Studies on the Rabies Virus RNA Polymerase : 2. Possible Relationships between the Two Forms of the Non- Catalytic Subunit (P Protein)

Fumihiko Takamatsu; Naoki Asakawa; Kinjiro Morimoto; Kenji Takeuchi; Yoshiro Eriguchi; Harufusa Toriumi; Akihiko Kawai

Fumihiko Takamatsu, Naoki Asakawa, Kinjiro Morimoto, Kenji Takeuchi, Yoshiro Eriguchi, Harufusa Toriumi, Akihiko Kawai

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Keywords: Rabies virus P protein, cDNA cloning, Gene expression, Non-catalytic subunit of rabies virus RNA polymerase, Rabies virion-associated 40-kDa polypeptide

JOURNAL FREE ACCESS

1998 Volume 42 Issue 11 Pages 761-771

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Published: November 20, 1998 Received: May 28, 1998 Available on J-STAGE: March 17, 2008 Accepted: August 10, 1998 Advance online publication: - Revised: -
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Date of correction: March 17, 2008 Reason for correction: - Correction: TITLE Details: Wrong : Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non-Catalytic Subunit (P Protein)
Right : Studies on the Rabies Virus RNA Polymerase : 2. Possible Relationships between the Two Forms of the Non- Catalytic Subunit (P Protein)

Date of correction: March 17, 2008 Reason for correction: - Correction: ABSTRACT Details: Wrong : We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [³H]leucine and [³²P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s).
Right : We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [³H] leucine and [³²P] orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme (s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor (s).

Date of correction: March 17, 2008 Reason for correction: - Correction: DTPUB Details: Right : 19981120

