Abstract
Poliovirus RNA-dependent RNA polymerase 3D and viral protein 3AB are both thought to be required for the initiation of RNA synthesis. These two proteins physically associate with each other and with viral RNA replication complexes found on virus-induced membranes in infected cells. An understanding of the interface between 3D and 3AB would provide a first step in visualizing the architecture of the multiprotein complex that is assembled during poliovirus infection to replicate and package the viral RNA genome. The identification of mutations in 3D that diminish 3D-3AB interactions without affecting other functions of 3D polymerase is needed to study the function of the 3D-3AB interaction in infected cells. We describe the use of the yeast two-hybrid system to isolate and characterize mutations in 3D polymerase that cause it to interact less efficiently with 3AB than wild-type polymerase. One mutation, a substitution of leucine for valine at position 391 (V391L), resulted in a 3AB-specific interaction defect in the two-hybrid system, causing a reduction in the interaction of 3D polymerase with 3AB but not with another viral protein or a host protein tested. In vitro, purified 3D-V391L polymerase bound to membrane-associated 3AB with reduced affinity. Poliovirus that contained the 3D-V391L mutation was temperature sensitive, displaying a pronounced conditional defect in RNA synthesis. We conclude that interaction between 3AB and 3D or 3D-containing polypeptides plays a role in RNA synthesis during poliovirus infection.
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- Barton D. J., Black E. P., Flanegan J. B. Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA. J Virol. 1995 Sep;69(9):5516–5527. doi: 10.1128/jvi.69.9.5516-5527.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barton D. J., Morasco B. J., Eisner-Smerage L., Collis P. S., Diamond S. E., Hewlett M. J., Merchant M. A., O'Donnell B. J., Flanegan J. B. Poliovirus RNA polymerase mutation 3D-M394T results in a temperature-sensitive defect in RNA synthesis. Virology. 1996 Mar 15;217(2):459–469. doi: 10.1006/viro.1996.0140. [DOI] [PubMed] [Google Scholar]
- Bienz K., Egger D., Pasamontes L. Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography. Virology. 1987 Sep;160(1):220–226. doi: 10.1016/0042-6822(87)90063-8. [DOI] [PubMed] [Google Scholar]
- Bienz K., Egger D., Rasser Y., Bossart W. Intracellular distribution of poliovirus proteins and the induction of virus-specific cytoplasmic structures. Virology. 1983 Nov;131(1):39–48. doi: 10.1016/0042-6822(83)90531-7. [DOI] [PubMed] [Google Scholar]
- Busby S., Irani M., Crombrugghe B. Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments. J Mol Biol. 1982 Jan 15;154(2):197–209. doi: 10.1016/0022-2836(82)90060-2. [DOI] [PubMed] [Google Scholar]
- Datta U., Dasgupta A. Expression and subcellular localization of poliovirus VPg-precursor protein 3AB in eukaryotic cells: evidence for glycosylation in vitro. J Virol. 1994 Jul;68(7):4468–4477. doi: 10.1128/jvi.68.7.4468-4477.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Diamond S. E., Kirkegaard K. Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperature-sensitive mutants defective in RNA synthesis. J Virol. 1994 Feb;68(2):863–876. doi: 10.1128/jvi.68.2.863-876.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elble R. A simple and efficient procedure for transformation of yeasts. Biotechniques. 1992 Jul;13(1):18–20. [PubMed] [Google Scholar]
- Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
- Golemis E. A., Brent R. Fused protein domains inhibit DNA binding by LexA. Mol Cell Biol. 1992 Jul;12(7):3006–3014. doi: 10.1128/mcb.12.7.3006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gyuris J., Golemis E., Chertkov H., Brent R. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell. 1993 Nov 19;75(4):791–803. doi: 10.1016/0092-8674(93)90498-f. [DOI] [PubMed] [Google Scholar]
- Kitamura N., Semler B. L., Rothberg P. G., Larsen G. R., Adler C. J., Dorner A. J., Emini E. A., Hanecak R., Lee J. J., van der Werf S. Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature. 1981 Jun 18;291(5816):547–553. doi: 10.1038/291547a0. [DOI] [PubMed] [Google Scholar]
- Lama J., Paul A. V., Harris K. S., Wimmer E. Properties of purified recombinant poliovirus protein 3aB as substrate for viral proteinases and as co-factor for RNA polymerase 3Dpol. J Biol Chem. 1994 Jan 7;269(1):66–70. [PubMed] [Google Scholar]
