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PubMed1985

Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses.

Although the genetic organization of tobacco mosaic virus (TMV) differs considerably from that of the tripartite viruses (alfalfa mosaic virus [AlMV] and brome mosaic virus [BMV]), all of these RNA plant viruses share three domains of homology among their nonstructural proteins. One such domain, common to the AlMV and BMV 2a proteins and the readthrough portion of TMV p183, is also homologous to the readthrough protein nsP4 of Sindbis virus (Haseloff et al., Proc. Natl. Acad. Sci. U.S.A. 81:4358-4362, 1984). Two more domains are conserved among the AlMV and BMV 1a proteins and TMV p126. We show here that these domains have homology with portions of the Sindbis proteins nsP1 and nsP2, respectively. These results strengthen the view that the four viruses share mechanistic similarities in their replication strategies and may be evolutionarily related. These results also suggest that either the AlMV 1a, BMV 1a, and TMV p126 proteins are multifunctional or Sindbis proteins nsP1 and nsP2 function together as subunits in a single complex.

Amino Acid Sequence↗
PubMed2003

A functional interaction between ribosomal proteins S7 and S11 within the bacterial ribosome.

In this study, we used site-directed mutagenesis to disrupt an interaction that had been detected between ribosomal proteins S7 and S11 in the crystal structure of the bacterial 30 S subunit. This interaction, which is located in the E site, connects the head of the 30 S subunit to the platform and is involved in the formation of the exit channel through which passes the 30 S-bound messenger RNA. Neither mutations in S7 nor mutations in S11 prevented the incorporation of the proteins into the 30 S subunits but they perturbed the function of the ribosome. In vivo assays showed that ribosomes with either mutated S7 or S11 were altered in the control of translational fidelity, having an increased capacity for frameshifting, readthrough of a nonsense codon and codon misreading. Toeprinting and filter-binding assays showed that 30 S subunits with either mutated S7 or S11 have an enhanced capacity to bind mRNA. The effects of the S7 and S11 mutations can be related to an increased flexibility of the head of the 30 S, to an opening of the mRNA exit channel and to a perturbation of the proposed allosteric coupling between the A and E sites. Altogether, our results demonstrate that S7 and S11 interact in a functional manner and support the notion that protein-protein interactions contribute to the dynamics of the ribosome.

Amino Acid Sequence↗
PubMed1999

Degradation of FinP antisense RNA from F-like plasmids: the RNA-binding protein, FinO, protects FinP from ribonuclease E.

Transfer of F-like plasmids is regulated by the FinOP system, which controls the expression of traJ, a positive regulator of the transfer operon. F FinP is a 79 base antisense RNA, composed of two stem-loops, complementary to the 5' untranslated leader of traJ mRNA. Binding of FinP to the traJ leader sequesters the traJ ribosome binding site, preventing its translation and repressing plasmid transfer. The FinO protein binds stem-loop II of FinP and traJ mRNA and promotes duplex formation in vitro. FinO stabilizes FinP, increasing its effective concentration in vivo. To determine how FinO protects FinP from decay, the degradation of FinP was examined in a series of ribonuclease-deficient strains. Using Northern blot analysis, full-length FinP was found to be stabilized sevenfold in an RNase E-deficient strain. The major site of RNase E cleavage was mapped on synthetic FinP, to the single-stranded region between stem-loops I and II. A secondary site near the 5' end ( approximately 10 bases) was also observed. A GST-FinO fusion protein protected FinP from RNase E cleavage at both sites in vitro. Two duplexes between FinP and traJ mRNA were detected in an RNase III-deficient strain. The larger duplex resulted from extension of the FinP transcript at its 3' end, suggesting readthrough at the terminator that corresponds to FinP stem-loop II. A point mutant of finP (finP305; C30U) that is unable to repress traJ in the presence of FinO was also characterized. The pattern of RNase E digestion of finP305 RNA differed from FinP, and GST-FinO did not protect finP305 RNA from cleavage in vitro. The half-life of finP305 RNA decreased more than tenfold in vivo, such that the steady-state levels of finP305 RNA, in the presence of FinO, were insufficient to significantly reduce the level of traJ mRNA available for translation, allowing derepressed levels of transfer.

Bacterial Outer Membrane Proteins↗
PubMed1990

Immunodetection in vivo of beet necrotic yellow vein virus-encoded proteins.

