Effects of an opal termination codon preceding the nsP4 gene sequence in the O'Nyong-Nyong virus genome on Anopheles gambiae infectivity

The genomic RNA of an alphavirus encodes four different nonstructural proteins, nsP1, nsP2, nsP3, and nsP4. The polyprotein P123 is produced when translation terminates at an opal termination codon between nsP3 and nsP4. The polyprotein P1234 is produced when translational readthrough occurs or when the opal termination codon has been replaced by a sense codon in the alphavirus genome. Evolutionary pressures appear to have maintained genomic sequences encoding both a stop codon (opal) and an open reading frame (arginine) as a general feature of the O'nyong-nyong virus (ONNV) genome, indicating that both are required at some point. Alternate replication of ONNVs in both vertebrate and invertebrate hosts may determine predominance of a particular codon at this locus in the viral quasispecies. However, no systematic study has previously tested this hypothesis in whole animals. We report here the results of the first study to investigate in a natural mosquito host the functional significance of the opal stop codon in an alphavirus genome. We used a full-length cDNA clone of ONNV to construct a series of mutants in which the arginine between nsP3 and nsP4 was replaced with an opal, ochre, or amber stop codon. The presence of an opal stop codon upstream of nsP4 nearly doubled (75.5%) the infectivity of ONNV over that of virus possessing a codon for the amino acid arginine at the corresponding position (39.8%). Although the frequency with which the opal virus disseminated from the mosquito midgut did not differ significantly from that of the arginine virus on days 8 and 10, dissemination did began earlier in mosquitoes infected with the opal virus. Although a clear fitness advantage is provided to ONNV by the presence of an opal codon between nsP3 and nsP4 in Anopheles gambiae, sequence analysis of ONNV RNA extracted from mosquito bodies and heads indicated codon usage at this position corresponded with that of the virus administered in the blood meal. These results suggest that while selection of ONNV variants is occurring, de novo mutation at the position between nsP3 and nsP4 does not readily occur in the mosquito. Taken together, these results suggest that the primary fitness advantage provided to ONNV by the presence of an opal codon between nsP3 and nsP4 is related to mosquito infectivity.     

Mutations which alter the level or structure of nsP4 can affect the efficiency of Sindbis virus replication in a host-dependent manner.

Two mutants of Sindbis virus have been isolated which grow inefficiently at 34.5 degrees C in mosquito cells yet replicate normally in chicken embryo fibroblast cells at the same temperature. In addition, these mutants exhibit temperature-sensitive growth in both cell types and are RNA- at the nonpermissive temperatures (K.J. Kowal and V. Stollar, Virology 114:140-148, 1981). To clarify the basis of this host restriction, we have mapped the causal mutations for these temperature-dependent, host-restricted mutants. Functional mapping and sequence analysis of the mutant cDNAs revealed several mutations which mapped to the amino terminus of nsP4, the putative polymerase subunit of the viral RNA replicase. These mutations resulted in the following amino acid changes in nsP4: leucine to valine at residue 48, aspartate to glycine at residue 142, and proline to arginine at residue 187. Virus containing any of these mutations was restricted in its ability to replicate in mosquito but not chicken embryo fibroblast cells at 34.5 degrees C. In addition to its temperature-dependent, host-restricted phenotype, virus derived from one cDNA clone also exhibited decreased levels of nsP34 and nsP4 yet contained only a silent change in its genome. This C-to-U mutation occurred at nucleotide 5751, the first nucleotide after the opal termination codon separating nsP3 and nsP4. Our results suggest that this substitution decreases readthrough of the opal codon and diminishes production of nsP34 and nsP4. Such a decrease in synthesis rates might lead to levels of these products which are insufficient for viral RNA replication in mosquito cells at the higher temperature. This work provides the first evidence that nsP4 function can be strongly influenced by the host environment.     

Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes.     

