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RNA-Dependent RNA Polymerase Consensus Sequence of the L-A Double-Stranded RNA Virus: Definition of Essential Domains

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RNA-Dependent RNA Polymerase Consensus Sequence of the L-A Double-Stranded RNA Virus: Definition of Essential Domains Proc. Nati. Acad. Sci. USA Vol. 89, pp. 2185-2189, March 1992 Biochemistry RNA-dependent RNA polymerase consensus sequence of the L-A double-stranded RNA virus: Definition of essential domains JUAN CARLOS RIBAS AND REED B. WICKNER Section on the Genetics of Simple Eukaryotes, Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, Building 8, Room 207, National Institutes of Health, Bethesda, MD 20892 Communicated by Herbert Tabor, November 27, 1991 (received for review October 2, 1991) ABSTRACT The L-A double-stranded RNA virus of Sac- lacking M1 (reviewed in refs. 10 and 18). M1 depends on L-A charomyces cerevisiac makes a gag-pol fusion protein by a -1 for its coat and replication proteins (19). MAK10 is one of ribosomal frameshift. The pol amino acid sequence includes three chromosomal genes needed for L-A virus propagation consensus patterns typical of the RNA-dependent RNA poly- within yeast cells (20). In a maklO host, L-A proteins merases (EC 2.7.7.48) of (+) strand and double-stranded RNA expressed from a cDNA clone of L-A support the replication viruses of animals and plants. We have carried out "alanine- of the M1 satellite virus but (for unknown reasons) do not scanning mutagenesis" of the region of L-A including the two support propagation of the L-A virus itself (21). Thus, while most conserved polymerase motifs, SG...T...NT..N (. = any L-A requires the MAK10 product itself, M1 requires MAK10 amino acid) and GDD. By constructing and analyzing 46 only because it requires the L-A-encoded proteins. We have different mutations in and around the RNA polymerase con- used this phenomenon to assay the importance for viral sensus regions, we have precisely dermed the extent of domains propagation of specific sequences encoded by L-A. Previous and specific residues essential for viral replication. Assuming work to examine the importance of the amino acid patterns that this highly conserved region has a common secondary conserved among RNA-dependent RNA polymerases has structure among different viruses, we predict a largely fl-sheet dealt with the enzymes encoded by Qf phage, poliovirus, and structure. brome mosaic virus (22-24). We have defined the regions surrounding the two most The (+) strand RNA viruses of animals and plants share highly conserved RNA polymerase consensus patterns that amino acid sequence patterns in their RNA-dependent RNA are necessary for viral propagation. We show that, although polymerase (EC 2.7.7.48) regions (1-3) (Fig. 1). The same these domains are highly conserved among a broad range of patterns are present in most of the dsRNA viruses whose viruses in both primary structure and predicted secondary polymerase segment has been sequenced, including the L-A structure, they are not interchangeable. virus of Saccharomyces cerevisiae (4), reovirus (5), blue- tongue virus (6), and rotavirus (7). Two other dsRNA viruses MATERIALS AND METHODS [46 phage (8) and infectious bursal disease virus (9)] show a less clear fit to the pattern. The extent of these common Strains and Media. YPAD, YPG, 4.7MB, SD, and synthetic sequence patterns suggests that there is a common structure complete medium (25) and LB medium (26) have been and function among the more than 50 viral enzymes for which described. S. cerevisiae strains 2955p0 (MATa trpl adel his3 sequence data are available and that information obtained maklO-1 L-A-o M-o p0), 2629 (MATa leul karl-i L-A-HNB about one of them may be applicable to others. While M1), and 5X47 (MATa/MATa hisl /+ trpl/+ ura3/+ [KIL- different enzymes must recognize different sites on viral o]) were used. DNA sequencing was done by the dideoxy RNA and interact with different viral and host proteins, there method of Sanger et al. (27) with deoxyadenosine 5'-[a- are also common functions which they must carry out, [35S]thio]triphosphate, using a Sequenase kit [United States including binding of Mg2' and rNTPs, holding onto the Biochemical (28)]. template RNA chain, and the chain elongation reaction itself. Site-Directed Mutagenesis. Mutagenesis of the L-A cDNA It is likely that these conserved sequence patterns are part of expression plasmid pI2L2 (21) was carried out as described domains responsible for such common functions. by Kunkel (29), using the Muta-Gene kit from Bio-Rad. All The L-A dsRNA virus ofyeast replicates by a conservative mutations were confirmed by sequencing. To ensure that loss mechanism with (+) and (-) strands made inside the viral of activity was due to the introduced mutation and not to particle at different points in the replication