Open reading frames identified on the four genomic RNAs of beet necrotic yellow vein virus were cloned into bacterial expression vectors and resulting cl-fusion proteins expressed in Escherichia coli were used to raise polyclonal antibodies. This set of antisera was used to show the presence of 7 of 9 predicted viral proteins in mechanically inoculated Chenopodium quinoa leaves by the Western blot technique. Viral coat protein (p22) and its readthrough protein p85 encoded by RNA-2 could be detected in all subcellular fractions. Two other RNA-2-encoded proteins, p42 and p13, are predominantly associated with membranous structures. Another RNA-2-encoded protein, p14, as well as the two polypeptides p25 and p31, encoded by RNA-3 and -4, respectively, are soluble proteins. The viral proteins could first be detected about the time lesions became visible and increased thereafter except for p85, in which case the amount of the soluble form decreased with time. No protein could be detected corresponding to the RNA-1-encoded p237 protein or to the p15 species encoded by open reading frame V of RNA-2.

Antibodies, Viral↗
PubMed1984

Complete nucleotide sequence of the genomic RNA of Sindbis virus.

The entire nucleotide sequence of the genomic RNA of the type virus of the alphavirus genus, Sindbis virus, has been determined. The genome is 11,703 nucleotides in length, exclusive of the 5' cap and the 3'-terminal poly(A) tract. After the 5'-terminal cap there are 59 nucleotides of 5' nontranslated nucleic acid followed by a reading frame of 7539 nucleotides that encodes the nonstructural polypeptides and which is open except for a single opal termination codon. Following 48 untranslated bases located in the junction region which separates the nonstructural and structural protein coding sequences, there is an open reading frame 3735 nucleotides long that encodes the structural proteins. Finally, the 3' untranslated region is 322 nucleotides long. The nonstructural proteins are translated from the genomic RNA as two polyprotein precursors. The first is 1896 amino acids in length and terminates at an opal codon at position 1897. This polyprotein is processed to produce three polypeptides called nsP1, nsP2, and nsP3. Sites of post-translational cleavage to produce these three proteins have been tentatively located using available N-terminal amino acid sequence data. In both cases cleavage probably occurs between the two alanine residues in the sequence Gly-Ala-Ala. The fourth nonstructural protein, nsP4, is produced when readthrough of the opal codon produces a second polyprotein precursor of length 2513 amino acids, which is also cleaved posttranslationally. The structural proteins are translated from a subgenomic message which begins at nucleotide 7598, is 4106 nucleotides in length (exclusive of the poly(A) tract), and is coterminal with the 3' end of the genomic RNA. The structural proteins are also translated as a polyprotein precursor which is cleaved to produce a nucleocapsid protein and two integral membrane glycoproteins as well as two small peptides not present in the mature virion. A replication strategy for Sindbis virus based upon the complete nucleotide sequence, as well as prior data, is presented.

Amino Acid Sequence↗
PubMed1999

Genetic study of interactions between the cytoskeletal assembly protein sla1 and prion-forming domain of the release factor Sup35 (eRF3) in Saccharomyces cerevisiae.

Striking similarities between cytoskeletal assembly and the "nucleated polymerization" model of prion propagation suggest that similar or overlapping sets of proteins may assist in both processes. We show that the C-terminal domain of the yeast cytoskeletal assembly protein Sla1 (Sla1C) specifically interacts with the N-terminal prion-forming domain (Sup35N) of the yeast release factor Sup35 (eRF3) in the two-hybrid system. Sla1C and several other Sup35N-interacting proteins also exhibit two-hybrid interactions with the poly-Gln-expanded N-proximal fragment of human huntingtin, which promotes Huntington disease-associated aggregation. The Sup35N-Sla1C interaction is inhibited by Sup35N alterations that make Sup35 unable to propagate the [PSI(+)] state and by the absence of the chaperone protein Hsp104, which is essential for [PSI] propagation. In a Sla1(-) background, [PSI] curing by dimethylsulfoxide or excess Hsp104 is increased, while translational readthrough and de novo [PSI] formation induced by excess Sup35 or Sup35N are decreased. These data show that, in agreement with the proposed function of Sla1 during cytoskeletal formation, Sla1 assists in [PSI] formation and propagation, but is not required for these processes. Sla1(-) strains are sensitive to some translational inhibitors, and some sup35 mutants, obtained in a Sla1(-) background, are sensitive to Sla1, suggesting that the interaction between Sla1 and Sup35 proteins may play a role in the normal function of the translational apparatus. We hypothesize that Sup35N is involved in regulatory interactions with intracellular structural networks, and [PSI] prion may be formed as a by-product of this process.

