INTRODUCTION

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Sindbis virus is the type member of the genus Alphavirus in the family Togaviridae, with a single-stranded plus-sense RNA of 11.7 kb (22). Four nonstructural proteins required for viral RNA replication and transcription, nsP1, nsP2, nsP3, and nsP4, are translated from the 5' two-thirds of the genome as two overlapping polyproteins, P123 and P1234, which are cleaved by a proteolytic activity present in the C-terminal half of nsP2 (10). Differential processing of these nonstructural polyproteins results in regulation of minus-strand and plus-strand RNA synthesis in infected cells, with the result that minus-strand RNA is made only early after infection (16, 20).

nsP4 is believed to be the viral RNA polymerase because it possesses certain sequence motifs characteristic of RNA polymerases (12, 13) and because a temperature-sensitive mutation in nsP4 is known that leads to cessation of RNA elongation upon a shift to a nonpermissive temperature (1, 8). Production and accumulation of nsP4 are regulated by three mechanisms in Sindbis-infected cells. First, translation of nsP4 occurs only upon readthrough of a UGA termination codon located 7 amino acids upstream from the nsP3/nsP4 boundary (21), and it occurs with an efficiency of 5 to 20% (4, 20). Wild-type virus grows more efficiently than a UGA-to-sense-codon mutant (17). Strauss and Strauss have postulated (22) that a function of the opal codon may be to produce sufficient quantities of the trans-acting protease to efficiently convert the initial replicase, which contains uncleaved P123 plus nsP4 and can synthesize only minus-strand RNA efficiently, to a replicase which contains cleaved products plus nsP4 and which can now make plus-strand RNA (16, 20). Another possibility is that the opal codon serves to regulate the ratio of capping or helicase activities present in P123 and its cleaved products to RNA polymerase activity present in nsP4. Second, nsP4 is produced only early in infection by cis cleavage of the nsP3/nsP4 bond in polyprotein P1234 (4, 19). Later in infection, accumulation of trans-acting protease leads to nascent cleavage at the nsP2/nsP3 bond such that P1234 cannot be produced and P12 and P34 are the primary translation products. P12 undergoes further cleavage, but P34 accumulates late in infection because the viral nonstructural proteinases present at this time are unable to cleave the nsP3/nsP4 bond (4). Third, nsP4 is metabolically unstable and vulnerable to a rapid degradation by the N-end rule pathway (5), in which the stability of a protein is dependent on the amino acid at its N terminus (23). The N-terminal amino acid of Sindbis virus nsP4 is Tyr, which is a destabilizing amino acid in mammalian cells, and it has been shown that while a fraction of nsP4 is stable, presumably because it is sequestered within RNA replication complexes (2), free nsP4 is rapidly degraded (9).

In this study, we characterized Sindbis mutants, as well as revertants of these mutants, that have different amino acids at the N terminus of nsP4, resulting in differences in the stabilities of nsP4, in an effort to elucidate the importance of the rapid degradation of nsP4 in viral replication. Our results show that efficient function of nsP4 is dependent upon the presence of an aromatic amino acid or histidine at the N terminus and that other N-terminal amino acids, whether stabilizing or destabilizing, lead to reduced activity.

	MATERIALS AND METHODS

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Cells and medium. Secondary chicken embryo fibroblast monolayers were cultured in Eagle's minimum essential medium containing 3% fetal calf serum and used for RNA transfection, virus growth, and plaque assay as described previously (20).

