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 (gag) and the gag-pol fusion Sindbis virus clone TotollOl (30) or a clone of reovirus protein (21). segment Li in pBR322 (5), and 100 pmol each of two Semiquantitative Killer Activity Assay. To determine not synthetic 55-mer primers (with the 5' 30 bases of each only ifa specific L-A cDNA mutation permits M1 propagation complementary to the L-A flanking sequences and the 3' 25 but also different degrees of activity when M1 is ultimately bases of each complementary to the ends of the reovirus or lost (see Results), we designed a semiquantitative method. Sindbis virus sequence to be amplified). The samples were The 2955p° transformants were grown for 4 days at 30°C, subjected to 35 cycles of amplification in a DNA thermal pooled and grown overnight, mixed in 0.1 ml of water with cycler (Perkin-Elmer/Cetus) as follows: 94°C for 1.5 min, approximately equal amounts of the freshly grown donor 37°C for 1.5 min, and 72°C for 2 min. Then, 2.5-10% of the strain, 2629, and poured onto a YPAD plate to allow cyto- product was used as template for a second identical PCR duction. After 6-8 hr at 30°C, the mating mixture was except that only the 55-mer primer having the L-A (-) strand streaked for single colonies on synthetic complete medium sequence was included. This generated an excess of the lacking leucine and tryptophan, to select against the donor strand to be used as a mutagenic oligonucleotide. Then, 40% strain and for cells carrying the L-A cDNA expression of the reaction products in 8 ,ul of H20 were 5' phosphory- plasmid. After 4 days, colonies were replica plated to SD lated with T4 polynucleotide kinase (BRL) and used for medium, on which only diploids can grow, to YPG, on which site-directed mutagenesis of the L-A cDNA expression plas- only diploids and cytoductants can grow, and on 4.7MB mid pI2L2 as described above. plates having a lawn of strain 5X47, to test (3 days, 22°C) for Assay of Altered L-A Expression Vectors. The L-A expres- the maintenance of M1 dsRNA by the killer phenotype. The sion vector pI2L2 (21) has the PGKI promoter (32), the L-A presence or absence ofM1 in the cytoductants is the indicator sequences from one ofour full-length clones (4), the origin of of whether the L-A proteins made from the mutant cDNA replication of the 2-,um DNA plasmid, the yeast TRPI gene clone were active or inactive. for selection in yeast, the fl phage replication origin for For each mutant cDNA, 50-100 cytoductants were exam- production of ssDNA, and pBR322 sequences (Fig. 2). ined and their killer zones were estimated as wild-type Amino acid residues in L-A's pol ORF are numbered starting (100%o), 1/2-size (50%), ¼/4-size (25%), dark blue color around with the Arg residue at base 1964, which is the first amino acid the colony but no clear zone (10%1), or no sign of killing (0%1). after the -1 ribosomal frameshift (17). The L-A expression The results obtained for different mutants having the same vector pI2L2, specifically mutated in the L-A coding se- amino acid change were quite similar. quences, was introduced by transformation (33) into yeast strain 2955p°. Into this strain was then introduced, by cyto- plasmic mixing [cytoduction (34)], M1 and L-A viruses. RESULTS Because this strain is defective in MAKIO, it is unable to It has been argued that for scanning a protein with amino acid maintain L-A dsRNA, even though the normal L-A proteins changes, mutagenesis to alanine is the most "neutral" change are supplied from the cDNA vector. M1 is stably maintained, that one can make (35). This amino acid is neither bulky nor however, and is present in viral particles (21). This provides particularly small. It is not a helix-breaker and is neither

L-A RNA polywras. SG...T...NT..NGDD Yeast PGK ORFI Yest ft Promoter Major Cost Prenin> ORF2 ,%- 2 N TRPI or RNJ 2 ADNAOr! Amp Oil I I aI ~~~~~~~I~~I