Carrier Proteins↗
PubMed2026

Long-read sequencing reveals widespread novel splicing and neojunction-derived neoantigens in nasopharyngeal carcinoma.

The widespread transcriptomic diversity driven by alternative splicing (AS) contributes to all hallmarks of cancer and represents a critical source of neoantigens for personalized immunotherapy. However, unlike other major malignancies, the full repertoire of AS in nasopharyngeal carcinoma (NPC) remains underexplored. Here, we employ long-read sequencing (LR-seq) to generate a high-resolution, isoform-level transcriptomic atlas from a cohort of 14 NPC tumor samples and four immortalized nasopharyngeal epithelial cell lines. We identify a substantial number of full-length novel transcripts (22,687; ∼44.38%), which reveal diverse splicing patterns and previously unannotated splicing events. By integrating short-read RNA-seq data to quantify isoform expression, we discover a subset of novel transcripts that are differentially expressed between tumor samples and immortalized nasopharyngeal epithelial cell lines. Furthermore, LR-seq enables precise identification of chimeric readthrough fusion transcripts, such as CLDN15-FIS1 and FOXRED2-TXN2 Finally, we develop a computational framework, tumor-specific splicing neoantigen detection (TS-SNAD), to predict neoantigens originating from novel exon-exon junctions (neojunctions) in tumor-specific novel transcripts. Using this framework, we identify neojunction-derived neoantigens and experimentally validate the immunogenicity of selected HLA-B*40:01-restricted neoantigens. These neojunction-derived peptides constitute a new class of noncanonical neoantigens with significant potential for developing personalized cancer vaccines for NPC.

Humans↗
PubMedDate not supplied

Synaptogenesis and myopathy under acetylcholinesterase overexpression.

Environmental, congenital, and acquired immunological insults perturbing neuromuscular junction (NMJ) activity may induce a variety of debilitating neuromuscular pathologies. However, the molecular elements linking NMJ dysfunction to long-term myopathies are unknown. Here, we report dramatically elevated levels of mRNA encoding c-Fos and the "readthrough" (R) variant of acetylcholinesterase (AChE) in muscles of transgenic mice overexpressing synaptic (S) AChE in motoneurons and in control mice treated with the irreversible cholinesterase inhibitor diisopropylfluorophosphonate (DFP). Tongue muscles from DFP-treated and AChE-S transgenic mice displayed exaggerated neurite branching and disorganized, wasting fibers. Moreover, diaphragm muscles from both transgenic and DFP-treated mice exhibited NMJ proliferation. 2'-O-methyl-protected antisense oligonucleotides targeted to AChE mRNA suppressed feedback upregulation of AChE and ameliorated DFP-induced NMJ proliferation. Our findings demonstrate common transcriptional responses to cholinergic NMJ stress of diverse origin, and implicate deregulated AChE expression in excessive neurite outgrowth, uncontrolled synaptogenesis, and myopathology.

Acetylcholinesterase↗
PubMed1997

Phenotypic heterogeneity of mutational changes at a conserved nucleotide in 16 S ribosomal RNA.