N-terminal mutants. pToto1101 (18), a full-length cDNA clone of Sindbis virus from which infectious RNA transcripts can be produced in vitro, was used as a parental clone for generating mutations in the N-terminal residue of nsP4. The wild-type Tyr codon (TAC, nucleotides [nt] 5769 to 5771, where nt 1 is the first nucleotide in the genomic RNA) was changed to Ala (GCC), Met (ATG), Leu (CTC), Arg (AGG), or Phe (TTC) by cassette mutagenesis as previously described (4). Changes to Cys (TGC), Thr (ACT), Asn (AAC), Gln (CAG), Glu (GAG), or Pro (CCC) codons were carried out by in vitro mutagenesis as follows. The region in pToto1101 from nt 5758 to 6085 was amplified by PCR with a plus-sense mutagenic primer annealing to nt 5758 to 5782, 5'-GGGTAGGTGGGXXXATATTTTCGAC-3' (where XXX is the codon for residue 1 of nsP4), and a minus-sense primer, YA16, annealing to nt 6069 to 6085, 5'-TACAATGGTTTCGGATA-3', and the resultant 0.3-kb DNA was purified on a commercial spin column (Qiagen Inc., Chatworth, Calif.). Similarly, the region from nt 5164 to 5782 was amplified with a minus-sense mutagenic primer annealing to nt 5758 to 5782, 5'-GTCGAAAATATX'X'X'CCCACCTACCC-3' (where X'X'X' is complementary to the codon for residue 1 of nsP4), and a plus-sense primer, YA19, annealing to nt 5164 to 5180, 5'-ATAACACCTCGCTTGAT-3', and the resultant 0.6-kb DNA was purified. The 0.3-kb DNA and 0.6-kb DNA were then fused by a fusion PCR approach, in which the two fragments were mixed, denatured, annealed, and subjected to a second cycle of PCR amplification with the YA16 and YA19 primers. The 0.9-kb fragment was purified and digested with SpeI (nt 5263) and EcoRI (nt 5870), and the 0.6-kb SpeI-EcoRI fragment was purified and cloned into an intermediate vector, pSIN34CE2, containing a 4.5-kb SpeI (nt 5263)-to-BssHII (nt 9805) insert from pToto1101 in a Proteus1 plasmid vector (18). The resulting insert was then removed and inserted into an SpeI- and BssHII-digested pToto1101 vector. More than three independent clones were examined for each mutant. The nsP4 N-terminal mutant constructs were called pToto1101.4X, where X indicates the mutated amino acid at the N terminus of nsP4.

Analysis of in vitro translation products. Miniprep DNA of pToto1101 constructs was prepared by a modified boiling method (11) and digested with XhoI. The linearized template DNA was transcribed with an SP6 RNA polymerase at 38°C for 1 h in the presence of a cap analog. Transcript RNA was translated in rabbit reticulocyte lysates (Promega Biotec, Madison, Wis.) in the presence of [³⁵S]Met (1,200 Ci/mmol) at 30°C for 90 min. Labeled translation products were separated in a sodium dodecyl sulfate-polyacrylamide gel and visualized by fluorography with sodium salicylate (3).

RNA transfection. Confluent monolayers of chicken cells in 35-mm plates were treated with DEAE-dextran and transfected with RNA transcripts diluted in phosphate-buffered saline. For direct plaque analysis, cells were overlaid with Eagle's minimal essential medium containing 1% agarose and 3% calf serum after transfection and incubated at 30 or 40°C for 2 days before being stained with neutral red. For observation of cytopathic effects and recovery of viable viruses, the transfected cells were incubated in a liquid medium for up to 7 days at 30 or 40°C. Viruses produced during this extended incubation were examined by plaque assay on confluent monolayers of chicken cells with incubation at 30 or 40°C for 2 days before being stained.

RT-PCR and nucleotide sequencing. RNA was extracted from 0.4 ml of the primary transfection medium with phenol-chloroform and precipitated with ethanol. cDNA was synthesized by reverse transcription with minus-sense primer YA16, and the region from nt 5164 to 6085 was amplified with primers YA16 and YA19. The 0.9-kb DNA was digested with SpeI and EcoRI, and the resultant 0.6-kb fragment was cloned into an SpeI- and EcoRI-digested pSIN34CE2. The nucleotide sequence of the nsP4 N-terminal region was determined from more than three independent clones by sequencing with Sequenase, using a minus-sense primer annealing to nt 5857 to 5876.

Testing of nsP4 mutations in a wild-type background. The Trp-nsP4 and His-nsP4 mutations were introduced into pToto1101 by replacing the BamHI fragment (nt 4634 to 7335) of pToto1101 with that of double-stranded cDNA made to the revertant virus RNA by the method of Gubler and Hoffman (7). The Trp-nsP4 and His-nsP4 mutations were also combined with the 2V mutation by replacing the BamHI fragment of pToto1101.2V with that from the mutant cDNA.

