Role of the alfalfa mosaic virus methyltransferase-like domain in negative-strand RNA synthesis

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AMV) encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, which has a putative role in capping the viral RNAs. Six residues in this domain that are highly conserved in the methyltransferase domains of alphavirus-like viruses were mutated individually in AMV P1. None of the mutants was infectious to plants. Mutant RNA 1 was coexpressed with wild-type (wt) RNAs 2 and 3 from transferred DNA vectors in Nicotiana benthamiana by agroinfiltration. Mutation of His-100 or Cys-189 in P1 reduced accumulation of negative- and positive-strand RNA in the infiltrated leaves to virtually undetectable levels. Mutation of Asp-154, Arg-157, Cys-182, or Tyr-266 in P1 reduced negative-strand RNA accumulation to levels ranging from 2 to 38% of those for the wt control, whereas positive-strand RNA accumulation by these mutants was 2% or less. The (transiently) expressed replicases of the six mutants were purified from the agroinfiltrated leaves. Polymerase activities of these preparations in vitro ranged from undetectable to wt levels. The data indicate that, in addition to its putative role in RNA capping, the methyltransferase-like domain of P1 has distinct roles in replication-associated functions required for negative-strand RNA synthesis. The defect in negative-strand RNA synthesis of the His-100 and Cys-189 mutants could be complemented in trans by coexpression of wt P1.     

A single-stranded loop in the 5' untranslated region of cucumber mosaic virus RNA 4 contributes to competitive translational activity

The 5' untranslated region (UTR) of cucumber mosaic virus (CMV) RNA 4 confers a highly competitive translational advantage on a heterologous luciferase open reading frame. Here we investigated whether secondary structure in the 5' UTR contributes to this translational advantage. Stabilization of the 5' UTR RNA secondary structure inhibited competitive translational activity. Alteration of a potential single-stranded loop to a stem by substitution mutations greatly inhibited the competitive translational activity. Tobacco plants infected with wild type virus showed a 2.5-fold higher accumulation of maximal coat protein than did plants infected with a loop-mutant virus. Amplification of viral RNA in these plants could not explain the difference in accumulation of coat protein. Phylogenetic comparison showed that potential single-stranded loops of 12-23 nucleotides in length exist widely in subgroups of CMV.     

5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.     

Mutation of host DnaJ homolog inhibits brome mosaic virus negative-strand RNA synthesis

The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.     

Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.     

Sindbis virus nsP1 functions in negative-strand RNA synthesis.

A mutation at nucleotide 1101 of Sindbis virus ts11 nsP1 caused temperature-sensitive negative-strand synthesis and suppressed the 24R phenotype, which is caused by a mutation in nsP4. Nonstructural proteins synthesized and accumulated by ts11 at 40 degrees C did not cause the reactivation of negative-strand synthesis upon return to 30 degrees C and did not prevent the formation of new replication complexes at 30 degrees C.     

Eukaryotic elongation factor 1A interacts with the upstream pseudoknot domain in the 3' untranslated region of tobacco mosaic virus RNA

The genomic RNA of tobacco mosaic virus (TMV), like that of other positive-strand RNA viruses, acts as a template for both translation and replication. The highly structured 3' untranslated region (UTR) of TMV RNAs plays an important role in both processes; it is not polyadenylated but ends with a tRNA-like structure (TLS) preceded by a conserved upstream pseudoknot domain (UPD). The TLS of tobamoviral RNAs can be specifically aminoacylated and, in this state, can interact with eukaryotic elongation factor 1A (eEF1A)/GTP with high affinity. Using a UV cross-linking assay, we detected another specific binding site for eEF1A/GTP, within the UPDs of TMV and crucifer-infecting tobamovirus (crTMV), that does not require aminoacylation. A mutational analysis revealed that UPD pseudoknot conformation and some conserved primary sequence elements are required for this interaction. Its possible role in the regulation of tobamovirus gene expression and replication is discussed.     

Structure of the 5'-terminal untranslated region of the genomic RNAs from two strains of alfalfa mosaic virus.