Alphavirus minus-strand RNA synthesis: identification of a role for Arg183 of the nsP4 polymerase

A partially conserved region spanning amino acids 142 to 191 of the Sindbis virus (SIN) nsP4 core polymerase is implicated in host restriction, elongation, and promoter recognition. We extended the analysis of this region by substituting Ser, Ala, or Lys for a highly conserved Arg183 residue immediately preceding its absolutely conserved Ser184-Ala-Val-Pro-Ser188 sequence. In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (ts) growth phenotype caused by a ts defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys). Plus-strand synthesis by nsP4 Arg183 mutant polymerases was unaffected when corrected for minus-strand numbers, although 26S mRNA synthesis was enhanced at the elevated temperature compared to wild type. The ts defect was not due to a failure to form or accumulate nsP4 at 40 degrees C. In contrast to their growth in chicken cells, the nsP4 Arg183 mutants replicated equally poorly, if at all, in mosquito cells. We conclude that Arg183 within the Pro180-Asn-Ile-Arg-Ser184 sequence of the SIN nsP4 polymerase contributes to the efficient initiation of minus strands or the formation of its replicase and that a host factor(s) participates in this event.     

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal,ochre, and amber suppressor tRNAs

Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNA^Cys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.     

Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells.

We have used site-specific mutagenesis to change the anticodon of a Xenopus laevis tyrosine tRNA gene so that it would recognize ochre codons. This tRNA gene is expressed when amplified in monkey cells as part of a SV40 recombinant and efficiently suppresses termination at both the ochre codon separating the adenovirus 2 hexon gene from a 23-kd downstream gene and the ochre codon at the end of the NS1 gene of influenza virus A/Tex/1/68. Termination at an amber codon of a NS1 gene of another influenza virus strain was not suppressed by the (Su+) ochre gene suggesting that in mammalian cells amber codons are not recognized by ochre suppressor tRNAs. Finally, microinjection into mammalian cells of both (Su+) ochre tRNA genes and selectible genes containing ochre nonsense mutations gives rise to colonies under selective conditions. We conclude that it should be possible to isolate a wide assortment of mammalian cell lines with ochre suppressor activity.     

Usage of the three termination codons in a single eukaryotic cell, the Xenopus laevis oocyte.

Oocytes from Xenopus laevis were injected with purified amber (UAG), ochre (UAA), and opal (UGA) suppressor tRNAs from yeasts. The radioactively labeled proteins translated from the endogenous mRNAs were then separated on two-dimensional gels. All three termination codons are used in a single cell, the Xenopus laevis oocyte. But a surprisingly low number of readthrough polypeptides were observed from the 600 mRNAs studied in comparison to uninjected oocytes. The experimental data are compared with the conclusions obtained from the compilation of all available termination sequences on eukaryotic and prokaryotic mRNAs. This comparison indicates that the apparent resistance of natural termination codons against readthrough, as observed by the microinjection experiments, cannot be explained by tandem or very close second stop codons. Instead it suggests that specific context sequences around the termination codons may play a role in the efficiency of translation termination.     

Evidence that the supE44 Mutation of Escherichia coli Is an Amber Suppressor Allele of glnX and that It Also Suppresses Ochre and Opal Nonsense Mutations

Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.Translation termination is mediated by one of the three stop codons (UAA, UAG, or UGA). When such stop codons arise in coding sequences due to mutations, referred to as nonsense mutations, they lead to abrupt arrest of the translation process. However, the termination efficiency of such nonsense codons is not 100%, as certain tRNAs have the ability to read these nonsense codons. Genetic code ambiguity is seen in several organisms. Stop codons have been shown to have alternate roles apart from translation termination. In organisms from all three domains of life, UGA encodes selenocysteine through a specialized mechanism. In Methanosarcinaceae, UAG encodes pyrrolysine (3). UAA and UAG are read as glutamine codons in some green algae and ciliates such as Tetrahymena and Diplomonads (24), and UAG alone encodes glutamine in Moloney murine leukemia virus (32). UGA encodes cysteine in Euplotes; tryptophan in some ciliates, Mycoplasma species, Spiroplasma citri, Bacillus, and tobacco rattle virus; and an unidentified amino acid in Pseudomicrothorax dubius and Nyctotherus ovalis (30). In certain cases the context of the stop codon in translational readthrough has been shown to play a role; for example, it has been reported that in vitro in tobacco mosaic virus, UAG and UAA are misread by tRNA^Tyr in a highly context-dependent manner (34, 9).Termination suppressors are of three types, i.e., amber, ochre, and opal suppressors, which are named based on their ability to suppress the three stop codons. Amber suppressors can suppress only amber codons, whereas ochre suppressors can suppress ochre codons (by normal base pairing) as well as amber codons (by wobbling) and opal suppressors can read opal and UGG tryptophan codon in certain cases. As described by Sambrook et al. (27), a few amber suppressors can also suppress ochre mutations by wobbling. The suppression efficiency varies among these suppressors, with amber suppressors generally showing increased efficiency over ochre and opal suppressors. supE44, an amber suppressor tRNA, is an allele of and is found in many commonly used strains of Escherichia coli K-12. Earlier studies have shown that supE44 is a weak amber suppressor and that its efficiency varies up to 35-fold depending on the reading context of the stop codon (8).Translational accuracy depends on several factors, which include charging of tRNAs with specific amino acids, mRNA decoding, and the presence of antibiotics such as streptomycin and mutations in ribosomal proteins which modulate the fidelity of the translational machinery. Among these, mRNA decoding errors have been reported to occur at a frequency ranging from about 10⁻³ to 10⁻⁴ per codon. Translational misreading errors also largely depend on the competition between cognate and near-cognate tRNA species. Poor availability of cognate tRNAs increases misreading (18).Several studies with E. coli and Saccharomyces cerevisiae have shown the readthrough of nonsense codons in suppressor-free cells. In a suppressor-free E. coli strain, it has been shown in vitro that glutamine is incorporated at the nonsense codons UAG and UAA (26). It has been reported that overexpression of wild-type tRNA^Gln in yeast suppresses amber as well as ochre mutations (25). In this study, we have confirmed the presence of an amber suppressor mutation in the glnX gene in a supE44 strain by sequence analysis. This was done essentially because we observed that supE44 could also suppress lacZ ochre mutations, albeit inefficiently. On further investigation using an in vivo luciferase reporter assay system for tRNA-mediated nonsense suppression (28), we found that the efficiency of suppression of amber lesion by supE44 is significantly higher than that reported previously in the literature. An increased ability to suppress ochre and opal nonsense mutations was observed in cells bearing supE44 compared to in the wild type. Such an effect was observed only in the stationary phase and was abolished in logarithmic phase.     

Modification of Asn374 of nsP1 suppresses a Sindbis virus nsP4 minus-strand polymerase mutant

Our recent study (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76:8632-8640, 2002) found minus-strand synthesis to be temperature sensitive in vertebrate and invertebrate cells when the Arg183 residue of the Sindbis virus nsP4 polymerase was changed to Ser, Ala, or Lys. Here we report the results of studies identifying an interacting partner of the region of the viral polymerase containing Arg183 that suppresses the Ser183 codon mutation. Large-plaque revertants were observed readily following growth of the nsP4 Ser183 mutant at 40 degrees C. Fifteen revertants were characterized, and all had a mutation in the Asn374 codon of nsP1 that changed it to either a His or an Ile codon. When combined with nsP4 Ser183, substitution of either His374 or Ile374 for Asn374 restored wild-type growth in chicken embryo fibroblast (CEF) cells at 40 degrees C. In Aedes albopictus cells at 34.5 degrees C, neither nsP1 substitution suppressed the nsP4 Ser183 defect in minus-strand synthesis. This argued that the nsP4 Arg183 residue itself is needed for minus-strand replicase assembly or function in the mosquito environment. The nsP1 His374 suppressor when combined with the wild-type nsP4 gave greater than wild-type levels of viral RNA synthesis in CEF cells at 40 degrees C ( approximately 140%) and in Aedes cells at 34.5 degrees C (200%). Virus producing nsP1 His374 and wild-type nsP4 Arg183 made more minus strands during the early period of infection and before minus-strand synthesis ceased at about 4 h postinfection. Shirako et al. (Y. Shirako, E. G. Strauss, and J. H. Strauss, Virology 276:148-160, 2000) identified amino acid substitutions in nsP1 and nsP4 that suppressed mutations that changed the N-terminal Tyr of nsP4. The nsP4 N-terminal mutants were defective also in minus-strand synthesis. Our study implicates an interaction between another conserved nsP1 region and an internal region, predicted to be in the finger domain, of nsP4 for the formation or activity of the minus-strand polymerase. Finally, the observation that a single point mutation in nsP1 results in minus-strand synthesis at greater than wild-type levels supports the concept that the wild-type nsP sequences are evolutionary compromises.     

Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system.

Amber, ochre, and opal nonsense suppressor tRNAs isolated from yeast were injected into Xenopus laevis oocytes together with purified mRNAs (globin mRNA from rabbit, tobacco mosaic virus-RNA). Yeast opal suppressor tRNA is able to read the UGA stop codon of the rabbit beta-globin mRNA, thus producing a readthrough protein. A large readthrough product is also obtained upon coinjection of yeast amber or ochre suppressor tRNA with TMV-RNA. The amount of readthrough product is dependent on the amount of injected suppressor tRNA. The suppression of the terminator codon of TMV-RNA is not susceptible to Mg++ concentration or polyamine addition. Therefore, the Xenopus laevis oocyte provides a simple, sensitive, and well buffered in vivo screening system for all three types of eukaryotic nonsense suppressor tRNAs.     

Nonsense Mutants in the rII A Cistron of Bacteriophage T4

After in vitro treatment of bacteriophage T4 with hydroxylamine (HA), 54 nonsense mutants in the rII A cistron were isolated. These mutants were characterized by growth on suppressor strains of Escherichia coli, and the mutational sites were mapped in the rII A cistron. Twenty-five (9 sites) were amber (UAG), 20 (6 sites) were opal (UGA), and 9 (6 sites) were ochre (UAA). Mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably involving mutations occurring in adjacent nucleotides. Based on the specificity of HA mutagenesis (GC → AT), the amino acid codons in which the mutations occurred have been inferred. It is suggested that the three amber-opal pairs arose in tryptophan codons (UGG) and the six ochre mutants arose in glutamine codons (CAA). The six unpaired ambers and the three unpaired opals have been tentatively assigned to glutamine codons (CAG) and arginine codons (CGA), respectively, in the wild-type phage.     

A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors

Summary A spontaneous mutant was isolated that harbors a weak suppressing activity towards a UAG mutation, together with an inability to grow at 43° C in rich medium. The mutation is shown to be associated with an increased misreading of UAG at certain codon contexts and UAA. UGA, missense or frameshift mutations do not appear to be misread to a similar extent. The mutation gives an increased efficiency to several amber tRNA suppressors with-out increasing their ambiguity towards UAA. The ochre suppressors SuB and Su5 are stimulated in their reading of both UAG and UAA with preference for UAG. An opal suppressor is not affected. The effect of the mutation on the efficiency of amber and ochre suppressors is dependent on the codon context of the nonsense codon.The mutated gene (uar) has been mapped and found to be recessive both with respect to suppressor-enhancing ability as well as for temperature sensitivity. The phenotype is partly suppressed by the ochre suppressor SuC. It is suggested that uar codes for a protein, which is involved in translational termination at UAG and UAA stop codons.     

Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells

We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.     