cycle (reviewed changes elsewhere in the L-A sequence or vector, two or in ref. 10). In vitro systems for replication, transcription, and three mutants were checked for each change, and 16 wild- packaging are available, and the signals for the replication type clones (nonmutant at the site where mutagenesis was step and for packaging have been defined (11-14). L-A attempted) isolated during attempts to make mutations were encodes its 70-kDa major coat protein (called gag) (4, 15) and tested. All mutants of a given type were either all inactive or a 170-kDa gag-pol fusion protein (4, 16) formed by a -1 all active, and all ofthe wild-type clones isolated were active. ribosomal frameshift indistinguishable in mechanism from This approach may be ofgeneral utility in extensive localized that used by retroviruses for a similar purpose (17). The pol mutagenesis projects when complete resequencing of each region of the gag-pol fusion protein (4) has all the character- mutant isolate or insertion of mutagenized segments into istic sequence patterns identified by Kamer and Argos (1) as unmutagenized vectors would be impractical. typical of (+) strand RNA viral RNA-dependent RNA poly- Substitution of Homologous Domains of Other Viruses by merases. Asymmetric PCR. Fragments of 81, 105, and 225 nucleotides The M1 satellite virus of L-A encodes a protein, the killer from reovirus segment Li (5) and of 87, 159, and 180 toxin, that is secreted by cells carrying M1 and kills cells nucleotides from Sindbis virus (30) were amplified in a PCR process as described (31), in two steps using the Gene Amp The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: ssRNA and dsRNA, single-stranded and double- in accordance with 18 U.S.C. §1734 solely to indicate this fact. stranded RNA; ORF, open reading frame; aa, amino acids. 2185 Downloaded by guest on September 24, 2021 2186 Biochemistry: Ribas and Wickner Proc. Natl. Acad. Sci. USA 89 (1992) + Strand RNA Viruses: Carnat 524 GCRNMDNNALGNCLLACLITKH.............. LIKIRSRLINNflRCVLI TobEch 2580 KGNNEQPSXVVDIMNVIIAMLY .... .... TCEKCGINKEEIVYYVNMDDLLIA BromMV 521 FQR~fDAFYFG=LVTNAMIAY ............ ASDLSDCDCAIFSGDSLII TobMV 1440 YQRPEfDVTTFIGVIIAACLAS ....NI........K IKGAFcGfClSLLY Al fal faMV585 FQRM=DALZYAWUIVTLACLCH .... ........ VYDLMDPNFVVASMSLIG CowpeaMV1492 CGIPEFP !IVHIFNEILIRYHYK. 9 aa. ELNVQSFDKLIGLVTYgIDNLIS FIG. 1. Consensus sequence patterns ofviral RNA- dependent RNA polymerases of (+) single-stranded Mdlbrg 804 AMNKKW4FLILFVML3F4IAS .... ...... RVLEERLTNSKCAAFIERRNIVH RNA (ssRNA) viruses (1, 3) and double-stranded RNA WNileF 3120 DQRGMQVVTYALIFTNIAVQKV.25 aa.RTWLFENGEERLSRMAVSgRRCVVK YellowF 3100 DQRGDIQVVXYALifITNLKVQLI.25 aa.EAWLTEHGCDRLKRNAVSfRCVVR (dsRNA) viruses (4-9). The region shown here has the SINDBIS 2327 AMKKMXFLTLFVZVUVVIASRVL .......... EERLKTSRCAAFIMflRNIIH most extensive homology among these proteins, but DengueV 3088 DQRGEQVG!YGLMFTNEUAQLIRQ. 23 aa.WLARVGRERLSRNAISfIRRCVVK conserved patterns extend well beyond these in both N-terminal and C-terminal directions (1, 3). The most FootMDV 2156 GGMPflSCSATSII=ILNINIYVLYAL ...... RRHYEGVELDTYTNISYRIVVA EMC 2121 GGLICAATSNIflIMNNIIIRAGLY ...... LTYKNFEFDDVKVLSYflRLLVA highly conserved residues are shown by asterisks. The Rhinol4 2006 GGMPECSGXSIFIXINNIIIRT ..... LILDAYKGIDLD. KLKILAYflRLIVS viral sequences shown here are not more homologous Hepat A 1191 GSMPMfSPC!AILMIIPNVNLYY... VFSKIFGKSPVFFCQALKILCY MVLIF to L-A than those of other (+) ssRNA and dsRNA Polio 2025 GGNPM2CSGZSIFNMNINNLIIRT ..... LLLKTYKGIDLD.HLKMIAYgRVIAS viruses. Carnat, carnation mottle virus; TobEch, to- Coxsac B 300 GGMPIfCSGZSIFIMMINNIIIRT..... LMLKVYKGIDLD.QFRMIAYEDDVIAS bacco etch virus; MV, mosaic virus; TobMV, tobacco dURNA Viruses: mosaic virus; Mdlbrg, Middleburg virus; F, fever virus; L-A 544 GTLLQWRLTTFMtVLNWAYMKLAGV .......... FDLDDVQDSVHNflDVMIS FootMDV, foot-and-mouth disease virus; EMC, en- BTV 715 DTHILENSZLIANIMHXNAIGTLIQRA.... VGREQPGILTFLSEQYVMUfTLFY cephalomyocarditis; BTV, bluetongue virus; IBDV, ROTA V 586 GAVAMEKQZKAA]gIAXLULIKTVLSRISN ...... KYSFATKIIRVDgBMYAV REOVIRUS 676 TTFPSSTAZSTEUANNSTMMETFLTV... 20 aa . QRNYVCQOfWiGLMI infectious bursal disease virus; aa, amino acids. Amino PHI6 390 VGLSMQGAZDlIMg!LLSITYLVMQLD.24 aa.... QGHEEIRQISKARAILG acid symbols shown in lowercase do not agree with the IBDV 476 YGQGUNAA!FINULIETLVLDQWNL... 20 aa . NFKIERSIDDIRGK consensus. kit (Perkin-Elmer/Cetus). The first reaction mixture con- an in vivo assay ofthe activity ofthe proteins encoded by the tained 100 mM Tris HCI at pH 8.3, 50 mM KCl, 1.5 mM L-A cDNA clone in the absence of the L-A virus itself. The MgCl2, 0.001% gelatin, each dNTP at 0.2 mM, 2.5 units of stable maintenance of M1 by the L-A cDNA clone requires Taq DNA polymerase, 10-4 pmol of CsCl-purified DNA of both the major coat protein
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