scholarly journals The predicted stem-loop structure in the 3’-end of the human norovirus antigenomic sequence is required for its genomic RNA synthesis by its RdRp

2021 ◽  
pp. 101225
Author(s):  
Takashi Shimoike ◽  
Tsuyoshi Hayashi ◽  
Tomoichiro Oka ◽  
Masamichi Muramatsu
Keyword(s):  
Rna Synthesis ◽  
Loop Structure ◽  
Genomic Rna ◽  
Human Norovirus ◽  
Stem Loop ◽  
Stem Loop Structure

scholarly journals The 3′ end of the human norovirus antigenomic sequence is required for its genomic RNA synthesis by RNA-dependent RNA polymerase

2021 ◽  
Author(s):  
Takashi Shimoike ◽  
Tsuyoshi Hayashi ◽  
Tomoichiro Oka ◽  
Masamichi Muramatsu
Keyword(s):  
Rna Polymerase ◽  
Genome Sequence ◽  
Rna Synthesis ◽  
Loop Structure ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
Human Norovirus ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Positive Sense

Norovirus genome is a single-stranded positive-strand RNA. To reveal the mechanism underlying the initiation of the norovirus genomic RNA synthesis by its RNA-dependent RNA polymerase (RdRp), we used an in vitro assay to detect the complementary RNA synthesis activity. Results showed that the purified recombinant RdRp synthesized the complementary positive-sense RNA from the 100 nt template corresponding to the 3′ end region of the viral antisense genome sequence, but that RdRp did not synthesize the antisense genomic RNA from the 100 nt template, corresponding to the 5′ end region of the positive-sense genome sequence. The 31 nt region at the 3′ end of the RNA antisense template was then predicted to form the stem-loop structure. Its deletion resulted in the loss of complementary RNA synthesis by RdRp. The connection of the 31 nt to the 3′ end of the positive-sense RNA template allowed to be recognized by the RdRp. Similarly, an electrophoretic mobility shift assay further revealed that RdRp bound to the antisense RNA specifically, but the 31 nt deletion at the 3′ end lost the binding to RdRp. Therefore, combining this observation with further deletion and mutation analysis, we concluded that the predicted stem-loop structure in the 31 nt and region close to the antisense viral genomic stem sequences are important for initiating the positive-sense human norovirus genomic RNA synthesis by its RdRp.

scholarly journals Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12008-12022 ◽  
Author(s):  
Brandon L. Walter ◽  
Todd B. Parsley ◽  
Ellie Ehrenfeld ◽  
Bert L. Semler
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Protein ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

ABSTRACT The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

scholarly journals An RNA Pseudoknot in the 3′ End of the Arterivirus Genome Has a Critical Role in Regulating Viral RNA Synthesis

2007 ◽  
Vol 81 (17) ◽  
pp. 9426-9436 ◽  
Author(s):  
Nancy Beerens ◽  
Eric J. Snijder
Keyword(s):  
Rna Synthesis ◽  
Critical Role ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Replicase Gene ◽  
Loop Structure ◽  
Minus Strand ◽  
Stem Loop ◽  
Loop Region ◽  
Stem Loop Structure

ABSTRACT In the life cycle of plus-strand RNA viruses, the genome initially serves as the template for both translation of the viral replicase gene and synthesis of minus-strand RNA and is ultimately packaged into progeny virions. These various processes must be properly balanced to ensure efficient viral proliferation. To achieve this, higher-order RNA structures near the termini of a variety of RNA virus genomes are thought to play a key role in regulating the specificity and efficiency of viral RNA synthesis. In this study, we have analyzed the signals for minus-strand RNA synthesis in the prototype of the arterivirus family, equine arteritis virus (EAV). Using site-directed mutagenesis and an EAV reverse genetics system, we have demonstrated that a stem-loop structure near the 3′ terminus of the EAV genome is required for RNA synthesis. We have also obtained evidence for an essential pseudoknot interaction between the loop region of this stem-loop structure and an upstream hairpin residing in the gene encoding the nucleocapsid protein. We propose that the formation of this pseudoknot interaction may constitute a molecular switch that could regulate the specificity or timing of viral RNA synthesis. This hypothesis is supported by the fact that phylogenetic analysis predicted the formation of similar pseudoknot interactions near the 3′ end of all known arterivirus genomes, suggesting that this interaction has been conserved in evolution.

scholarly journals Inhibition of dengue virus translation and RNA synthesis by a morpholino oligomer targeted to the top of the terminal 3′ stem–loop structure

Virology ◽  
2006 ◽  
Vol 344 (2) ◽  
pp. 439-452 ◽  
Author(s):  
Katherine Lynn Holden ◽  
David A. Stein ◽  
Theodore C. Pierson ◽  
Asim A. Ahmed ◽  
Karen Clyde ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Synthesis ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

scholarly journals The 5′ Untranslated Region of Alfalfa Mosaic Virus RNA 1 Is Involved in Negative-Strand RNA Synthesis

