Nucleic Acids Research, Vol. 20, No. 21 5843

The phosphoprotein (P) gene of the rhabdovirus Piry: its cloning, sequencing, and expression in Escherichia coli Sailen Barik Department of Molecular Biology, NC2-130, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA Received September 22, 1992; Accepted September 30, 1992 The phosphoprotein (P) and the large protein (L) constitute the RNA-dependent RNA polymerase of the rhabdoviruses (1). We have recently shown that sequential phosphorylation by cellular casein kinase II (CKII) and an L-associated protein kinase is essential for the transcriptional activity of the P protein of vesicular stomatitis virus (VSV), a prototype rhabdovirus (2, 3). In order to investigate whether this is a general feature of all rhabdoviral P proteins, I have cloned and sequenced the P protein gene of Piry virus, a close relative of VSV. The Piry P protein, as found in the virion and in Piry-infected cells, is highly phosphorylated (4). Piry virus (a kind gift from Dr Robert Shope, Yale University) was grown in BHK-21 cells. Viral mRNAs were amplified by reverse transcription and PCR (RT-PCR) using oligo(dT) and various degenerate oligonucleotides as primers. Following several rounds of sequencing, RT-PCR, and hybridization with labeled Piry genomic RNA, prospective PCR products were cloned into pET-3a as described (5). The final P clone was characterized by the following criteria: i) The 984 bp insert contained a single continuous open reading frame that could code for a protein of 328 amino acids with a calculated MW of 35711, slightly larger than the VSV P protein (4); ii) Upon expression in E.coli (5), this clone synthesized a protein of Mr -49 k in SDS-PAGE, identical to that of Piry virion P protein (Figure 1). iii) The recombinant protein reacted specifically with anti-Piry antibody (data not shown). The deduced amino acid sequences of P proteins of Piry and VSV were highly dissimilar; however, the hydropathicity patterns were similar (Figure 2A), particularly in the N-terminal 200 residues, indicating that the overall secondary structure of P proteins may be conserved (6). Remarkably, both proteins contained a highly acidic N-tenninal domain (Figure 2B). Earlier, we had shown that Ser59 and Ser6l of the VSV P protein (6) were sites for CKII-mediated phosphorylation (3). Although Ser and Thr account for 20% of all amino acids in the Piry P protein, only three Ser residues (Ser72, 89, 94) in the acidic region conform to the consensus CKII recognition motif. Mapping of the sites of phosphorylation of Piry P protein and identification of the cognate protein kinase(s) are in progress.

REFERENCES 1. Banerjee,A.K. and Barik,S. (1992) Virology 188, 417-428. 2. Barik,S. and Banerjee,A.K. (1992) J. Virol. 66, 1109-1118. 3. Barik,S. and Banerjee,A.K. (1992) Proc. Natl. Acad. Sci. USA 89, 6570-6574. 4. Bell,J.C. and Prevec,L. (1979) J. Virol. 30, 56-63. 5. Barik,S. and Banerjee,A.K. (1991) J. Virol. 65, 1719-1726. 6. Gill,D.S. and Banerjee,A.K. (1985) J. Virol. 55, 60-66. 7. Marck,C. (1988) Nucleic Acids Res. 16, 1829-1836.

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Figure 1. Identification of Piry P protein. Total proteins of E.coli BL21 (DE3) containing pET-3a-P-Piry clone with (+) or without (-) induction by IPTG were analyzed by SDS-PAGE and Coomassie blue staining. Piry virion proteins (V) are included for comparison; P polypeptide is indicated by arrowhead.

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Fig. 2. Structurl analyses of Piry and VSV P proteins. Deduced primary sequences of Piry P (this study) and VSV P (6) proteins were analyzed for hydropathicity (Kyte-Doolittle) (panel A) and acid-base composition (panel B) by using the DNA strider program (7).

The phosphoprotein (P) gene of the rhabdovirus Piry: its cloning, sequencing, and expression in Escherichia coli.

Nucleic Acids Research, Vol. 20, No. 21 5843 The phosphoprotein (P) gene of the rhabdovirus Piry: its cloning, sequencing, and expression in Escheric...
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