Marker Effects on Reversion of T4rII Mutants

The frequencies of 2-aminopurine- and 5-bromouracil-induced A:T leads to G:C transitions were compared at nonsense sites throughout the rII region of bacteriophage T4. These frequencies are influenced both by adjacent base pairs within the nonsense codons and by extracodonic factors. Following 2AP treatment, they are high in amber (UAG) and lower in opal (UGA) codons than in allelic ochre (UAA) codons. In general, 5BU-induced transitions are more frequent in both amber and opal codons than in the allelic ochre codons. 2AP- and 5BU-induced transition frequencies in the first and third positions of opal codons are correlated with those in the corresponding positions of the allelic ochre codons. Similarly, the frequencies of 2AP-induced transition in the first and second positions of amber codons and their ochre alleles are correlated. However, there is little correlation between the frequencies of 5BU-induced transitions in the first and second positions of allelic amber and ochre codons.     

RNA cleavage linked with ribosomal action

Ribonuclease LS in Escherichia coli is a potential antagonist of bacteriophage T4. When T4 dmd is mutated, this RNase efficiently cleaves T4 mRNAs and leads to the silencing of late genes, thus blocking T4 growth. We previously found that, when two consecutive ochre codons were placed in the open reading frame of T4 soc, RNase LS cleaved soc mRNA at a specific site downstream of the ochre codons. Here, we demonstrate that RNase LS cleaves soc RNA at the same site even when only a single ochre codon is present or is replaced with either an amber or an opal codon. On the other hand, disruption of the Shine-Dalgarno sequence, a ribosome-binding site required for the initiation of translation, eliminates the cleavage. These results strongly suggest that RNase LS cleaves in a manner dependent on translation termination. Consistent with this suggestion, the cleavage dependency on an amber codon was considerably reduced in the presence of amber-codon-suppressing tRNA. Instead, two other cleavages that depend on translation of the region containing the target sites occurred farther downstream. Additional analysis suggests that an interaction of the ribosome with a stop codon might affect the site of cleavage by RNase LS in an mRNA molecule. This effect of the ribosome could reflect remodeling of the high-order structure of the mRNA molecule.     

Studies on the bacteriophage MS2: XLI. Nature of the azure mutation

Azure (or reverse amber) mutants grow normally on wild-type Escherichia coli but not on host strains harbouring a strong UAG suppressor mutation. Three different bacteriophage MS2 azure mutants obtained by treatment with nitrous acid have been characterized at the nucleotide sequence level. The 3′-end fragment of the ³²P-labelled mutant genomes was isolated by DNA:RNA hybridization and treatment with nuclease S₁, and was analyzed by mini-fingerprinting of the RNA. It is known that the wild-type MS2 polymerase gene ends with a UAG codon, followed seven triplets further by an in-phase UAA triplet. All three azure mutants contained an A → G transition in this UAA second stop codon of the polymerase gene, resulting in a second suppressible UAG (amber) codon. Analysis of revertants demonstrated that the azure mutation can be counteracted either by a true site reversion at the second stop or by the creation of a new stop signal for the polymerase gene, either UAA (ochre) or UGA (opal), before or at the first stop, or beyond the second stop. On the basis of these results, a mechanism for the azure mutation is proposed. Silent mutations (one in the coding region, three in the untranslated 3′-terminal sequence) have also been observed in these phage stocks.     

Intragenic localization of chain-terminating triplets arising by transitions and transversions

Mutational changes involving transitions can convert only one sense codon to ochre, two codons to amber, and two codons to UGA. One codon, UGG for tryptophan, can be converted by transitions to either amber or UGA. By transversion changes 15 other codons can be converted to ochre and/or amber and/or UGA. Ten amino acids can never be replaced by chain termination as a result of transition and transversion mutagenesis of single base-pairs. For two systems (bacteriophage T4 lysozyme and Escherichia coli K12 tryptophan synthetase A protein) in which the poly-peptide gene product has been completely sequenced one can construct predictive intra-genic distribution maps for the location of all possible chain-terminating mutations arising as a result of transitions and transversions.