Date of correction: March 17, 2008 Reason for correction: - Correction: CITATION Details: Wrong : 1) Banerjee, A.K., and Barik, S. 1992. Gene expression of vesicular stomatitis virus genome RNA. Virology 188: 417-428.
2) Barik, S., and Banerjee, A.K. 1992. Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P. Proc. Natl. Acad. Sci. U.S.A. 89: 6570-6574.
3) Barik, S., and Banerjee, A.K. 1992. Sequential phosphorylation of the phosphoprotein of vesicular stomatitis virus by cellular and viral protein kinases is essential for transcription activation. J. Virol. 66: 1109-1118.
4) Bhown, A.S., and Bennett, J.C. 1983. High-sensitivity sequence analysis of proteins recovered from sodium dodecyl sulfate gels. Methods Enzymol. 91: 450-455.
5) Brown, F., Bishop, D.H., Crick, J., Francki, R.I., Holland, J.J., Hull, R., Johnson, K., Martelli, G., Murphy, F.A., Obijeski, J.F., Peters, D., Pringle, C.R., Reichmann, M.E., Schneider, L.D., Shope, R.E., Simpson, D.I., Summers, D.F., and Wagner, R.R. 1979. Rhabdoviridae. Report of the rhabdovirus study group, International Committee on Taxonomy of Viruses. Intervirology 12: 1-7.
6) Chen, J.L., Das, T., and Banerjee, A.K. 1997. Phosphorylated states of vesicular stomatitis virus P protein in vitro and in vivo. Virology 228: 200-212.
7) Chen, C., and Okayama, H. 1987. High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7: 2745-2752.
8) Chenik, M., Chebli, K., and Blondel, D. 1995. Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism. J. Virol. 69: 707-712.
9) Chenik, M., Chebli, K., Gaudin, Y., and Blondel, D. 1994. In vivo interaction of rabies virus phosphoprotein (P) and nucleoprotein (N): existence of two N-binding sites on P protein. J. Gen. Virol. 75: 2889-2896.
10) Clinton, G.M., Burge, B.W., and Huang, A.S. 1978. Effects of phosphorylation and pH on the association of NS protein with vesicular stomatitis virus cores. J. Virol. 27: 340-346.
11) Conzelmann, K.-K., Cox, J.H., Schneider, L.G., and Thiel, H.-J. 1990. Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B 19. Virology 175: 485-499.
12) Cooper, J.A., Sefton, B.M., and Hunter, T. 1983. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 99: 387-402.
13) Cox, J.H. 1982. The structural proteins of rabies virus. Comp. Immunol. Microbiol. Infect. Dis. 5: 21-25.
14) De, B.P., and Banerjee, A.K. 1985. Requirements and functions of vesicular stomatitis virus L and NS proteins in the transcription process in vitro. Biochem. Biophys. Res. Commun. 126: 40-49.
15) Fu, Z.F., Zheng, Y., Wunner, W.H., Koprowski, H., and Dietzschold, B. 1994. Both the N- and the C-terminal domains of the nominal phosphoprotein of rabies virus are involved in binding to the nucleoprotein. Virology 200: 590-597.
16) Kawai, A. 1977. Transcriptase activity associated with rabies virion. J. Virol. 24: 826-835.
17) Kawai, A., Anzai, J., Honda, Y., Morimoto, K., Takeuchi, K., Kohno, T., Wakisaka, K., Goto, H., and Minamoto, N. 1997. Monoclonal antibody #5-2-26 recognizes the phosphatase-sensitive epitope of rabies virus nucleoprotein. Microbiol. Immunol. 41: 33-42.
18) Kawai, A., Matsumoto, S., and Tanabe, K. 1975. Characterization of rabies viruses recovered from persistently infected BHK cells. Virology 67: 520-533.
19) Kawai, A., and Morimoto, K. 1994. Functional aspects of lyssavirus proteins. Curr. Top. Microbiol. Immunol. 187: 27-42.
20) Kawai, A., and Takeuchi, K. 1992. Temperature-sensitivity of the replication of rabies virus (HEP-Flury strain) in BHK-21 cells. I. Alteration of viral RNA synthesis at the elevated temperature. Virology 186: 524-532.
21) Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
22) Larson, J.K., Otvos, L., Jr., and Ertl, H.C. 1992. Posttranslational side chain modification of a viral epitope results in diminished recognition by specific T cells. J. Virol. 66: 3996-4002.
23) Larson, J.K., and Wunner, W.H. 1990. Nucleotide and deduced amino acid sequences of the nominal nonstructural phosphoprotein of the ERA, PM and CVS-11 strains of rabies virus. Nucleic Acids Res. 18: 7172.
24) Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.
25) Maki, K., Sagara, J., and Kawai, A. 1991. A cationic detergent, cetyltrimethylammonium bromide (CTAB), selectively dissociates the intermediate filament of the fibroblast. Biochem. Biophys. Res. Commun. 175: 768-774.
26) Masters, P.S., and Banerjee, A.K. 1988. Complex formation with vesicular stomatitis virus phosphoprotein NS prevents binding of nucleocapsid protein N to nonspecific RNA. J. Virol. 62: 2658-2664.
27) Morimoto, K., Ohkubo, A., and Kawai, A. 1989. Structure and transcription of the glycoprotein gene of attenuated HEP-Flury strain of rabies virus. Virology 173: 465-477.
28) Morimoto, K., Akamine, T., Takamatsu, F., and Kawai, A. 1998. Studies on the rabies virus RNA polymerase: 1. cDNA cloning of the catalytic subunit of the avirulent HEP-Flury strain and its expression in animal cells. Microbiol. Immunol. 42: 485-496.
29) Naito, S., and Ishihama, A. 1975. Function and structure of RNA polymerase from vesicular stomatitis virus. J. Biol. Chem. 251: 4307-4314.
30) Naito, S., and Matsumoto, S. 1978. Identification of cellular actin within the rabies virus. Virology 91: 151-163.
31) Pattnaik, A.K., Hwang, L., Li, T., Englund, N., Mathur, M., Das, T., and Banerjee, A.K. 1997. Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication. J. Virol. 71: 8167-8175.
32) Prehaud, C., Nel, K., and Bishop, D.H. 1992. Baculovirus-expressed rabies virus M1 protein is not phosphorylated: it forms multiple complexes with expressed rabies N protein. Virology 189: 766-770.
33) Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185: 60-89.
34) Tordo, N., Poch, O., Ermine, A., Keith, G., and Rougeon, F. 1986. Walking along the rabies genome: is the large G-L intergenic region a remnant gene? Proc. Natl. Acad. Sci. U.S.A. 83: 3914-3918.
35) Tuffereau, C., Fischer, S., and Flamand, A. 1985. Phosphorylation of the N and M1 proteins of rabies virus. J. Gen. Virol. 66: 2285-2289.