We report the sequences of the 5'-terminal regions of the 3 Alfalfa Mosaic Virus genomic RNAs for the Strasbourg strain (AlMV-S) and for a new isolate, AlMV-B; they are compared to similar data obtained by Koper-Zwarthoff et al. (Nucleic Acids Res. 1980, 8, 5635-5647) for strain 425. The structure of these leaders is highly conserved in RNAs 1 and 2. The length of the leader is 102, 100 and 101 nucleotides in RNA1 for strains S, B and 425 respectively; 55 and 56 in RNA2 for strains S and B respectively. In RNA3 however, there are important differences near the 5'-terminus between strain S and the other two: The total leader length is 258 nucleotides for strain S and 242 for strain B. The secondary structure models show a conserved hairpin near the 5'-end of each genomic RNA of AlMV-S. This hairpin is inexistent in RNA3 of the B and 425 strains. The degree of base-pairing increases with leader length. The initiator codon is located in a single stranded region in RNA2 whereas it is found in a hairpin stem in RNA 3.     

The F1 motif of dengue virus polymerase NS5 is involved in promoter-dependent RNA synthesis

The mechanism by which viral RNA-dependent RNA polymerases (RdRp) specifically amplify viral genomes is still unclear. In the case of flaviviruses, a model has been proposed that involves the recognition of an RNA element present at the viral 5' untranslated region, stem-loop A (SLA), that serves as a promoter for NS5 polymerase binding and activity. Here, we investigated requirements for specific promoter-dependent RNA synthesis of the dengue virus NS5 protein. Using mutated purified NS5 recombinant proteins and infectious viral RNAs, we analyzed the requirement of specific amino acids of the RdRp domain on polymerase activity and viral replication. A battery of 19 mutants was designed and analyzed. By measuring polymerase activity using nonspecific poly(rC) templates or specific viral RNA molecules, we identified four mutants with impaired polymerase activity. Viral full-length RNAs carrying these mutations were found to be unable to replicate in cell culture. Interestingly, one recombinant NS5 protein carrying the mutations K456A and K457A located in the F1 motif lacked RNA synthesis dependent on the SLA promoter but displayed high activity using a poly(rC) template. Promoter RNA binding of this NS5 mutant was unaffected while de novo RNA synthesis was abolished. Furthermore, the mutant maintained RNA elongation activity, indicating a role of the F1 region in promoter-dependent initiation. In addition, four NS5 mutants were selected to have polymerase activity in the recombinant protein but delayed or impaired virus replication when introduced into an infectious clone, suggesting a role of these amino acids in other functions of NS5. This work provides new molecular insights on the specific RNA synthesis activity of the dengue virus NS5 polymerase.     

Kissing of the two predominant hairpin loops in the coxsackie B virus 3' untranslated region is the essential structural feature of the origin of replication required for negative-strand RNA synthesis.

Higher-order RNA structures in the 3' untranslated region (3'UTR) of enteroviruses are thought to play a pivotal role in viral negative-strand RNA synthesis. The structure of the 3'UTR was predicted by thermodynamic calculations using the STAR (structural analysis of RNA) computer program and experimentally verified using chemical and enzymatic probing of in vitro-synthesized RNA. A possible pseudoknot interaction between the 3D polymerase coding sequence and domain Y and a "kissing" interaction between domains X and Y was further studied by mutational analysis, using an infectious coxsackie B3 virus cDNA clone (domain designation as proposed by E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V.I. Agol (Nucleic Acids Res. 20:1739-1745, 1992). The higher-order RNA structure of the 3'UTR appeared to be maintained by an intramolecular kissing interaction between the loops of the two predominant hairpin structures (X and Y) within the 3'UTR. Disturbing this interaction had no effect on viral translation and processing of the polyprotein but exerted a primary effect on viral replication, as was demonstrated in a subgenomic coxsackie B3 viral replicon, in which the capsid P1 region was replaced by the luciferase gene. Mutational analysis did not support the existence of the pseudoknot interaction between hairpin loop Y and the 3D polymerase coding sequence. Based on these experiments, we constructed a three-dimensional model of the 3'UTR of coxsackie B virus that shows the kissing interaction as the essential structural feature of the origin of replication required for its functional competence.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 1.