FIG. 2. The L-A expression vector pI2L2, which was the starting point for the mutations described here. PGK, phosphoglycerate kinase; ORF, open reading frame; ori, replication origin. Downloaded by guest on September 24, 2021 Biochemistry: Ribas and Wickner Proc. Natl. Acad. Sci. USA 89 (1992) 2187

particularly hydrophobic nor polar. These ideas have been A Domain2 Downstream encapsulated in the expression, "alanine-scanning mutagen- Plasmid Activity Wild-type = pl2L2 aa 579 G D D aa 707 G D D 100% esis," and we have adopted this approach to simplify the task M4 A E E G DD 0.4 of defining essential regions in the L-A pol ORF. M5 G D D A £ F 100 L-A pol Domains. We substituted alanine for pairs ofamino Domain 1 acids or single residues as shown in Fig. 3. The most highly B conserved residues [544SG, 549T, 579GDD (the number Wild-type = pl2L2 S GWR L T T F M N T V L N 100% M53 SGWRLTTFMNSVLN 100 always indicates the first residue)] were all essential for M54 SGWRLTTFMNTVL_ 2 activity (<1%), but the less strictly conserved 553NT (which poliolC S G.QC Q T 2 F NTV L N 0.3 reolC SG TT I f N T VL N 0.4 is NS, HT, or GT in some viruses) was 4% active when mdl SGMFLTL F V N T M L N 2 changed to AA, and 557N (which is T, M, S, . . . in other viruses) was 17% when changed to A. 554T is often S in other viruses, and this works in L-A as well, but 557N cannot be C M55 LNWADLDD 18% changed to T (Fig. 4B). The eight nonconserved residues L-A domain I L-A domain 2 inside the SG...T...NT..N motif were essential, but the rates AMAAAAA of loss of M1 appeared to be slower than for the adjacent FIG. 4. (A) Effect of changing 579GDD to AEE and 707GDD to conserved residues (except for 557N). AEE. (B) Changes in domain 1: Effect of changing 554T to S and We sought to establish borders of putative functional 557N to T and replacement of residues within L-A's RNA polymer- domains. The SG ...T... NT..N domain comprised 22 amino ase SG...T...NT..N motifwith homologous domains from poliovirus acids from residue 540 to residue 561, and the 579GDD (polio 1C), reovirus (reolC), and Middleburg virus (mdl). Changed domain was 29 amino acids from residue 565 to residue 593. residues are indicated by underlining. (C) Expansion of the interdo- Having localized the limits for both domains, we constructed main region of L-A with six alanine residues inactivates it. single-residue changes of each couple to ascribe this limit to mid, though less dramatically than a conserved residue one specific amino acid. In the couple 54OGT, only the T was that this distance is important. lethal, while in 560YM, either alone was lethal. Thus, the mutation, indicating When full killer colonies from -- AA and 562KL SG...T...NT..N domain (domain 1) comprises 21 amino acids 538LQ AA, and weak killer colonies from 562GV -- AA, 573QD from 541T to 561M. Although 565GV -k AA was less than AA, 575SV -- AA, 584IS -* AA, and 586LN -- AA were 25% active, both 565G -* A and 566V -- A were fully active, suggesting that each is partially necessary. Thus, the GDD restreaked for single colonies on media selecting retention of domain is 29 residues from 565G to 593V (domain 2). the L-A expression plasmid, all of the colonies from mutant There are two GDD sequences in the L-A pol ORF, one groups that did not affect the killer activity in the first assay starting at residue 579 and the other at residue 707. While any remained full killers, but those from mutant groups with of the substitutions in the GDD starting at 579 were lethal, reduced activity in the first assay now had no killer activity there was no effect of 707GDD -> AEE (Fig. 4A). Between at all. Thus, the former maintain M1 as stably as the wild type, the two domains, there are 3 to 5 residues that were not and the latter will ultimately completely lose M1, though they essential for activity (at least 562KLA). Since the number of may vary in their rate of its loss. residues between the domains is quite variable among various Effect of Partial and Full Domain Substitutions. We re- viruses, we sought to determine if this distance is critical by placed the L-A domains with homologous, but nonidentical, inserting 6 alanine residues at this point (Fig. 4C). This regions from other viruses. We first substituted the noncon- mutation largely, but not completely, inactivated the plas- served residues within the SG...T...NT..N motif with the Domain la Domain 2 -I1 100- Domain lb I Ir

>,75-

0)

. 1:11 25- t 1v RM~~~~~~~nX~ 0 .