Right : 1) Banerjee, A.K., and Barik, S. 1992. Gene expression of vesicular stomatitis virus genome RNA. Virology 188 : 417-428.
2) Barik, S., and Banerjee, A.K. 1992. Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P. Proc. Natl. Acad. Sci. U.S.A. 89 : 6570-6574.
3) Barik, S., and Banerjee, A.K. 1992. Sequential phosphorylation of the phosphoprotein of vesicular stomatitis virus by cellular and viral protein kinases is essential for transcription activation. J. Virol. 66 : 1109-1118.
4) Bhown, A.S., and Bennett, J.C. 1983. High-sensitivity sequence analysis of proteins recovered from sodium dodecyl sulfate gels. Methods Enzymol. 91 : 450-455.
5) Brown, F., Bishop, D.H., Crick, J., Francki, R.I., Holland, J.J., Hull, R., Johnson, K., Martelli, G., Murphy, F.A., Obijeski, J.F., Peters, D., Pringle, C.R., Reichmann, M.E., Schneider, L.D., Shope, R.E., Simpson, D.I., Summers, D.F., and Wagner, R.R. 1979. Rhabdoviridae. Report of the rhabdovirus study group, International Committee on Taxonomy of Viruses. Intervirology 12 : 1-7.
6) Chen, J.L., Das, T., and Banerjee, A.K. 1997. Phosphorylated states of vesicular stomatitis virus P protein in vitro and in vivo. Virology 228 : 200-212.
7) Chen, C., and Okayama, H. 1987. High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7 : 2745-2752.
8) Chenik, M., Chebli, K., and Blondel, D. 1995. Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism. J. Virol. 69 : 707-712.
9) Chenik, M., Chebli, K., Gaudin, Y., and Blondel, D. 1994. In vivo interaction of rabies virus phosphoprotein (P) and nucleoprotein (N) : existence of two N-binding sites on P protein. J. Gen. Virol. 75 : 2889-2896.
10) Clinton, G.M., Burge, B.W., and Huang, A.S. 1978. Effects of phosphorylation and pH on the association of NS protein with vesicular stomatitis virus cores. J. Virol. 27 : 340-346.
11) Conzelmann, K.-K., Cox, J.H., Schneider, L.G., and Thiel, H.-J. 1990. Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B19. Virology 175 : 485-499.
12) Cooper, J.A., Sefton, B.M., and Hunter, T. 1983. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 99 : 387-402.
13) Cox, J.H. 1982. The structural proteins of rabies virus. Comp. Immunol. Microbiol. Infect. Dis. 5 : 21-25.
14) De, B.P., and Banerjee, A.K. 1985. Requirements and functions of vesicular stomatitis virus L and NS proteins in the transcription process in vitro. Biochem. Biophys. Res. Commun. 126 : 40-49.
15) Fu, Z.F., Zheng, Y., Wunner, W.H., Koprowski, H., and Dietzschold, B. 1994. Both the N- and the C-terminal domains of the nominal phosphoprotein of rabies virus are involved in binding to the nucleoprotein. Virology 200 : 590-597.
16) Kawai, A. 1977. Transcriptase activity associated with rabies virion. J. Virol. 24 : 826-835.
17) Kawai, A., Anzai, J., Honda, Y., Morimoto, K., Takeuchi, K., Kohno, T., Wakisaka, K., Goto, H., and Minamoto, N. 1997. Monoclonal antibody #5-2-26 recognizes the phosphatasesensitive epitope of rabies virus nucleoprotein. Microbiol. Immunol. 41 : 33-42.
18) Kawai, A., Matsumoto, S., and Tanabe, K. 1975. Characterization of rabies viruses recovered from persistently infected BHK cells. Virology 67 : 520-533.