	RESULTS

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Stabilities of mutant nsP4s in vitro. To examine the stabilities of the nsP4s bearing different amino acids at the N terminus, RNA transcripts from pToto1101 derivatives containing the nsP4 N-terminal mutations were translated in rabbit reticulocyte lysates (Fig. 1). The transcripts also contained the 2V mutation, which abolishes cleavage at the nsP2/nsP3 site, so that a P23 polyprotein was produced instead of nsP2 and nsP3, the latter of which interferes with visualization of nsP4 because of their similar migration rates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1, lane 13). We have previously shown that production of nsP4 is not influenced by the 2V mutation (19).

View larger version (55K): [in this window] [in a new window]   FIG. 1.   Analysis of Sindbis virus nsP4 N-terminal mutants by translation in rabbit reticulocyte lysates. The N terminus of nsP4, which is Tyr in the wild type (lane 11), was changed to Ala (lane 1), Cys (lane 2), Leu (lane 3), Met (lane 4), Thr (lane 5), Asn (lane 6), Gln (lane 7), Arg (lane 8), Glu (lane 9), Phe (lane 10), or Pro (lane 12) in pToto1101.2V, which has a GlyVal mutation at the P2 position of the nsP2/nsP3 cleavage site. Lane 13 shows translation products from the wild-type pToto1101 transcripts. In vitro transcripts of the different RNAs were translated in rabbit reticulocyte lysates at 30°C for 90 min in the presence of [35S]methionine. The cleavage efficiency at the nsP3/nsP4 site and the stability of the mutant nsP4s, as determined from the upper panel, are shown below. Cleavage efficiency: ++, efficient; +, less efficient; , not detectable. nsP4 stability: +, stable; , unstable; nd, not determined.

Plaque phenotype of nsP4 N-terminal mutants. We examined the ability of the mutants carrying different N-terminal residues on nsP4 to form a plaque, and the results are shown in Fig. 2. In this experiment, cells were transfected with RNA, overlaid with medium containing agarose, incubated at 30 or 40°C, and stained with neutral red. The Phe-nsP4 mutant and the wild-type Tyr-nsP4 formed large plaques at both 30 and 40°C. The Met-nsP4 mutant formed small plaques at 30°C and minute plaques at 40°C but produced similar numbers of plaques at the two temperatures, so that it is not temperature sensitive for plaque formation. The other nine mutants did not form visible plaques at either temperature, and the mutations are thus lethal by this criterion.

View larger version (10K): [in this window] [in a new window]   FIG. 2.   Growth of mutants with different N-terminal residues in nsP4. The N terminus of nsP4 (Tyr = Y in the wild-type virus) was changed to Phe (F), Met (M), Ala (A), Cys (C), Leu (L), Thr (T), Asn (N), Gln (Q), Arg (R), Glu (E), or Pro (P). RNA transcripts were transfected into chicken cells, and the transfected cells were overlaid with medium containing agarose, incubated at 30 or 40°C, and stained with neutral red to visualize plaques. The sizes of the plaques formed are shown in millimeters; a minus sign indicates that no plaques were visible. A second set of cells were transfected with the different mutants, overlaid with liquid medium, and incubated at 30 or 40°C for 7 days or until cytopathology was obvious. +, cytopathology developed and viable virus could be isolated from the culture; , no cytopathology was visible after 7 days, and no virus could be demonstrated in the culture.