RNA sites that contain unpaired or mismatched nucleotides can be interaction sites for other macromolecules. C1054, a virtually universally conserved nucleotide in the 16 S (small subunit) ribosomal RNA of Escherichia coli, is part of a highly conserved bulge in helix 34, which has been located at the decoding site of the ribosome. This helix has been implicated in several translational events, including peptide chain termination and decoding accuracy. Here, we observed interesting differences in phenotype associated with the three base substitutions at, and the deletion of, nucleotide C1054. The phenotypes examined include suppression of nonsense codons on different media and at different temperatures, lethality conditioned by temperature and level of expression of the mutant rRNA, ribosome profiles upon centrifugation through sucrose density gradients, association of mutant 30 S subunits with 50 S subunits, and effects on the action of tRNA suppressor mutants. Some of our findings contradict previously reported properties of individual mutants. Particularly notable is our finding that the first reported 16 S rRNA suppressor of UGA mutations was not a C1054 deletion but rather the base substitution C1054A. After constructing deltaC1054 by site-directed mutagenesis, we observed, among other differences, that it does not suppress any of the trpA mutations previously reported to be suppressed by the original UGA suppressor. In general, our results are consistent with the suggestion that the termination codon readthrough effects of mutations at nucleotide 1054 are the result of defects in peptide chain termination rather than of decreases in general translational accuracy. The phenotypic heterogeneity associated with different mutations at this one nucleotide position may be related to the mechanisms of involvement of this nucleotide, the two-nucleotide bulge, and/or helix 34 in particular translational events. In particular, previous indications from other laboratories of conformational changes associated with this region are consistent with differential effects of 1054 mutations on RNA-RNA or RNA-protein interactions. Finally, the association of a variety of phenotypes with different changes at the same nucleotide may eventually shed light on speculations about the coevolution of parts of ribosomal RNA with other translational macromolecules.

Base Sequence↗
PubMed2003

The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis.

Alternative splicing induces, under abnormal cholinergic neurotransmission, overproduction of the rare "readthrough" acetylcholinesterase variant AChE-R. We explored the pathophysiological relevance of this phenomenon in patients with myasthenia gravis (MG) and rats with experimental autoimmune MG (EAMG), neuromuscular junction diseases with depleted acetylcholine receptors. In MG and EAMG, we detected serum AChE-R accumulation. In EAMG, we alleviated electromyographic abnormalities by nanomolar doses of EN101, an antisense oligonucleotide that selectively lowers AChE-R in blood and muscle yet leaves unaffected the synaptic variant AChE-S. Whereas animals treated with placebo or conventional anticholinesterases continued to deteriorate, a 4 wk daily oral administration of EN101 improved survival, neuromuscular strength and clinical status in moribund EAMG rats. The efficacy of targeting only one AChE splicing variant highlights potential advantages of mRNA-targeted therapeutics for chronic cholinergic malfunctioning.

Acetylcholinesterase↗
PubMed1997

Genome structure of cucumber leaf spot virus: sequence analysis suggests it belongs to a distinct species within the Tombusviridae.

The complete nucleotide sequence of cucumber leaf spot virus (CLSV) has been determined and the sizes and locations of predicted viral proteins deduced. The genome consists of 4432 nucleotides and contains five long ORFs. The 5' proximal ORF encodes a 25 kDa product that terminates in an amber codon which may be readthrough to produce an 84 kDa protein (ORF 2). ORF 3 codes for the 41 kDa coat protein (CP). ORFs 4 and 5 are completely overlapping at the 3' terminus and code for 27 and 17 kDa products, respectively. The CLSV genome structure is similar to that of tombusviruses and nearly identical to pothos latent virus (PoLV), a newly proposed, atypical, member of the Tombusviridae. It is proposed that CLSV and PoLV be considered strains of a new tombusvirus species. Amino acid sequence comparisons of the CLSV CP and the CPs of several small spherical plant viruses suggest that CLSV is most closely related to melon necrotic spot carmovirus (MNSV), red clover necrotic mosaic dianthovirus (RCNMV) and cucumber necrosis tombusvirus (CNV). These viruses, like CLSV, are transmitted by the soil inhabiting fungus, Olpidium bornovanus.

Amino Acid Sequence↗
PubMed1997

An NMR and mutational study of the pseudoknot within the gene 32 mRNA of bacteriophage T2: insights into a family of structurally related RNA pseudoknots.

NMR methods were used to investigate a series of mutants of the pseudoknot within the gene 32 messenger RNA of bacteriophage T2, for the purpose of investigating the range of sequences, stem and loop lengths that can form a similar pseudoknot structure. This information is of particular relevance since the T2 pseudoknot has been considered a representative of a large family of RNA pseudoknots related by a common structural motif, previously referred to as 'common pseudoknot motif 1' or CPK1. In the work presented here, a mutated sequence with the potential to form a pseudoknot with a 6 bp stem2 was shown to adopt a pseudoknot structure similar to that of the wild-type sequence. This result is significant in that it demonstrates that pseudoknots with 6 bp in stem2 and a single nucleotide in loop1 are indeed feasible. Mutated sequences with the potential to form pseudoknots with either 5 or 8 bp in stem2 yielded NMR spectra that could not confirm the formation of a pseudoknot structure. Replacing the adenosine nucleotide in loop1 of the wild-type pseudoknot with any one of G, C or U did not significantly alter the pseudoknot structure. Taken together, the results of this study provide support for the existence of a family of similarly structured pseudoknots with two coaxially stacked stems, either 6 or 7 bp in stem2, and a single nucleotide in loop1. This family includes many of the pseudoknots predicted to occur downstream of the frameshift or readthrough sites in a significant number of viral RNAs.