- Ma J., Ptashne M. A new class of yeast transcriptional activators. Cell. 1987 Oct 9;51(1):113–119. doi: 10.1016/0092-8674(87)90015-8. [DOI] [PubMed] [Google Scholar]
- McBride A. E., Schlegel A., Kirkegaard K. Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2296–2301. doi: 10.1073/pnas.93.6.2296. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Molla A., Harris K. S., Paul A. V., Shin S. H., Mugavero J., Wimmer E. Stimulation of poliovirus proteinase 3Cpro-related proteolysis by the genome-linked protein VPg and its precursor 3AB. J Biol Chem. 1994 Oct 28;269(43):27015–27020. [PubMed] [Google Scholar]
- Novak J. E., Kirkegaard K. Coupling between genome translation and replication in an RNA virus. Genes Dev. 1994 Jul 15;8(14):1726–1737. doi: 10.1101/gad.8.14.1726. [DOI] [PubMed] [Google Scholar]
- Pata J. D., Schultz S. C., Kirkegaard K. Functional oligomerization of poliovirus RNA-dependent RNA polymerase. RNA. 1995 Jul;1(5):466–477. [PMC free article] [PubMed] [Google Scholar]
- Paul A. V., Cao X., Harris K. S., Lama J., Wimmer E. Studies with poliovirus polymerase 3Dpol. Stimulation of poly(U) synthesis in vitro by purified poliovirus protein 3AB. J Biol Chem. 1994 Nov 18;269(46):29173–29181. [PubMed] [Google Scholar]
- Pfister T., Egger D., Bienz K. Poliovirus subviral particles associated with progeny RNA in the replication complex. J Gen Virol. 1995 Jan;76(Pt 1):63–71. doi: 10.1099/0022-1317-76-1-63. [DOI] [PubMed] [Google Scholar]
- Plotch S. J., Palant O. Poliovirus protein 3AB forms a complex with and stimulates the activity of the viral RNA polymerase, 3Dpol. J Virol. 1995 Nov;69(11):7169–7179. doi: 10.1128/jvi.69.11.7169-7179.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Racaniello V. R., Baltimore D. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4887–4891. doi: 10.1073/pnas.78.8.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Richards O. C., Baker S., Ehrenfeld E. Mutation of lysine residues in the nucleotide binding segments of the poliovirus RNA-dependent RNA polymerase. J Virol. 1996 Dec;70(12):8564–8570. doi: 10.1128/jvi.70.12.8564-8570.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Richards O. C., Hanson J. L., Schultz S., Ehrenfeld E. Identification of nucleotide binding sites in the poliovirus RNA polymerase. Biochemistry. 1995 May 16;34(19):6288–6295. doi: 10.1021/bi00019a005. [DOI] [PubMed] [Google Scholar]
- Sarnow P. Role of 3'-end sequences in infectivity of poliovirus transcripts made in vitro. J Virol. 1989 Jan;63(1):467–470. doi: 10.1128/jvi.63.1.467-470.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
- Schlegel A., Giddings T. H., Jr, Ladinsky M. S., Kirkegaard K. Cellular origin and ultrastructure of membranes induced during poliovirus infection. J Virol. 1996 Oct;70(10):6576–6588. doi: 10.1128/jvi.70.10.6576-6588.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shih H. M., Goldman P. S., DeMaggio A. J., Hollenberg S. M., Goodman R. H., Hoekstra M. F. A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the coactivator CBP. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13896–13901. doi: 10.1073/pnas.93.24.13896. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Towner J. S., Ho T. V., Semler B. L. Determinants of membrane association for poliovirus protein 3AB. J Biol Chem. 1996 Oct 25;271(43):26810–26818. doi: 10.1074/jbc.271.43.26810. [DOI] [PubMed] [Google Scholar]
- Vidal M., Brachmann R. K., Fattaey A., Harlow E., Boeke J. D. Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10315–10320. doi: 10.1073/pnas.93.19.10315. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vidal M., Braun P., Chen E., Boeke J. D., Harlow E. Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10321–10326. doi: 10.1073/pnas.93.19.10321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xiang W., Cuconati A., Paul A. V., Cao X., Wimmer E. Molecular dissection of the multifunctional poliovirus RNA-binding protein 3AB. RNA. 1995 Nov;1(9):892–904. [PMC free article] [PubMed] [Google Scholar]
- van der Werf S., Bradley J., Wimmer E., Studier F. W., Dunn J. J. Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2330–2334. doi: 10.1073/pnas.83.8.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]