-41- .-.ri F-.. 41 WVLDPDTKEWYRLOGTLLSGWRLTTFMNITVLNWAYMKLAGVFDLDDVODSVHNGDDVMI SLNRVSTAVRIMDAMHRII.. L-A------seauence * * * *** _ _ ___AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAEEAAAAAAAAAAAAAAAAhAAAAAC___ _ mutantt aa 7rlt- 3 -IT 4 O4252 1 1 22X 3 ER30L3 1 4 4 0 ° No. of mutants 2 2 3 2 2 3 2 4 1 2 2 3 3 3 3 2 3 2 2 2 2 3 3 2 1 1 3 3 2 2 2 6 3 2 3 3 tested

FIG. 3. Alanine-scanning mutagenesis of the L-A RNA polymerase region. The regions necessary to efficiently replicate the satellite virus M1 extend substantially beyond the conserved residues. Percent killer activity reflects the rate of loss of M1. Only mutants with 100%t killer activity here can stably maintain M1. Downloaded by guest on September 24, 2021 2188 Biochemistry: Ribas and Wickner Proc. Natl. Acad. Sci. USA 89 (1992)

Reovirus Ll domain 1 border ofa domain in L-A, with residues from the other virus 21 aa (+ 3 aa on each end) which might be involved in the function ofits (possibly larger) MR1 ¶FPfQSTAISTEHIANNSTMMETF domain (MS). In some cases, we inserted extra amino acid YRLQl3TLLMWRLIFMIVLNWAY GV L-A domainl residues from the other virus at the borders of the L-A 21 aa domains (MR). In none of these substitutions did we obtain substantial activity (Fig. 5). For reovirus the distance be- Reovirus LI domain 2 29as (+ 3aaon each end) tween its SG...T...NT..N and GDD motifs is 16 amino acid MR2 LMLRKMIKSL1QRNWCQ LRGG IIDG1rAGK>JN residues greater than for L-A. Ifthis distance were important, YMKL>GVFDLDDV DSVHNGDDVMISLNRVSTAV IMDA as it seems to be for L-A, then we may have failed to see L-A domain 2 29 aa activity for this reason. Sindbis virus has the same distance between the SG..T. ..NT..N and GDD motifs as does L-A. Reovirus Li domains 1 +2 69 (+3 aa on each end) The failure of these substitutions to work may be because tjQ interactions among more than just the two domains are KADIMMl +L ZE13 7 IYHL TLJ ...... VSTA .I MDA critical or because one or both domains are involved in L-A domains 1 + 2 site-specific binding to the template. 53 aa Consensus Secondary Structures. The presence of consen- sus patterns for viral RNA in Sindbis domains la + 2 RNA-dependent polymerases 6O aa such a large group of animal, plant, and fungal viruses ...... MS1a+2 PTGRFKFGAMMKSI SDKEMAq indicates that these enzymes must have a common secondary VLIrUJI rWcYLU ...... VtSjAVfIMUAMHV structure in these regions. Although the secondary structure L-A domains Ia +2 prediction programs currently available are only about 60%6 60 aa accurate when predicting the structure of a single residue of Sindbis domains lb + 2 a single protein (36), if used on a homologous set of proteins 53 aa with the assumption that they have a common secondary TGTRFKFU MK1% ...... SDKEMIAE MS1b+2 structure in the region under consideration, the consensus VLDPDTKEWYRIQGMI5.VSTAV,I DAMH prediction should be far more accurate. We have applied the L-A domains lb +2 method ofRobson and Garnier (37) to 42 known or presumed 53 aa viral RNA-dependent RNA polymerases, aligning the struc- Sindbis domain 2 tures by using the regions of homology (Fig. 6). The results 29 aa RVLEERLKTSRCMFIGNIIHGWSDKEMAE show a remarkable degree ofagreement among the structures MS2 12% predicted for various viruses, suggesting that this approach YM=GVFDLDDVODSVHNVMISLNRVSTAVRv DAMH may be valid. The most conserved regions are predicted to L-A domain 2 have 3-sheet structure with turns at the most conserved 29 aa residues, a result similar to that reported previously for a FIG. 5. Substitutions of reovirus (MR) and Sindbis virus (MS) slightly different domain for a set of RNA-dependent poly- domains for RNA polymerase consensus domains in L-A and effect merases that overlaps with those we have studied (3). on activity. wt, Wild type. corresponding residues from poliovirus, reovirus, and Mid- DISCUSSION dleburg virus (Fig. 4B). None of these substitutions left any RNA-dependent RNA polymerases are unique to viruses. substantial activity. We then constructed substitution mu- This means that such enzymes should be possible targets for tants in which all of domain 1, all of domain 2, or both were antiviral drugs. Fortunately, the RNA-dependent RNA poly- substituted from homologous regions of other viruses into merases of (+) strand ssRNA viruses and dsRNA viruses L-A to determine if they were interchangeable. While the share substantial common sequence patterns (1-3, 38). If one borders of domain 2 in Fig. 3 seem clear, the left border of could determine the specific function of these residues, it domain 1 appeared ambiguous, and so in some experiments, might be possible to design drugs, based on the substrate, that we made substitutions of domain la and of lb (MS). We also would act to block the replication of whole classes of animal replaced residues that did not seem to be essential, at the and plant viruses. It was with this admittedly ambitious goal