2003 ◽  
Vol 77 (20) ◽  
pp. 11284-11289 ◽  
Author(s):  
A. Corina Vlot ◽  
John F. Bol
Keyword(s):  
Mosaic Virus ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Alfalfa Mosaic Virus ◽  
Loop Structure ◽  
Stem Loop ◽  
Genomic Rnas ◽  
Negative Strand ◽  
Stem Loop Structure ◽  
Virus Rna

ABSTRACT The three genomic RNAs of alfalfa mosaic virus each contain a unique 5′ untranslated region (5′ UTR). Replacement of the 5′ UTR of RNA 1 by that of RNA 2 or 3 yielded infectious replicons. The sequence of a putative 5′ stem-loop structure in RNA 1 was found to be required for negative-strand RNA synthesis. A similar putative 5′ stem-loop structure is present in RNA 2 but not in RNA 3.

scholarly journals RNA signals in the 3′ terminus of the genome of Equine arteritis virus are required for viral RNA synthesis

2006 ◽  
Vol 87 (7) ◽  
pp. 1977-1983 ◽  
Author(s):  
Nancy Beerens ◽  
Eric J. Snijder
Keyword(s):  
Secondary Structure ◽  
Virus Replication ◽  
Rna Secondary Structure ◽  
Rna Synthesis ◽  
Full Length ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

RNA virus genomes contain cis-acting sequences and structural elements involved in virus replication. Both full-length and subgenomic negative-strand RNA synthesis are initiated at the 3′ terminus of the positive-strand genomic RNA of Equine arteritis virus (EAV). To investigate the molecular mechanism of EAV RNA synthesis, the RNA secondary structure of the 3′-proximal region of the genome was analysed by chemical and enzymic probing. Based on the RNA secondary structure model derived from this analysis, several deletions were engineered in a full-length cDNA copy of the viral genome. Two RNA domains were identified that are essential for virus replication and most likely play a key role in viral RNA synthesis. The first domain, located directly upstream of the 3′ untranslated region (UTR) (nt 12610–12654 of the genome), is mainly single-stranded but contains one small stem–loop structure. The second domain is located within the 3′ UTR (nt 12661–12690) and folds into a prominent stem–loop structure with a large loop region. The location of this stem–loop structure near the 3′ terminus of the genome suggests that it may act as a recognition signal during the initiation of minus-strand RNA synthesis.

scholarly journals The Red Clover Necrotic Mosaic Virus RNA2 trans-Activator Is Also a cis-Acting RNA2 Replication Element

2005 ◽  
Vol 79 (2) ◽  
pp. 978-986 ◽  
Author(s):  
Masahiro Tatsuta ◽  
Hiroyuki Mizumoto ◽  
Masanori Kaido ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno
Keyword(s):  
Rna Synthesis ◽  
Red Clover ◽  
Loop Structure ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Stem Loop ◽  
Protein Coding ◽  
Subgenomic Rna ◽  
Stem Loop Structure ◽  
Rna Interaction

ABSTRACT The expression of the coat protein gene requires RNA-mediated trans-activation of subgenomic RNA synthesis in Red clover necrotic mosaic virus (RCNMV), the genome of which consists of two positive-strand RNAs, RNA1 and RNA2. The trans-acting RNA element required for subgenomic RNA synthesis from RNA1 has been mapped previously to the protein-coding region of RNA2, whereas RNA2 is not required for the replication of RNA1. In this study, we investigated the roles of the protein-coding region in RNA2 replication by analyzing the replication competence of RNA2 mutants containing deletions or nucleotide substitutions. Our results indicate that the same stem-loop structure (SL2) that functions as a trans-activator for RNA-mediated coat protein expression is critically required for the replication of RNA2 itself. Interestingly, however, disruption of the RNA-RNA interaction by nucleotide substitutions in the region of RNA1 corresponding to the SL2 loop of RNA2 does not affect RNA2 replication, indicating that the RNA-RNA interaction is not required for RNA2 replication. Further mutational analysis showed that, in addition to the stem-loop structure itself, nucleotide sequences in the stem and in the loop of SL2 are important for the replication of RNA2. These findings suggest that the structure and nucleotide sequence of SL2 in RNA2 play multiple roles in the virus life cycle.

scholarly journals In Vitro Synthesis of Minus-Strand RNA by an Isolated Cereal Yellow Dwarf Virus RNA-Dependent RNA Polymerase Requires VPg and a Stem-Loop Structure at the 3′ End of the Virus RNA

2006 ◽  
Vol 80 (21) ◽  
pp. 10743-10751 ◽  
Author(s):  
Toba A. M. Osman ◽  
Robert H. A. Coutts ◽  
Kenneth W. Buck
Keyword(s):  
Rna Polymerase ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Loop Structure ◽  
Minus Strand ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Virus Rna ◽  
Yellow Dwarf Virus

ABSTRACT Cereal yellow dwarf virus (CYDV) RNA has a 5′-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3′-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3′ terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3′ end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3′-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.

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