Animals↗
PubMed1991

Rescue of Sindbis virus-specific RNA replication and transcription by using a vaccinia virus recombinant.

A heterologous system expressing functional Sindbis virus nonstructural proteins (nsPs) has several possible uses for studying Sindbis virus-specific RNA replication and transcription in vivo and in vitro. Of the many possible approaches, vaccinia virus offers an attractive transient expression system given that Sindbis virus replication can occur in cells which have been previously infected by vaccinia virus. In this report, a vaccinia virus recombinant (called vSINNS), which contains the cDNA encoding the Sindbis virus nsPs under the control of either the vaccinia virus 7.5K promoter or the bacteriophage T7 promoter, has been constructed and characterized. Upon infection of several cell types with vSINNS, Sindbis virus nsP precursors and processed forms, including nsP1, nsP2, and both phosphorylated and nonphosphorylated forms of nsP3, were synthesized. Proteins containing the putative RNA-dependent RNA polymerase domain (nsP4 and nsP34), which are normally produced in small amounts by readthrough of an opal termination codon, were not detected in vSINNS-infected cells. However, all nsP functions necessary for Sindbis virus-specific RNA synthesis must have been expressed, since both replication and subgenomic mRNA transcription of an engineered Sindbis virus defective interfering RNA in cells infected with vSINNS was observed. Furthermore, vSINNS could be used as a helper virus to amplify, to relatively high titers, a replication-defective Sindbis virus mutant containing an in-frame deletion in the conserved N-terminal domain of nsP3. These data, as well as the observation that normal yields of parental Sindbis virus are produced in cells which have been previously infected with vSINNS, indicate that expression of Sindbis virus nsPs, in the absence of Sindbis virus-specific RNA replication, is not sufficient to block the formation of active RNA replication complexes by superinfecting Sindbis virus.

Animals↗
PubMed1999

A sequence in the Escherichia coli fdhF "selenocysteine insertion Sequence" (SECIS) operates in the absence of selenium.

The UGA codon context of the Escherichia coli fdhF mRNA includes an element called the selenocysteine insertion sequence (SECIS) that is responsible for the UGA-directed incorporation of the amino acid selenocysteine into a protein. Here, we describe an extended fdhF SECIS that includes the information for an additional function: the prevention of UGA readthrough under conditions of selenium deficiency. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively, and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Here, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium, the SECIS enables one of two alternatives for the translational machinery: either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.

Arginine↗
PubMed2000

Functions of the 126- and 183-kDa proteins of tobacco mosaic virus.

Tobacco mosaic virus produces two proteins that contain domains similar to the methyltransferase (MT) and helicase (HEL)-like domains of the replicase-associated proteins of other RNA viruses. The more abundant 126-kDa protein contains only the MT and HEL-like domains, whereas the 183-kDa readthrough protein additionally contains the polymerase domain. We examined the functions of these proteins by constructing a bipartite system to express the 126- and 183-kDa proteins from separate RNAs. Mutants expressing the 183-kDa protein recognized promoters for negative- and positive-stranded RNA synthesis, transcribed subgenomic mRNAs, capped RNAs, synthesized proteins, moved cell to cell within the plant, and replicated defective RNAs (dRNAs). The principal function of the 126-kDa protein was to increase the rate of replication approximately tenfold. The 126-kDa protein appeared to function primarily in cis, and production of the 126-kDa protein in trans did not enhance replication of the helper virus. dRNAs producing a functional 126-kDa protein were replicated efficiently by helper viruses that produced only the 183-kDa protein but not by wild-type virus, suggesting that efficient replication required the 183-kDa protein to form a heterodimer with the 126-kDa protein already bound to the target dRNA.