L-A ORF2: E W Y R L Q G T LL S G W R LT T F M N T V L E W A Y M K L L-A Predicted HH EE E E T EEE C T E EE E EE E EE HH HHHH H H E Helix 151210 8 7 8 5 4 4 4 5 5 5 6 9 8 9 9111216211921222424262219 Sheet 192629313229101714 5 3 9162827272626232217181614181514121215 Turn 5 1 0 0 1 3251915161612 6 0 1 0 2 2 2 2 1 0 0 0 0 0 0 0 0 0 Coil 0 1 1 1 0 0 0 0 714161413 6 3 5 3 3 4 4 6 1 5 4 0 0 0 0 0 0 Consensus conformation: EEE E E E T TTT CCEE E E E E E EE HHHHH HH HH

L-A ORF2 A G V F D L D D V 0 D S V H N G D V M I S L N R V S T A V L-A Predicted E E E E H HHH HH E EE CC T T EE EE E E E H H H E E E * .** ** * ** ** * ** * ** ** .*** Helix 1715191724303431292516 8 911101112171512 9121320262623252829 Sheet 12 9111410 4 4 7 9142323128j11 3 81825293128311812 810141110 Turn 3 4 5 6 6 3 3 3 2 2 2 0 1 2172620 6 0 0 1 1 0 1 2 2 3 0 0 0 Coil 5 9 5 2 0 3 1 0 1 0 0 0 0 1 3 1 1 1 2 0 0 0 0 1 0 4 4 1 1 1 Consensus conformation: H HHH H H HHH H E E E E T T T E E E E E E HH H HH H H FIG. 6. Conservation of secondary structure among many RNA-dependent RNA polymerases from diverse viruses. The program ofRobson and Gamier (37) was used to predict the conformations of 42 known and presumed RNA-dependent RNA polymerases of (+) ssRNA viruses and dsRNA viruses. The output was aligned by using the conserved residues (double underlining), and the number of sequences predicted to have the indicated conformation is tabulated. Asterisk, L-A prediction agrees with consensus; dot, almost agrees; E, ,-sheet; H, a-helix; T, turn; and C, coil. Domains 1 and 2 are underlined. Downloaded by guest on September 24, 2021 Biochemistry: Ribas and Wickner Proc. Natl. Acad. Sci. USA 89 (1992) 2189 that we began to define domains in the L-A virus pol ORF 1. Kamer, G. & Argos, P. (1984) Nucleic Acids Res. 12, 7269- surrounding the conserved RNA-dependent RNA polymer- 7282. 2. Argos, P. (1988) Nucleic Acids Res. 16, 9909-9916. ase sequence patterns that are essential for viral propagation. 3. Poch, O., Sauvaget, I., Delarue, M. & Tordo, N. (1989) EMBO Previous studies of these regions in other viruses have been J. 8, 3867-3874. limited to mutation of the G residue of the GDD motif in 4. Icho, T. & Wickner, R. B. (1989) J. Biol. Chem. 264, 6716- poliovirus and Q83 phage (22, 23) and mutation in brome 6723. mosaic virus of several residues in and around the D... [YF]D 5. Wiener, J. R. & Joklik, W. K. (1989) Virology 169, 194-203. 6. Roy, P., Fukusho, A., Ritter, G. D. & Lyon, D. (1988) Nucleic consensus pattern that is N-terminal to the SG...T...NT..N Acids Res. 16, 11759-11767. motif (24), all resulting in decreased or temperature-sensitive 7. Cohen, J., Charpilienne, A., Chilmonczyk, S. & Estes, M. K. activity. One study of QJ3 replicase found that insertions in (1989) Virology 171, 131-140. most parts of the molecule inactivate it (39). In the studies of 8. Mindich, L., Nemhauser, I., Gottlieb, P., Romantschuk, M., poliovirus (23), polymerase was produced in Escherichia coli Carton, J., Frucht, S., Strassman, J., Bamford, D. H. & in Kalkkinen, N. (1988) J. Virol. 