INTRODUCTION

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Rubella virus (RUB) is the sole member of the Rubivirus genus of the Togaviridae family of animal viruses. This family also includes the Alphavirus genus, whose type species is Sindbis virus (SIN). In many regards, RUB is a typical representative of the togaviruses, although important differences between RUB and the alphaviruses have been discovered (reviewed in reference 7; the molecular biology of alphaviruses is reviewed in reference 31). As with the alphaviruses, the RUB virion contains an icosahedral nucleocapsid consisting of the single-stranded, plus-polarity genomic RNA of approximately 10,000 nucleotides (nt) and multiple copies of a single capsid protein C. The genomic RNA is capped and polyadenylated. The nucleocapsid is surrounded by a lipid envelope containing two viral glycoproteins, E1 and E2. In the cytoplasm of infected cells, the genomic RNA serves as an mRNA for translation of a 240-kDa polyprotein precursor that is posttranslationally cleaved by a viral papain-like cysteine protease into P150 and P90, two nonstructural proteins (NSPs) thought to function in virus RNA replication (1, 5, 15). The NSPs are encoded by an open reading frame (ORF) that covers the 5' terminal two-thirds of the genome. It is in the processing of the NSP precursor that RUB differs most from alphaviruses. The processing of the alphavirus precursor involves a sophisticated cascade of temporally regulated cleavages resulting in production of four NSPs as well as a number of specific products of incomplete proteolysis. This process is believed to orchestrate the course of virus RNA synthesis in which the different polypeptides perform different specific functions (reviewed in reference 31). In contrast, the single cis cleavage mediated by the RUB NS protease would not allow such an elaborate regulation.

The RUB genomic RNA next functions as the template for the synthesis of a complementary genome-length, minus-polarity RNA which serves in turn as the template for production of new genomic plus strands as well as a subgenomic RNA. The subgenomic RNA contains sequences from the 3'-terminal one-third of the genome and is the mRNA for translation of the structural proteins (SPs; C, E2, and E1) encoded by the SP ORF. The individual SPs are generated by cotranslational cleavage of the polyprotein precursor by cellular signalase. Synthesis of the subgenomic RNA is initiated on the minus-strand template at a site located between the two ORFs.

One of the most intriguing similarities between alphaviruses and RUB is the conservation of three regions of homologous nucleotide sequence and/or structure which are thought to be regulatory signals for viral replication (3). One of these is a stem-and-loop structure at the 5' end of the genomic RNA [5'(+)SL]. The RUB 5'(+)SL can stimulate translation of reporter genes both in vitro and in vivo and was shown to bind a number of cellular proteins, one of which is the La autoantigen (18, 24, 25). The functional significance of the cell protein binding is unknown.

In togaviruses, the complementary equivalent of the 5'(+)SL present at the 3' end of viral minus-strand RNA [3'()SL] is thought to serve as a promoter for initiation of genomic plus-strand RNA by the viral replicase (31). The 3'()SL of both SIN and RUB have been shown to bind cellular proteins (18, 20, 21); in the case of SIN, one of these was identified as mosquito analog of the La autoantigen (22). In SIN, the 5'(+)SL and 3'()SL occur at the exact ends of the plus- and minus-strand RNAs. Site-directed mutagenesis of these structures conducted with an infectious clone of SIN revealed that only deletions at the immediate end of the genomic RNA (nt 5 or nt 2 to 4), which are located at the bottom of the stem, were lethal (19). Deletions downstream from this site of 1 to 15 nt resulted in viable viruses, some of which grew less well than the parental virus. Although the effect of these mutations on the viral macromolecular synthesis was not examined, these observations are currently considered as evidence of the importance of the 3'()SL for initiation of genomic RNA synthesis. In terms of this model, RUB is a convenient counterpart of SIN in that the 5'(+)SL is located 14 nt downstream from the exact 5' end. Therefore, it should be possible to perform similar mutagenic analysis of RUB to distinguish whether the exact 5' nucleotides, the stem-and-loop structure, or both are crucial for virus viability.

Another intriguing feature at the 5' end of the RUB genome is the presence of a short ORF between nt 3 and 54 that could encode a 17-amino-acid peptide. This ORF overlaps the start of the NSP ORF at nt 41 in an alternate translational frame and thus could also potentially downregulate translation of the NSP ORF. In this study, we determined the 5'-terminal sequences of additional strains of RUB to ascertain that both the secondary structure and the short ORF were conserved. Then we used Robo302, an infectious clone of RUB (27), to mutagenize the genomic 5' sequences to study the potential functional significance of these features.