Double-stranded cDNA of alfalfa mosaic virus (AlMV) RNA 1 has been cloned and sequenced. From clones with overlapping inserts, and other sequence data, the complete primary sequence of the 3644 nucleotides of RNA 1 was deduced: a long open reading frame for a protein of Mr 125,685 is flanked by a 5'-terminal sequence of 100 nucleotides and a 3' noncoding region of 163 nucleotides, including the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common. The two UGA-termination codons halfway RNA 1, that were postulated by Van Tol et al. (FEBS Lett. 118, 67-71, 1980) to account for partial translation of RNA 1 in vitro into Mr 58,000 and Mr 62,000 proteins, were not found in the reading frame of the Mr 125,685 protein.     

An RNA domain within the 5' untranslated region of the tomato bushy stunt virus genome modulates viral RNA replication

The terminal half of the 5' untranslated region (UTR) in the (+)-strand RNA genome of tomato bushy stunt virus was analyzed for possible roles in viral RNA replication. Computer-aided thermodynamic analysis of secondary structure, phylogenetic comparisons for base-pair covariation, and chemical and enzymatic solution structure probing were used to analyze the 78 nucleotide long 5'-terminal sequence. The results indicate that this sequence adopts a branched secondary structure containing a three-helix junction core. The T-shaped domain (TSD) formed by this terminal sequence is closed by a prominent ten base-pair long helix, termed stem 1 (S1). Deletion of either the 5' or 3' segment forming S1 (coordinates 1-10 or 69-78, respectively) in a model subviral RNA replicon, i.e. a prototypical defective interfering (DI) RNA, reduced in vivo accumulation levels of this molecule approximately 20-fold. Compensatory-type mutational analysis of S1 within this replicon revealed a strong correlation between formation of the predicted S1 structure and efficient DI RNA accumulation. RNA decay studies in vivo did not reveal any notable changes in the physical stabilities of DI RNAs containing disrupted S1s, thus implicating RNA replication as the affected process. Further investigation revealed that destabilization of S1 in the (+)-strand was significantly more detrimental to DI RNA accumulation than (-)-strand destabilization, therefore S1-mediated activity likely functions primarily via the (+)-strand. The essential role of S1 in DI RNA accumulation prompted us to examine the 5'-proximal secondary structure of a previously identified mutant DI RNA, RNA B, that lacks the 5' UTR but is still capable of low levels of replication. Mutational analysis of a predicted S1-like element present within a cryptic 5'-terminal TSD confirmed the importance of the former in RNA B accumulation. Collectively, these data support a fundamental role for the TSD, and in particular its S1 subelement, in tombusvirus RNA replication.     

Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA.

RNA 3 of alfalfa mosaic virus (AIMV) encodes the movement protein P3 and the viral coat protein which is translated from the subgenomic RNA 4. The 5'-leader sequences of RNA 3 of AIMV strains S, A, and Y differ in length from 314 to 392 nucleotides and contain a variable number of internal control regions of type 2 (ICR2 motifs) each located in a 27 nt repeat. Infectious cDNA clones were used to exchange the leader sequences of the three strains. This revealed that the leader sequence controls the specific ratio in which RNAs 3 and 4 are synthesized for each strain. In addition, it specifies strain specific differences in the kinetics of P3 accumulation in plants. Subsequent deletion analysis revealed that a 5'-sequence of 112 nt containing one ICR2 motif was sufficient for a 10 to 20% level of RNA 3 accumulation in protoplasts and a delayed accumulation in plants. An additional leader sequence of maximally 114 nt, containing two ICR2 motifs, was required to permit wildtype levels of RNA 3 accumulation. The effect of deletions in the leader sequence on P3 synthesis in vitro and in vivo was investigated.