scholarly journals Requirements at the 3′ End of the Sindbis Virus Genome for Efficient Synthesis of Minus-Strand RNA

2005 ◽  
Vol 79 (8) ◽  
pp. 4630-4639 ◽  
Author(s):  
Richard W. Hardy ◽  
Charles M. Rice
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Genome Replication ◽  
Minus Strand ◽  
Wild Type ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Type 3

ABSTRACT The 3′-untranslated region of the Sindbis virus genome is 0.3 kb in length with a 19-nucleotide conserved sequence element (3′ CSE) immediately preceding the 3′-poly(A) tail. The 3′ CSE and poly(A) tail have been assumed to constitute the core promoter for minus-strand RNA synthesis during genome replication; however, their involvement in this process has not been formally demonstrated. Utilizing both in vitro and in vivo analyses, we have examined the role of these elements in the initiation of minus-strand RNA synthesis. The major findings of this study with regard to efficient minus-strand RNA synthesis are the following: (i) the wild-type 3′ CSE and the poly(A) tail are required, (ii) the poly(A) tail must be a minimum of 11 to 12 residues in length and immediately follow the 3′ CSE, (iii) deletion or substitution of the 3′ 13 nucleotides of the 3′ CSE severely inhibits minus-strand RNA synthesis, (iv) templates possessing non-wild-type 3′ sequences previously demonstrated to support virus replication do not program efficient RNA synthesis, and (v) insertion of uridylate residues between the poly(A) tail and a non-wild-type 3′ sequence can restore promoter function to a limited extent. This study shows that the optimal structure of the 3′ component of the minus-strand promoter is the wild-type 3′ CSE followed a poly(A) tail of at least 11 residues. Our findings also show that insertion of nontemplated bases can restore function to an inactive promoter.

scholarly journals Modification of the 5′ Terminus of Sindbis Virus Genomic RNA Allows nsP4 RNA Polymerases with Nonaromatic Amino Acids at the N Terminus To Function in RNA Replication

2003 ◽  
Vol 77 (4) ◽  
pp. 2301-2309 ◽  
Author(s):  
Yukio Shirako ◽  
Ellen G. Strauss ◽  
James H. Strauss
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Minus Strand ◽  
Genomic Rna ◽  
Wild Type ◽  
N Terminus

ABSTRACT We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40°C upon addition of AU, AUA, or AUU to the 5′ terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3′-UA-5′ is required at the 3′ terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5′ end of the genomic RNA provides unpaired 3′-UA-5′ at the 3′ end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3′-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3′ terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.

scholarly journals Alphavirus RNA Genome Repair and Evolution: Molecular Characterization of Infectious Sindbis Virus Isolates Lacking a Known Conserved Motif at the 3′ End of the Genome

2000 ◽  
Vol 74 (20) ◽  
pp. 9776-9785 ◽  
Author(s):  
Jyothi George ◽  
Ramaswamy Raju
Keyword(s):  
Sindbis Virus ◽  
Repeat Sequence ◽  
Genome Replication ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Long Term Stability ◽  
Nontranslated Region ◽  
Viral Promoters ◽  
Sequence Elements

ABSTRACT The 3′ nontranslated region of the genomes of Sindbis virus (SIN) and other alphaviruses carries several repeat sequence elements (RSEs) as well as a 19-nucleotide (nt) conserved sequence element (3′CSE). The 3′CSE and the adjoining poly(A) tail of the SIN genome are thought to act as viral promoters for negative-sense RNA synthesis and genome replication. Eight different SIN isolates that carry altered 3′CSEs were studied in detail to evaluate the role of the 3′CSE in genome replication. The salient findings of this study as it applies to SIN infection of BHK cells are as follows: i) the classical 19-nt 3′CSE of the SIN genome is not essential for genome replication, long-term stability, or packaging; ii) compensatory amino acid or nucleotide changes within the SIN genomes are not required to counteract base changes in the 3′ terminal motifs of the SIN genome; iii) the 5′ 1-kb regions of all SIN genomes, regardless of the differences in 3′ terminal motifs, do not undergo any base changes even after 18 passages; iv) although extensive addition of AU-rich motifs occurs in the SIN genomes carrying defective 3′CSE, these are not essential for genome viability or function; and v) the newly added AU-rich motifs are composed predominantly of RSEs. These findings are consistent with the idea that the 3′ terminal AU-rich motifs of the SIN genomes do not bind directly to the viral polymerase and that cellular proteins with broad AU-rich binding specificity may mediate this interaction. In addition to the classical 3′CSE, other RNA motifs located elsewhere in the SIN genome must play a major role in template selection by the SIN RNA polymerase.