19) Kawai, A., and Morimoto, K. 1994. Functional aspects of lyssavirus proteins. Curr. Top. Microbiol. Immunol. 187 : 27-42.
20) Kawai, A., and Takeuchi, K. 1992. Temperature-sensitivity of the replication of rabies virus (HEP-Flury strain) in BHK-21 cells. I. Alteration of viral RNA synthesis at the elevated temperature. Virology 186 : 524-532.
21) Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 : 680-685.
22) Larson, J.K., Otvos, L., Jr., and Ertl, H.C. 1992. Posttranslational side chain modification of a viral epitope results in diminished recognition by specific T cells. J. Virol. 66 : 3996-4002.
23) Larson, J.K., and Wunner, W.H. 1990. Nucleotide and deduced amino acid sequences of the nominal nonstructural phosphoprotein of the ERA, PM and CVS-11 strains of rabies virus. Nucleic Acids Res. 18 : 7172.
24) Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 : 265-275.
25) Maki, K., Sagara, J., and Kawai, A. 1991. A cationic detergent, cetyltrimethylammonium bromide (CTAB), selectively dissociates the intermediate filament of the fibroblast. Biochem. Biophys. Res. Commun. 175 : 768-774.
26) Masters, P.S., and Banerjee, A.K. 1988. Complex formation with vesicular stomatitis virus phosphoprotein NS prevents binding of nucleocapsid protein N to nonspecific RNA. J. Virol. 62 : 2658-2664.
27) Morimoto, K., Ohkubo, A., and Kawai, A. 1989. Structure and transcription of the glycoprotein gene of attenuated HEP-Flury strain of rabies virus. Virology 173 : 465-477.
28) Morimoto, K., Akamine, T., Takamatsu, F., and Kawai, A. 1998. Studies on the rabies virus RNA polymerase : 1. cDNA cloning of the catalytic subunit of the avirulent HEP-Flury strain and its expression in animal cells. Microbiol. Immunol. 42 : 485-496.
29) Naito, S., and Ishihama, A. 1975. Function and structure of RNA polymerase from vesicular stomatitis virus. J. Biol. Chem. 251 : 4307-4314.
30) Naito, S., and Matsumoto, S. 1978. Identification of cellular actin within the rabies virus. Virology 91 : 151-163.
31) Pattnaik, A.K., Hwang, L., Li, T., Englund, N., Mathur, M., Das, T., and Banerjee, A.K. 1997. Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication. J. Virol. 71 : 8167-8175.
32) Prehaud, C., Nel, K., and Bishop, D.H. 1992. Baculovirusexpressed rabies virus M1 protein is not phosphorylated : it forms multiple complexes with expressed rabies N protein. Virology 189 : 766-770.
33) Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185 : 60-89.
34) Tordo, N., Poch, O., Ermine, A., Keith, G., and Rougeon, F. 1986. Walking along the rabies genome : is the large G-L intergenic region a remnant gene? Proc. Natl. Acad. Sci. U.S.A. 83 : 3914-3918.
35) Tuffereau, C., Fischer, S., and Flamand, A. 1985. Phosphorylation of the N and M1 proteins of rabies virus. J. Gen. Virol. 66 : 2285-2289.

Abstract

We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [³H] leucine and [³²P] orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme (s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor (s).

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