62, 1180-1185. soluble form and the mutants produced were examined for 9. Morgan, M. M., Macreadie, I. G., Harley, V. R., Hudson, some of their enzymatic properties. Although some substi- P. J. & Azad, A. A. (1988) Virology 163, 240-242. tutions resulted in decreased activity, and not simply a 10. Wickner, R. B. (1991) in Molecular and Cellular Biology ofthe "dead" enzyme, no qualitative changes in the enzyme were Yeast Saccharomyces, eds. Jones, E. W., Pringle, J. & Broach, found that would suggest the nature of the defect. Some J. R. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. mutations of the human immunodeficiency virus reverse 263-2%. 11. Fujimura, T. & Wickner, R. B. (1988) J. Biol. Chem. 263, transcriptase domains have also been analyzed (40). Al- 454-460. though in vitro systems for L-A replication, transcription, 12. Fujimura, T. & Wickner, R. B. (1989) J. Biol. Chem. 264, and a partial packaging reaction have been developed (11, 10872-10877. 12), we were unable to assay these activities in particles 13. Esteban, R., Fujimura, T. & Wickner, R. B. (1989) EMBO J. 8, produced from the L-A expression vector. Thus our studies 947-954. have been initially restricted to measuring the effects of the 14. Fujimura, T., Esteban, R., Esteban, L. M. & Wickner, R. B. (1990) Cell 62, 819-828. mutations on overall viral propagation, assuming that 15. Hopper, J. E., Bostian, K. A., Rowe, L. B. & Tipper, D. J. changes in this region of pol are affecting mostly RNA- (1977) J. Biol. Chem. 252, 9010-9017. dependent RNA polymerase activity. 16. Fujimura, T. & Wickner, R. (1988) Cell 55, 663-671. This is the most extensive study made to date of these 17. Dinman, J. D., Icho, T. & Wickner, R. B. (1991) Proc. Natl. domains. We found that all of the consensus residues exam- Acad. Sci. USA 88, 174-178. ined were essential. Moreover, residues in the surrounding 18. Bussey, H. (1988) Yeast 4, 17-26. 19. Bostian, K. A., Sturgeon, J. A. & Tipper, D. J. (1980) J. areas were also necessary, in various degrees, for viral Bacteriol. 143, 463-470. propagation. Our experiments confirm part of the "consen- 20. Sommer, S. S. & Wickner, R. B. (1982) Cell 31, 429-441. sus" approach in that the most stringently required residues 21. Wickner, R. B., Icho, T., Fujimura, T. & Widner, W. R. (1991) were, in fact, those which are most conserved ones, except J. Virol. 65, 155-161. perhaps for the NT and N residues of the SG...T...NT..N 22. Inokuchi, V. & Hirashima, A. (1987) J. Virol. 61, 3946-3949. motif. However, the domain sizes predicted (3) were 28 and 23. Jablonski, S. A., Luo, M. & Morrow, C. D. (1991) J. Virol. 65, 11 residues for the SG...T...NT..N and GDD motifs, respec- 4564-4572. 24. Kroner, P., Richards, D., Traynor, P. & Ahlquist, P. (1989) J. tively, but we found 21 and 29 residues as their actual extents. Virol. 