	MATERIALS AND METHODS

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Cells and viruses. Vero cells obtained from the American Type Culture Collection were maintained in Dulbecco's modified Eagle medium (Gibco/BRL) containing 5% fetal bovine serum and gentamicin (10 µg/ml) at 35°C under 5% CO₂. Propagation and titration of RUB by plaque assay was done as previously described (5, 9, 34).

Construction of mutants. Standard recombinant DNA techniques (13) were used in these protocols, with minor modifications. Restriction enzymes and T4 DNA ligase were obtained from New England BioLabs or Boehringer Mannheim Biochemicals. Sequencing of DNA was done by using dideoxy-chain termination sequencing kits from United States Biochemicals (Cleveland, Ohio).

In vitro transcription, transfection of cells, and confirmation of mutations. In vitro transcriptions using SP6 RNA polymerase in the presence of m⁷G(5')ppp(5')G cap analog and Lipofectin-mediated transfections of Vero cells with the resulting transcripts were done as described elsewhere (27, 29). Successful transcription was confirmed by agarose gel electrophoresis of an aliquot of the transcription reaction in the presence of ethidium bromide. The efficiency of in vitro transcription of the mutant templates was comparable to that of Robo302, with the exception of the S3 mutant, which produced approximately 10 times fewer transcripts. Generally, duplicate plates of cells were transfected with each transcription reaction; one plate was overlaid with agar, and the cells in the other plate were maintained in growth medium. Five to six days later, characteristic plaques (if present) were picked, and virus was eluted and amplified once in Vero cells to produce a stock for sequencing to confirm the mutation and for subsequent analysis of the mutant phenotype. If no plaques were observed, the growth medium from the duplicate transfected culture was harvested and used as the mutant virus stock. Sequencing to determine the genomic 5' ends of different strains of RUB and to confirm the presence of mutations in the majority of mutants was done as described previously (27). Briefly, a 120-nt primer extension product (PEP) complementary to the 5' end was synthesized on virion RNA template, poly(A) tailed with terminal deoxynucleotidyltransferase, and PCR amplified. The resulting PCR product was cloned into pGEM1 or pGEM2 plasmid vector. To confirm the D_SL3, S_SL2, S_SL3, and S_SL4 mutations, the protocol was modified such that oligonucleotide 73 (5'-CAAGGATCCAGAACCTCATCTAGGAG; the BamHI site used for cloning into pGEM1 vector is underlined) was used in the PCR amplification step. This oligonucleotide, complementary to nt 50 to 66, is closer to the 5' end than oligonucleotides 36 and 292, used previously, and its use was found to increase the specificity of PCR amplification of the poly(A)-tailed PEP. To confirm the D_SL2, D_SL4, D_SL5, S_SL1, I_SL1, and D_SL1 mutations, the 5' PEP was not poly(A) tailed and was instead directly PCR amplified by using oligonucleotide 74, containing nt 1 to 18 of the genome (the first five of these mutations were downstream from nt 18 and thus A tailing was not necessary; this method was also useful for D_SL1 due to the 3'-5' exonuclease activity of Deep Vent polymerase used). For each mutation, several plasmid clones containing the complete 5' terminus were identified and sequenced.

Analysis of virus macromolecular synthesis. RUB-specific RNA and protein production was examined in cultures infected simultaneously. Vero cells grown in 35- and 60-mm-diameter plates (Corning Glass Works, Corning, N.Y.) and eight-chamber slides (Nunc, Naperville, Ill.) (for immunofluorescence assay [IFA]) were infected with Robo302 or mutant viruses at a multiplicity of infection (MOI) of 0.5 PFU/cell. At indicated times postinfection, total intracellular RNA was extracted from the infected monolayers in the 35-mm-diameter plates with TRI reagent (Molecular Research Center, Cincinnati, Ohio) according to the manufacturer's protocol, redissolved in 40 µl of H₂O, and stored at 70°C. Aliquots of 4 µl were used for Northern blotting, done as described previously (2). [³²P]dCTP nick-translated Robo102 plasmid (34) was used as a probe for total RUB-specific RNA, and [³²P]-UTP-labeled RNA transcripts synthesized from template plasmid pRUB-SP-ORF, which contains the 3'-terminal 3,292 nt of the RUB genome (14), were used as strand-specific probes. pRUB-SP-ORF was linearized with HindIII and transcribed with T7 RNA polymerase or linearized with EcoRI and transcribed with SP6 RNA polymerase to synthesize the minus- or plus-strand specific probe, respectively.