Frameshift Mutation↗
PubMed1998

Antisense RNA: function and fate of duplex RNA in cells of higher eukaryotes.

There is ample evidence that cells of higher eukaryotes express double-stranded RNA molecules (dsRNAs) either naturally or as the result of viral infection or aberrant, bidirectional transcriptional readthrough. These duplex molecules can exist in either the cytoplasmic or nuclear compartments. Cells have evolved distinct ways of responding to dsRNAs, depending on the nature and location of the duplexes. Since dsRNA molecules are not thought to exist naturally within the cytoplasm, dsRNA in this compartment is most often associated with viral infections. Cells have evolved defensive strategies against such molecules, primarily involving the interferon response pathway. Nuclear dsRNA, however, does not induce interferons and may play an important posttranscriptional regulatory role. Nuclear dsRNA appears to be the substrate for enzymes which deaminate adenosine residues to inosine residues within the polynucleotide structure, resulting in partial or full unwinding. Extensively modified RNAs are either rapidly degraded or retained within the nucleus, whereas transcripts with few modifications may be transported to the cytoplasm, where they serve to produce altered proteins. This review summarizes our current knowledge about the function and fate of dsRNA in cells of higher eukaryotes and its potential manipulation as a research and therapeutic tool.

Animals↗
PubMed1987

Theoretical analysis of single-round transcription experiments on trp leader region.

A kinetic model is proposed to reproduce the time courses of the concentration change in paused leader RNA, terminated leader RNA, and readthrough RNA in the single-round transcription experiments on trp leader region of Escherichia coli and its mutants, L132, L75, and L75L135 (Winkler, M. E., and C. Yanofsky, 1981, Biochemistry, 20:3738-3744; Fisher, R., and C. Yanofsky, 1983, J. Biol. Chem., 258:9208-9212). This model fits the experimental results well and also captures the essential aspects of the processes of transcriptional pausing and termination. In the wild type template, under optimal conditions, it is found that the transcription rate at the pause and attenuation sites is of the same order of magnitude, 10,000-fold lower than the transcription rate at the other sites, and the high termination level at the attenuation site is attributable to a higher dissociation rate. This analysis also provides a clue as to how the template base change, various concentrations of ribonucleoside triphosphates, and the presence or absence of L-factor affect the transcription and dissociation rates to yield different termination levels at the pause or attenuation site. It also discusses the molecular mechanism of the transcriptional pausing and termination.

DNA-Directed RNA Polymerases↗
PubMed1992

Characterization of an internal element in turnip crinkle virus RNA involved in both coat protein binding and replication.

The major coat-protein-binding element of turnip crinkle virus RNA was previously mapped in the region of the UAG termination codon in the viral polymerase gene. This region encompasses two of the high-affinity coat-protein-binding sites (Fa and Ff) that we suggested were physically associated in a stem-loop in a ribonucleoprotein complex involved in assembly initiation (Wei, Heaton, Morris, and Harrison, J. Mol. Biol. 214, 85-95, 1990). We have also demonstrated that this RNA element was capable of specific coat protein binding in vitro (Wei and Morris, J. Mol. Biol. 222, 437-443, 1991). We now provide physical evidence, by in vitro chemical and enzymatic probing of the viral RNA, that support the suggestion that the two coat-protein-binding sites base pair to form a stem structure (A/F stem) surrounding the UAG terminator in wild-type RNA. We have shown here that a mutant with seven conservative nucleotide substitutions in Fa does not accumulate to detectable levels in plants or protoplasts and that the A/F stem structure is drastically altered in this mutant. We suggest that the primary effect of this mutation is on replication rather than on a reduction in RNA stability resulting from a defect in encapsidation of the virion RNA because previous results have shown that encapsidation-deficient mutants have little or no effect on viral RNA replication (Hacker, Petty, Wei, and Morris, Virology 186, 1-8, 1992). The analysis of the A/F stem was extended by construction and characterization of a series of mutants and revertants that displayed variable levels of replication deficiency but minimal concomitant defect in encapsidation efficiency. The extent of the replication defect correlated with the predicted destabilization of the A/F stem structure. We conclude from these results that this RNA element is involved in viral replication, and we tentatively suggest that the A/F stem structure may be functionally involved in the readthrough translation of the viral polymerase.

Base Sequence↗