scholarly journals Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

1998 ◽  
Vol 72 (8) ◽  
pp. 6546-6553 ◽  
Author(s):  
Julie A. Lemm ◽  
Anders Bergqvist ◽  
Carol M. Read ◽  
Charles M. Rice
Keyword(s):  
Rna Polymerase ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Functional Assay ◽  
Minus Strand ◽  
Virus Rna ◽  
A Genome ◽  
Strand Initiation

ABSTRACT Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

scholarly journals Alphavirus Minus-Strand RNA Synthesis: Identification of a Role for Arg183 of the nsP4 Polymerase

2002 ◽  
Vol 76 (17) ◽  
pp. 8632-8640 ◽  
Author(s):  
Cori L. Fata ◽  
Stanley G. Sawicki ◽  
Dorothea L. Sawicki
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Temperature Sensitive ◽  
Minus Strand ◽  
Mrna Synthesis ◽  
Wild Type ◽  
Lethal Temperature ◽  
Host Restriction ◽  
Mosquito Cells ◽  
Promoter Recognition

ABSTRACT 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°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.

scholarly journals Requirements for Brome Mosaic Virus Subgenomic RNA Synthesis In Vivo and Replicase-Core Promoter Interactions In Vitro

2004 ◽  
Vol 78 (12) ◽  
pp. 6091-6101 ◽  
Author(s):  
K. Sivakumaran ◽  
Seung-Kook Choi ◽  
Masarapu Hema ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Wild Type ◽  
Subgenomic Rna ◽  
The Core ◽  
Rna Hairpin

ABSTRACT Based solely on in vitro results, two contrasting models have been proposed for the recognition of the brome mosaic virus (BMV) subgenomic core promoter by the replicase. The first posits that the replicase recognizes at least four key nucleotides in the core promoter, followed by an induced fit, wherein some of the nucleotides base pair prior to the initiation of RNA synthesis (S. Adkins and C. C. Kao, Virology 252:1-8, 1998). The second model posits that a short RNA hairpin in the core promoter serves as a landing pad for the replicase and that at least some of the key nucleotides help form a stable hairpin (P. C. J. Haasnoot, F. Brederode, R. C. L. Olsthoorn, and J. Bol, RNA 6:708-716, 2000; P. C. J. Haasnoot, R. C. L. Olsthoorn, and J. Bol, RNA 8:110-122, 2002). We used transfected barley protoplasts to examine the recognition of the subgenomic core promoter by the BMV replicase. Key nucleotides required for subgenomic initiation in vitro were found to be important for RNA4 levels in protoplasts. In addition, additional residues not required in vitro and the formation of an RNA hairpin within the core promoter were correlated with wild-type RNA4 levels in cells. Using a template competition assay, the core promoter of ca. 20 nucleotides was found to be sufficient for replicase binding. Mutations of the key residues in the core promoter reduced replicase binding, but deletions that disrupt the predicted base pairing in the proposed stem retained binding at wild-type levels. Together, these results indicate that key nucleotides in the BMV subgenomic core promoter direct replicase recognition but that the formation of a stem-loop is required at a step after binding. Additional functional characterization of the subgenomic core promoter was performed. A portion of the promoter for BMV minus-strand RNA synthesis could substitute for the subgenomic core promoter in transfected cells. The comparable sequence from Cowpea Chlorotic Mottle Virus (CCMV) could also substitute for the BMV subgenomic core promoter. However, nucleotides in the CCMV core required for RNA synthesis are not identical to those in BMV, suggesting that the subgenomic core promoter can induce the BMV replicase in interactions needed for subgenomic RNA transcription in vivo.

scholarly journals Rubella virus-induced superinfection exclusion studied in cells with persisting replicons

2007 ◽  
Vol 88 (10) ◽  
pp. 2769-2773 ◽  
Author(s):  
Claudia Claus ◽  
Wen-Pin Tzeng ◽  
Uwe G. Liebert ◽  
Teryl K. Frey
Keyword(s):  
Virus Infection ◽  
Rubella Virus ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Vero Cells ◽  
Minus Strand ◽  
Wild Type ◽  
Site Mutation ◽  
Superinfection Exclusion ◽  
First Time

For the first time, homologous superinfection exclusion was documented for rubella virus (RUB) by using Vero cells harbouring persisting RUB replicons. Infection with wild-type RUB was reduced by tenfold, whereas Sindbis virus infection was unaffected. Replication following infection with packaged replicons and transfection with replicon transcripts was also restricted in these cells, indicating that restriction occurred after penetration and entry. Translation of such ‘supertransfecting’ replicon transcripts was not impaired, but no accumulation of supertransfecting replicon RNA could be detected. We tested the hypothesis favoured in the related alphaviruses that superinfection exclusion is mediated by cleavage of the incoming non-structural precursor by the pre-existing non-structural (NS) protease, resulting in an inhibition of minus-strand RNA synthesis. However, cleavage of a precursor translated from a supertransfecting replicon transcript with an NS protease catalytic-site mutation was not detected and the event in the replication cycle at which superinfection exclusion is executed remains to be elucidated.