Computer analyses. Optimal and suboptimal RNA secondary structures were determined by using the FoldRNA and Mfold programs; the output files of these programs were plotted by using the Squiggles program (Genetics Computer Group, Madison, Wis.).

	RESULTS

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5'-terminal structure of the RUB genomic RNA. The 5'-terminal stem-and-loop structure [5'(+)SL] shown in Fig. 1A was initially predicted by computer (3). Its existence in the RUB genomic RNA is supported by the finding that primer extension reactions near the 5' end of the genomic RNA result in two strong-stop cDNA bands corresponding to the last residue of the 5'(+)SL (nt 65) and the 5' end of the genome (3). The 5'(+)SL follows a 14-nt single-stranded leader (ss-leader). We call the vertical part of the stem-and-loop formed by pairing of nt 15 to 29 and nt 51 to 65 the stem; the region consisting of nt 30 to 50 is called the upper loop. The predicted secondary structure of the genomic 5' end of SIN is shown for comparison in Fig. 1B. Also shown in Fig. 1A is the second element of interest in this study, the short ORF located between nt 3 and 54, which overlaps the start of the NSP ORF at nt 41 in an alternate frame.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Structures of the ends of the RNAs of RUB and SIN. The 5' terminus of the RUB genomic RNA (A) contains a 14-nt ss-leader followed by a stem-and-loop structure [5'(+)SL] composed of nt 15 to 65. The 5'(+)SL of the genomic RNA of SIN (B) is located at the exact 5' end (nt 1 to 44). Start and stop codons for the short ORF of RUB are underlined, and the initiation codons for the NSP ORF of both RUB and SIN are shaded. A potential pseudoknot formed by the RUB 5' terminus is also shown (this pseudoknot was not taken onto consideration to determine the given G value). The predicted complementary structure, 3'()SL, located at the 3' end of the RUB minus-strand RNA, is shown in panel C (numbering of nucleotides is from the 3' end).

Comparison of the 5' ends of six RUB strains. Previously, the sequence of the exact 5' terminus of the RUB genome had been determined only for the w-Therien (wild-type [wt]) and RA27/3 (vaccine) strains (28, 34). We determined the 5' sequences of four additional strains: f-Therien, a laboratory derivative of w-Therien selected for clear-plaque morphology (30); HPV-77, an independent vaccine strain (7); Hasnas, a wt strain isolated in the United States in 1990; and Machado, a wt strain isolated in Great Britain in 1992 (8). An alignment of these sequences is shown in Fig. 2. While the HPV-77 sequence is identical to that of w-Therien, the other strains differed by up to 3 nt within the 5'-terminal 100 nt. Interestingly, the Hasnas genome starts with an additional A residue. The Hasnas and Machado strains contain two of the three differences found between RA27/3 (G₇, U₃₄, and G₄₈) and w-Therien. The short ORF is preserved in all of these strains. However, the nucleotide differences within the short ORF would all result in amino acid substitutions in the predicted peptide (N₁₆S in f-Therien; E₂G, P₁₁L, and N₁₆D in RA27/3; and E₂G and P₁₁L in Hasnas and Machado).

View larger version (9K): [in this window] [in a new window]   FIG. 2.   Sequence alignment of the genomic 5' ends of different strains of RUB. Abbreviations: w-Th, w-Therien; f-Th, f-Therien; RA, RA27/3; Has, Hasnas; Mach, Machado. Start and stop codons for the short ORF are boxed, and the initiation site of the NSP ORF is indicated by the arrow.

Mutagenesis of the ss-leader sequence. A number of deletions of 1 to 6 nt (D1 to D11) and point mutations (S1 to S10) were introduced into the ss-leader of Robo302 (Fig. 3). Surprisingly, the most profound effects were produced by mutations that altered both A residues at nt 2 and 3. Deletions that led to removal of both A residues (D3, D4, D5, and D6), as well as substitution of the AA with CC (S1), were lethal. With the lethal mutants, no plaques were detected following transfection of Vero cells. Additionally, no virion RNA of mutants D3 and D4 was detected in the media by reverse transcription-PCR, and no intracellular RUB-specific RNA of mutants S1 and D6 was detected by Northern hybridization in Vero cells in which the media from the initially transfected cells had been passaged (data not shown). The other mutants were viable, and most grew to titers comparable to that of Robo302 (Table 1). Mutations which produced low titers following initial amplifications of plaques were reassayed by using the amplified stock to infect Vero cells, with harvest 2.5 days postinfection. Mutant D8 (nt 6 to 8 deleted) was found to produce titers 10-fold lower than those of Robo302. Roughly half of the mutants produced plaques with altered morphologies. With two exceptions (D8 and D10, in which nt 6 to 8 and nt 9 to 11, respectively, were deleted), all of these had mutations within the first 6 nt. Deletions within this region invariably gave rise to virus with altered plaque morphology, whereas many of the base substitutions produced virus with wt plaques.