Recovery of viable viruses from transfected cells. Although no distinct plaques were formed after RNA transfection and agarose overlay, cells transfected with Ala-, Cys-, Leu-, Thr-, Asn-, Gln-, or Arg-nsP4 mutant transcripts showed cytopathology after incubation in liquid medium at 30°C for 2 to 7 days, depending on the experiment, and viable viruses could be recovered from these culture fluids (Fig. 2). To study the appearance of viable viruses, several plates of cells were independently transfected with RNA from each of the mutants so as to generate multiple independent stocks of viruses from each mutant, and these independent stocks were characterized in various ways (Table 1).

nsP4 N-terminal amino acid in rescued viruses. Since the original Ala-, Cys-, Leu-, Thr-, Asn-, Gln-, and Arg-nsP4 mutants did not form plaques at either 30 or 40°C, we assumed that the viable viruses recovered at 30°C resulted from reversion of the original mutation or from suppression by a second-site mutation. To determine whether the original mutations were still present in the recovered viruses, RNA was extracted from virus preparations and a region encoding the nsP3/nsP4 cleavage site was amplified by RT-PCR, cloned, and sequenced. More than three independent RT-PCR clones were sequenced from each revertant stock to ensure that a consensus sequence was obtained. The results are shown in Table 1.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Possible amino acid substitutions at the N terminus of the nsP4 mutants. For eight of the nsP4 mutants that were examined, the amino acid substitutions that are possible with a single nucleotide change in the mutant codon are shown. Boxed substitutions (in boldface type) were observed in one or more revertants, whereas none of the other substitutions were found. The shaded substitutions are possible changes to the six amino acids that were not directly tested at the N terminus of nsP4; the fact that these substitutions were never observed in revertant viruses indicates that these six amino acids are not acceptable N-terminal residues. Y, pyrimidine; P, purine (used in third codon positions where degeneracy exists).

	DISCUSSION

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Requirement for Tyr, Trp, Phe, or His at the nsP4 N terminus. These studies show that only nsP4s bearing Tyr, Trp, Phe, or His at the N terminus possess wild-type or near-wild-type function. These amino acids have in common an unsaturated ring, which is fully conjugated in the case of the three aromatic amino acids. Virus with N-terminal Met, which shares with these amino acids the property of being bulky and hydrophobic, is also viable but forms small plaques and is thus attenuated. All the other amino acids tested at the N terminus are lethal, but for at least Ala, Leu, Thr, Asn, Gln, and Arg (and probably Cys), the lethality can be suppressed by mutations elsewhere in the genome. The resulting virus does not replicate as efficiently as the wild type, however, as shown by the small-plaque phenotype and the temperature sensitivity of most of these viruses. Pro is probably lethal because cleavage to produce nsP4 is very limited or nonexistent and RNA replication is too limited to allow revertants to arise. Glu is also lethal and seems to impair function so completely that suppressors cannot arise. The remaining six amino acids not tested in this study, Ser, Ile, Lys, Val, Asp, and Gly, appear to be unacceptable for nsP4 function, because they could all have arisen by a single nucleotide substitution in one or more of the mutants tested (Fig. 3), and the fact that no revertants with these amino acids were found suggests that they are also lethal. Although these six amino acids must be lethal, it is unknown whether they could be suppressed by mutations elsewhere in the genome.

Emergence of viable viruses. The emergence of viable viruses from many of the nonviable nsP4 N-terminal mutants must mean that limited RNA replication does occur after transfection with these mutant RNAs and that although the rate of replication is too low to permit the formation of a plaque, it is sufficient to allow mutations during replication that result in the appearance of revertants that overgrow the culture. Such RNAs have been called quasi-infectious by Gmyl et al. (6).

Rapid turnover and nsP4 function. Our results show that only Tyr, Trp, Phe, or His as nsP4 N-terminal residues lead to full activity, and these residues are all destabilizing for the N-end rule pathway. The other 16 residues, many of which were tested directly but some only indirectly by a failure to appear in revertants, lead to nsP4 that has reduced activity. The fact that Cys or Arg at the N terminus destabilizes nsP4 but renders it inactive whereas Met stabilizes nsP4 but results in partial activity indicates that there is no correlation between instability of nsP4 and its activity and that rapid turnover of nsP4 is a result of the requirement for an aromatic amino acid or histidine for full activity. We suggest that the primary role of the N-terminal tyrosine is to interact with other residues in nsP4, so that it folds properly, or to be involved in the interaction of nsP4 with other viral nonstructural proteins or cellular factors and that these interactions may be required for the recognition of viral promoters during RNA synthesis.