHK-21, clone 13) adapted to suspension culture and purified as described (11).Conditions for In Vitro Synthesis of Full-Length Positive Strand. Purified VSV (1.6 mg) was disrupted with Triton N-101 (0.05%) in 0.05 M Tris-HCl, pH 8/0.1 M NaCl/5 mM MgCl2/4 mM dithiothreitol/I mM ATP/0.5 mM CTP (total volume, 2 ml). The mixture was incubated for 30 min at 30'C. The reaction mixture was then chilled to 40C and layered onto 2 ml of solution containing 20% (wt/vol) glycerol in 0.01 M Tris.HCl, pH 7.4/0.01 M KCI/0.1 mM MgCl2/0.1 M NaCl/1 m...

In vitro Phosphorylation of NS Protein by the L Protein of Vesicular Stomatitis virus

“…Since 8-azido-ATP is not a substrate for RNA synthesis in vitro (data not shown), it appears that 8-azido-ATP may indeed have inactivated the protein kinase site which, in turn, inactivated the transcription initiation site of the L protein. These results are consistent with the previous observations that various phosphorylated states of NS have differential effects on transcription (Clinton et al, 1978;Hsu et al, 1982: Kingsford & Emerson, 1980Sinacore & Lucas-Lenard, 1982;Testa et al, 1980: Watanabe et al, 1974Witt & Summers, 1980). Future experiments along these lines will shed light on the role of NS phosphorylation on the transcription process in vitro.…”

“…t ND, Not determined• Lucas- Lenard, 1982;Testa et al, 1980; W a t a n a b e et W i t t & Summers, 1980), it was of interest to study whether inhibition of N S phosphorylation has any effect on R N A transcription in vitro. W e photoreacted the L protein with unlabelled 8-azido-ATP and studied its effect on phosphorylation of N S protein.…”

“…Since NS protein, in association with the L protein, constitutes the RNA polymerase complex (Emerson & Yu, 1975;Naito & Ishihama, 1976) that transcribes the nucleoprotein (N)-bound genome RNA template, regulation of phosphorylation of this protein may play a key role in the replicative process of the virus. A number of studies in vitro and in vivo have strongly suggested that the degree of phosphorylation of NS protein plays a regulatory role in transcription processes in vitro (Clinton et al, 1978 ;Hsu et al, 1982;Kingsford & Emerson, 1980;Sinacore & Lucas-Lenard, 1982;Testa et al, 1980: Watanabe et al, 1974Witt& Summers, 1980).…”

Vesicular Stomatitis Virus Growth in Drosophila melanogaster Cells. II. Modifications of Viral Protein Phosphorylation

“…Results of phosphoamino acid analysis are presented in Table 1; they were close to those described by Clinton & Huang (1981) in BHK cells. While NS phosphorylation sites remained unchanged, as well as the nature and ratio of the phosphoamino acids, the equilibrium between the two states of phosphorylation was different in the mature virions grown in Drosophila cells; if this occurred also in the cytoplasm of these cells, it could affect the regulation of viral RNA synthesis, via a mechanism of phosphorylation and dephosphorylation (Witt & Summers, 1980;Kingsford & Emerson, 1980;Testa et al, 1980). The results from M protein digestion were more complex.…”

“…Vesicular stomatitis virus (VSV), a negative-strand RNA-containing enveloped virus, possesses two phosphoproteins, NS and M (Clinton et al, 1978a), which are involved in viral RNA synthesis (Carroll & Wagner, 1979;Clinton et al, 1978b;Martinet et al, 1979;Testa et al, 1980;Kingsford & Emerson, 1980). As phosphorylation depends on the host cell (Imblum & Wagner, 1974;Moyer & Summers, 1974) and could alter virus multiplication, it might be related to the control mechanism which strongly modifies VSV growth in insect cells where VSV (and also alphaviruses) is not cytopathogenic (Mudd et al, 1973;Richardson et al, 1980).…”