View larger version (47K): [in this window] [in a new window]   FIG. 3.   Mutations introduced into the 5' terminus of Robo302. The wt (Robo302) sequence is boxed. Mutations in the ss-leader are shown above the wt sequence, and mutations in the 5'(+)SL are shown below the wt sequence. Mutations that were lethal are shaded, while viable mutations are indicated by + in the "Viability" column. Deviations from the wt plaque morphology exhibited by some of the mutants are indicated in the "Plaque phenotype" column (Robo302 forms large, clear plaques). In the "Method" column, the procedure of generation of each of the mutants is indicated: in E (express method), the ligation mixture was used directly for in vitro transcription followed by transfection of Vero cells, while in P (plasmid method), plasmids containing the mutation were generated prior to transcription and transfection. With both methods, mutations were verified by 5' sequencing of mutant virion RNAs. The mutants marked by asterisks were found to contain second site mutations (see Table 1 for sequences).

Mutagenesis of the 5'(+)SL. Five deletion mutations which were intended to make gross changes in the structure were introduced into the left side of the 5'(+)SL (Fig. 3): nt 15 to 21 at the bottom of the stem (D_SL1), nt 25 to 29 at the top of the stem (D_SL2), nt 15 to 29 (the entire left side of the stem; D_SL3), nt 30 to 35 in the lower part of the upper loop region (D_SL4), and nt 36 to 40 in the bulge of the upper loop region preceding the A₄₁UG initiation codon for the NSP ORF (D_SL5). No mutations were introduced downstream from the AUG initiation codon for the NSP ORF (nt 41 to 43) situated at the top of the 5'(+)SL, because the choice of mutations in this region would have been restricted to a few 1-nt silent substitutions to prevent alteration of the NSP amino acid sequence. Also, a 3-nt substitution (G₂₂CUAUC) was made to sew up the bulge at the middle of the stem (S_SL1), and a 4-nt (GUUU) insertion was introduced between nt 29 and 30 to straighten and sew up the upper part of the 5'(+)SL (I_SL1). The computer-predicted effects of the mutations on the 5'(+)SL are shown in Fig. 4A. As expected, the mutations that eliminated bulges are stronger than the wt structure (e.g., 30 kcal/mol for I_SL1 versus 20.7 kcal/mol in Robo302). Interestingly, the mutants that deleted regions of the 5'(+)SL formed alternate stem-and-loop structures utilizing nucleotides from the ss-leader. However, these structures were weaker as well as unstable: the 5'-terminal sequences were paired with different downstream regions when longer 5'-terminal segments were folded (data not shown).

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Predicted 5'-terminal structures of RNAs containing mutations within the 5'(+)SL. (A) Effects of the five deletions (DSL1 to 5), the mutation that sews up the bulge in the stem (SSL1), and the 4-nt insertion in the upper loop (ISL1) are shown. ISL1d virus is a clear-plaque derivative selected during passage of ISL1 mutant in Vero cells. (B) Effects of the three multiple-point mutations are shown. In both panels, the localization of deletions is indicated by arrowhead and inserted or substituted nucleotides are shaded. The NSP ORF initiation codon is boxed.

Effects of selected mutations on the virus macromolecular synthesis. Monolayers of Vero cells were infected at an MOI of 0.5 PFU/cell with the following mutants: S2, S3, D7, D8, and D9, which contain mutations in the ss-leader (Fig. 3); and I_SL1, I_SL1d, D_SL1, D_SL2, S_SL2, S_SL3, and S_SL4, which contain mutations in the 5'(+)SL. Virus RNA and protein synthesis were analyzed at both 48 and 72 h postinfection, with similar results at both time points; analysis at 72 h postinfection is presented here.