scholarly journals An Attenuating Mutation in nsP1 of the Sindbis-Group Virus S.A.AR86 Accelerates Nonstructural Protein Processing and Up-Regulates Viral 26S RNA Synthesis

2003 ◽  
Vol 77 (2) ◽  
pp. 1149-1156 ◽  
Author(s):  
Mark T. Heise ◽  
Laura J. White ◽  
Dennis A. Simpson ◽  
Christopher Leonard ◽  
Kristen A. Bernard ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Adult Mouse ◽  
Nonstructural Protein ◽  
Growth Curves ◽  
Protein Processing ◽  
Wild Type ◽  
Rnase Protection ◽  
Nonstructural Protein 1

ABSTRACT The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.

scholarly journals Replicase-Binding Sites on Plus- and Minus-Strand Brome Mosaic Virus RNAs and Their Roles in RNA Replication in Plant Cells

2004 ◽  
Vol 78 (24) ◽  
pp. 13420-13429 ◽  
Author(s):  
S.-K. Choi ◽  
M. Hema ◽  
K. Gopinath ◽  
J. Santos ◽  
C. Kao
Keyword(s):  
Mosaic Virus ◽  
Binding Sites ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
The Core ◽  
Strand Initiation

ABSTRACT The cis-acting elements for Brome mosaic virus (BMV) RNA synthesis have been characterized primarily for RNA3. To identify additional replicase-binding elements, nested fragments of all three of the BMV RNAs, both plus- and minus-sense fragments, were constructed and tested for binding enriched BMV replicase in a template competition assay. Ten RNA fragments containing replicase-binding sites were identified; eight were characterized further because they were more effective competitors. All eight mapped to noncoding regions of BMV RNAs, and the positions of seven localized to sequences containing previously characterized core promoter elements (C. C. Kao, Mol. Plant Pathol. 3:55-62, 2001), thus suggesting the identities of the replicase-binding sites. Three contained the tRNA-like structures that direct minus-strand RNA synthesis, three were within the 3′ region of each minus-strand RNA that contained the core promoter for genomic plus-strand initiation, and one was in the core subgenomic promoter. Single-nucleotide mutations known previously to abolish RNA synthesis in vitro prevented replicase binding. When tested in the context of the respective full-length RNAs, the same mutations abolished BMV RNA synthesis in transfected barley protoplasts. The eighth site was within the intercistronic region (ICR) of plus-strand RNA3. Further mapping showed that a sequence of 22 consecutive adenylates was responsible for binding the replicase, with 16 being the minimal required length. Deletion of the poly(A) sequence was previously shown to severely debilitate BMV RNA replication in plants (E. Smirnyagina, Y. H. Hsu, N. Chua, and P. Ahlquist, Virology 198:427-436, 1994). Interestingly, the B box motif in the ICR of RNA3, which has previously been determined to bind the 1a protein, does not bind the replicase. These results identify the replicase-binding sites in all of the BMV RNAs and suggest that the recognition of RNA3 is different from that of RNA1 and RNA2.

scholarly journals DIFFERENTIATION OF DROSOPHILA CELLS LACKING RIBOSOMAL DNA, IN VITRO

Genetics ◽  
1973 ◽  
Vol 73 (3) ◽  
pp. 429-434
Author(s):  
J James Donady ◽  
R L Seecof ◽  
M A Fox
Keyword(s):  
Drosophila Melanogaster ◽  
Ribosomal Rna ◽  
Ribosomal Dna ◽  
Rna Synthesis ◽  
Wild Type ◽  
Drosophila Cells

ABSTRACT Drosophila melanogaster embryos that lacked ribosomal DNA were obtained from appropriate crosses. Cells were taken from such embryos before overt differentiation took place and were cultured in vitro. These cells differentiated into neurons and myocytes with the same success as did wild-type controls. Therefore, ribosomal RNA synthesis is not necessary for the differentiation of neurons and myocytes in vitro.

scholarly journals Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

2000 ◽  
Vol 74 (24) ◽  
pp. 11671-11680 ◽  
Author(s):  
T. A. M. Osman ◽  
C. L. Hemenway ◽  
K. W. Buck
Keyword(s):  
Rna Polymerase ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Central Core ◽  
Minus Strand ◽  
Stem Loop ◽  
Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

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