63, 5302-5309. Of course, our work assumes that failure of viral propagation 25. Wickner, R. B. & Leibowitz, M. J. (1976) Genetics 82, 429- is due to failure ofRNA polymerase activity, and we have not 442. yet been able to measure this directly. Ifsequences with other 26. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular functions are interspersed among the RNA polymerase mo- Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold tifs, it would confuse our results. It will be of interest to Spring Harbor, NY), pp. 250-251. compare the results obtained here with similar studies from 27. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. other systems when they become available. 28. Tabor, S. & Richardson, C. C. (1987) Proc. Natl. Acad. Sci. We could locate a three- to five-amino acid region between USA 84, 4767-4771. the SG.. .T...NT..N and GDD motifs that was not essential, 29. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. but even this region could not be freely changed in length. 30. Rice, C. M., Levis, R., Strauss, J. H. & Huang, H. V. (1987) Insertion of six alanine residues prevented viral propagation. J. Virol. 61, 3809-3819. The importance of this interdomain distance may, in some 31. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, cases, have prevented our seeing activity with substitutions R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science of we 239, 487-491. sequences from other viruses. Although focused on the 32. Hitzeman, R. A., Leung, D. W., Perry, L. J., Kohr, W. J., two most conserved domains, less conserved motifs in other Levine, H. L. & Goeddel, D. V. (1983) Science 219, 620-625. parts of the molecule have been recognized (1, 3), and 33. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983) J. specific interactions of these with the two domains we Bacteriol. 153, 163-168. studied could have made substitution mutants inactive. Also 34. Conde, H. & Fink, G. R. (1976) Proc. Natl. Acad. Sci. USA 73, potentially critical may be some template sequence-specific 3651-3655. information encoded in these domains. Although we argue 35. Cunningham, B. C. & Wells, J. A. (1989) Science 244, 1081- that this is it is not and exam- 1085. (above) unlikely, impossible, 36. Gamier, J. (1990) Biochemie 72, 513-524. ination, in the L-A system, of where such specificity resides, 37. Robson, B. & Gamier, J. (1986) Introduction to Proteins and may be enlightening. Protein Engineering (Elsevier, Amsterdam). 38. Delarue, M., Poch, O., Tordo, N., Moras, D. & Argos, P. We thank Charles Rice, Wolfgang Joklik, and Bert Semler for (1990) Protein Eng. 3, 461-467. generously providing clones of Sindbis virus, reovirus, and poliovi- 39. Mills, D. R., Priano, C., DiMauro, P. & Binderow, B. D. (1988) rus RNA polymerases, respectively. J.C.R. acknowledges postdoc- J. Mol. Biol. 205, 751-764. toral fellowship support from the Ministerio de Educaci6n y Ciencia, 40. Larder, B. A., Kemp, S. D. & Purifoy, D. J. M. (1989) Proc. Spain. Natl. Acad. Sci. USA 86, 4803-4807. Downloaded by guest on September 24, 2021