View larger version (65K): [in this window] [in a new window]   FIG. 5.   Effects of selected mutations on virus macromolecular synthesis. Vero cells were infected with viruses indicated above each lane at an MOI of 0.5 PFU/cell. At 72 h postinfection, total intracellular RNAs were extracted and plus-strand RNAs were assayed by Northern hybridization (A) or the cells were radiolabeled with [35S]methionine and the SPs (B) and NSPs (C) were immunoprecipitated and resolved by polyacrylamide gel electrophoresis. In panel A, G signifies position of the genomic RNA band and SG signifies the subgenomic RNA band. In panels B and C, positions of the RUB-specific proteins and molecular weight markers (in kilodaltons) are indicated on the left and right, respectively.

View larger version (55K): [in this window] [in a new window]   FIG. 6.   Relative rates of the NSP synthesis in cells infected with the selected mutants. Amounts of radioactivities in the SP and NSP bands in Fig. 5B and C were quantitated with a phosphorimager. Total radioactivities in the NSP bands normalized to the radioactivity in the SP bands for each virus are given (SP synthesis was found to be roughly equivalent in all mutants).

	DISCUSSION

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In this study, we report the first use of the RUB infectious clone for major genetic manipulation. Extensive mutagenesis was done on the 5' genomic sequences, and this mutagenesis was tolerated by the virus in that the majority of the mutants were viable. Thus, the infectious clone will be useful for site-directed mutagenesis studies.

As the first step in this study, we determined the genomic 5' sequences of four additional strains of RUB (f-Therien, HPV, Hasnas, and Machado) for comparative purposes since the 5' ends extending to the 5'-most nucleotides of only two RUB strains, w-Therien and RA27/3, had been sequenced previously (28, 34). The two major elements of the 5'-terminal secondary structure, the 14-nt ss-leader and the 5'(+)SL, are present in all these strains. The stem part of the 5'(+)SL was completely conserved in that no differences were found within the regions (nt 15 to 29 and nt 51 to 65) that compose the stem. The six strains differed at up to 3 nt localized in the ss-leader and the upper loop part (nt 30 to 50) of the 5'(+)SL, and the differences in the upper loop led to predicted structural differences. This finding is consistent with results of Johnstone et al. (10), who sequenced nt 18 to 540 of seven different strains and found no difference in the stem but variation in the upper loop that led to variability in the predicted structure. The maintenance of the stem and the variability of the upper loop correlated with the mutagenesis results in that mutations in the stem resulted in more dramatic effects on the virus growth and plaque morphology compared to deletions in the upper loop.

In addition to the secondary structure, an interesting feature at the 5' terminus addressed in this study is the short ORF located between nt 3 and 54 of the RUB genome that overlaps the NSP ORF in an alternate translation frame. We found that this ORF was preserved in the six independent strains; however, its coding sequence was not as highly conserved as would be expected of a short peptide that plays a significant function in the virus replication cycle since up to three substitutions within the encoded 17 amino acid residues were found in some strains. Subsequently, we made mutations that interfered with or excluded translation of this ORF, and the resulting mutant viruses were found to be viable and have wild-type growth and plaque morphology. Therefore, this short ORF is not required for the virus replication.

Mutagenesis of the 5'(+)SL was initially approached by deleting portions of the stem and upper loop and yielded the overall result that none of these mutations, including a 15-nt deletion of the entire left side of the conserved stem (mutant D_SL3), were lethal, although this latter mutant was severely crippled. The deletions in the stem had a greater effect on virus titer and plaque morphology than did the deletions in the upper loop. Computer-assisted refolding of these deletions indicated that stem structures could still be formed, sometimes with nucleotides in the ss-leader; however, these structures were both weaker and less stable. The mutations that resulted in the weakest stem structure had the greatest effect on plaque morphology. To profoundly modify the appearance of the 5' secondary structure without changing the length of the 5' untranslated region, we made three multiple-point mutations (S_SL2, S_SL3, and S_SL4) which were predicted to completely alter the 5'(+)SL. All three of these mutants exhibited altered plaque morphology, and two grew to titers that approached Robo302 titers. The mutant that grew roughly 10 times less well than Robo302 and had the severest alteration in plaque morphology (S_SL3) had the least stable 5' structure. Therefore, there appears to be a requirement for a stem structure with some degree of stability at the 5' end of the RUB genome for efficient replication. Overall, these findings are in accordance with the results of a similar study conducted on the 5' end of SIN in which it was shown that various deletions within the 5'(+)SL of SIN of up to 15 nt in length introduced downstream from nt 5 were not lethal but had variable effects on virus replication (19). In the SIN study, deletions within the first 5' nt of the 5'(+)SL were lethal (except for deletion of nt 1, which was viable). However, as discussed below, we believe that this was due to the fact that these nucleotides are the first 5 nt of the SIN genome and not because they are part of the 5'(+)SL.

At the molecular level, the only parameter that differed among different mutants was production of NSPs, which was reduced by the mutations in the 5'(+)SL but not in the ss-leader. Thus, the 5'(+)SL is important for efficient initiation of translation of the NSP ORF. This finding is consistent with previous observations of a stimulatory effect of this structure on translation of reporter genes in vitro and in vivo (18, 24, 25). The La autoantigen has been shown to bind the 5'(+)SL, and some evidence indicates that La binding increases the efficiency of translation of the NSP ORF (4). It will be interesting to measure the effect of our 5'(+)SL mutations on La binding. Especially profound suppression of NSP synthesis was observed in mutants D_SL1 and D_SL2, which formed barely detectable plaques, and no viral proteins were detectable in mutant D_SL3, which failed to form plaques. This observation is consistent with our recent findings indicating that the level of NSP synthesis can be an important determinant of RUB cytopathogenicity (27).

Computer folding demonstrated that the 3'()SL at the 3' end of the minus-strand RNA of RUB is weaker than the 5'(+)SL and was destabilized by the mutations introduced into the 5'(+)SL. Since these mutations did not preclude viability and did not significantly affect plus-strand RNA synthesis, we conclude that the 3'()SL is not a negative-strand promoter necessary for initiation of genomic plus strands. As discussed above, large alterations in the 5'(+)SL of the SIN infectious clone were tolerated, although the effect of these mutations on plus-strand RNA synthesis was not studied (19). The other published data that argue against the idea of the 3'()SL being a promoter are the findings that the 5' noncoding sequences in defective interfering RNAs of both Semliki Forest virus (12, 23) and SIN (16, 17, 32, 33) are highly rearranged, with large portions of the 5'(+)SL being deleted and/or replaced by heterologous sequences of both viral and cellular origin. Computer analysis failed to recognize any common 5'-terminal secondary structure among such defective interfering RNAs of SIN (33). In this regard, minus-strand RNA is completely complexed in double-stranded replicative forms and replicative intermediates in togavirus-infected cells which are the templates for plus-strand RNA synthesis (11). It is not known if these RNAs are unwound by themselves to any extent, and thus it is possible that formation of any secondary structure by the minus-strand RNA, such as the 3'()SL, does not occur. In this case, initiation of plus-strand RNA could occur by template switching by the replicase from the plus-strand to the minus-strand template or by recognition of a double-stranded RNA feature. However, if such unwinding indeed takes place, then the possibility remains that a specific, more complex tertiary structure of a larger 3' segment of the minus-strand RNA serves as a promoter for the plus-strand RNA.

Perhaps the most intriguing finding in this study was that only a few nucleotides at the extreme 5' end of the genome were essential for RUB viability. Specifically, mutations that removed or altered the two A residues at nt 2 and 3 simultaneously (but not mutations that altered only one of these residues) were lethal. Mutations that changed the first A residue resulted in viable viruses, which however contained secondary mutations that moved an A residue within the first 3 nt of the genome. This finding coincides with the SIN mutagenesis study in which deletions in the first 5 nt were found to be lethal (19). Since the crucial AA dinucleotide in the RUB genome is located in the ss-leader, this indicates that in both RUB and SIN the 5'-most nucleotides are critical in virus replication, but not because in SIN they belong to the bottom of the 5'(+)SL as was previously suggested (19). It is possible that these residues constitute a core element of a short signal at the 5' end of genomic RNA (or 3' end of the minus-strand RNA) which is absolutely necessary for RNA replication. However, the signal is not as simple as one or more A's near the 5' end of the genome, since D5, which moved the A₇A₈ dinucleotide to the exact 5' end, was lethal. It should be noted that D8, which lacked G₆A₇A₈, gave rise to a small-plaque phenotype virus, while D9, which lacked nt 6 to 11, gave rise to a virus with a duplicated 5' end. Thus, nt 6 to 8 as well as some other surrounding nucleotides may also play a role in this putative signal. Alternatively, it has been shown that alphavirus genomic RNAs cyclize through interactions of nucleotides near the 5' and 3' ends of the molecule (6). Although the function of cyclization, as well as whether or not the RUB genomic RNA cyclizes, is not known, the exact 5' nucleotides of the togavirus genomes could be involved in cyclization. Further analysis is required to resolve these possibilities.