A 3′ terminal stem–loop structure in Nodamura virus RNA2 forms an essential cis-acting signal for RNA replication

ABSTRACT The parts of the RNA genome of infectious bronchitis virus (IBV) required for replication and packaging of the RNA were investigated using deletion mutagenesis of a defective RNA (D-RNA) CD-61 (6.1 kb) containing a chloramphenicol acetyltransferase reporter gene. A D-RNA with the first 544, but not as few as 338, nucleotides (nt) of the 5′ terminus was replicated; the 5′ untranslated region (UTR) comprises 528 nt. Region I of the 3′ UTR, adjacent to the nucleocapsid protein gene, comprised 212 nt and could be removed without impairment of replication or packaging of D-RNAs. A D-RNA with the final 338 nt, including the 293 nt in the highly conserved region II of the 3′ UTR, was replicated. Thus, the 5′-terminal 544 nt and 3′-terminal 338 nt contained the necessary signals for RNA replication. Phylogenetic analysis of 19 strains of IBV and 3 strains of turkey coronavirus predicted a conserved stem-loop structure at the 5′ end of region II of the 3′ UTR. Removal of the predicted stem-loop structure abolished replication of the D-RNAs. D-RNAs in which replicase gene 1b-derived sequences had been removed or replaced with all the downstream genes were replicated well but were rescued poorly, suggesting inefficient packaging. However, no specific part of the 1b gene was required for efficient packaging.

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

Journal of Virology ◽

10.1128/jvi.76.23.12008-12022.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 12008-12022 ◽

Cited By ~ 100

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.

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Identification of a specific exon sequence that is a major determinant in the selection between a natural and a cryptic 5' splice site.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4581 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4581-4590 ◽

Cited By ~ 21

Author(s):

L Domenjoud ◽

H Gallinaro ◽

L Kister ◽

S Meyer ◽

M Jacob

Keyword(s):

Splice Site ◽

Adenovirus Type ◽

Loop Structure ◽

Stem Loop ◽

Cis Acting ◽

Stem Loop Structure ◽

Natural Site ◽

Proximal Site ◽

Exon 1 ◽

Long Stem

The first intron of the early region 3 from adenovirus type 2 contains a cryptic 5' splice site, Dcr1, 74 nucleotides downstream from the natural site D1. The cryptic site can be activated when the natural site is inactivated by mutagenesis. To investigate the basis for selection between a natural and a cryptic 5' splice site, we searched for cis-acting elements responsible for the exclusive selection of the natural site. We show that both the relative intrinsic strength of the sites and the sequence context affect the selection. A 120-nucleotide segment located at the 3' end of exon 1 enhances splicing at the proximal site D1; in its absence the two sites are used according to their strength. Thus, three cis-acting elements are involved in the silencing of the cryptic site: the sequence of D1, the sequence of Dcr1, and an upstream exonic sequence. We show that the exonic element folds, in solution, into a 113-nucleotide-long stem-loop structure. We propose that this potential stem-loop structure which is located 6 nucleotides upstream of the exon 1-intron junction is responsible for the preferential use of the natural 5' splice site.

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Role of the 5′-Proximal Stem-Loop Structure of the 5′ Untranslated Region in Replication and Translation of Hepatitis C Virus RNA

Journal of Virology ◽

10.1128/jvi.77.5.3312-3318.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3312-3318 ◽

Cited By ~ 59

Author(s):

Guangxiang Luo ◽

Shaojie Xin ◽

Zhaohui Cai

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Hcv Rna ◽

Loop Structure ◽

Nucleotide Substitutions ◽

Stem Loop ◽

Stem Loop Structure ◽

Compensatory Mutations ◽

Cell Colony

ABSTRACT Sequences of the untranslated regions at the 5′ and 3′ ends (5′UTR and 3′UTR) of the hepatitis C virus (HCV) RNA genome are highly conserved and contain cis-acting RNA elements for HCV RNA replication. The HCV 5′UTR consists of two distinct RNA elements, a short 5′-proximal stem-loop RNA element (nucleotides 1 to 43) and a longer element of internal ribosome entry site. To determine the sequence and structural requirements of the 5′-proximal stem-loop RNA element in HCV RNA replication and translation, a mutagenesis analysis was preformed by nucleotide deletions and substitutions. Effects of mutations in the 5′-proximal stem-loop RNA element on HCV RNA replication were determined by using a cell-based HCV replicon replication system. Deletion of the first 20 nucleotides from the 5′ end resulted in elimination of cell colony formation. Likewise, disruption of the 5′-proximal stem-loop by nucleotide substitutions abolished the ability of HCV RNA to induce cell colony formation. However, restoration of the 5′-proximal stem-loop by compensatory mutations with different nucleotides rescued the ability of the subgenomic HCV RNA to replicate in Huh7 cells. In addition, deletion and nucleotide substitutions of the 5′-proximal stem-loop structure, including the restored stem-loop by compensatory mutations, all resulted in reduction of translation by two- to fivefold, suggesting that the 5′-proximal stem-loop RNA element also modulates HCV RNA translation. These findings demonstrate that the 5′-proximal stem-loop of the HCV RNA is a cis-acting RNA element that regulates HCV RNA replication and translation.

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Identification of a specific exon sequence that is a major determinant in the selection between a natural and a cryptic 5' splice site

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4581-4590.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4581-4590

Author(s):

L Domenjoud ◽

H Gallinaro ◽

L Kister ◽

S Meyer ◽

M Jacob

Keyword(s):

Splice Site ◽

Adenovirus Type ◽

Loop Structure ◽

Stem Loop ◽

Cis Acting ◽

Stem Loop Structure ◽

Natural Site ◽

Proximal Site ◽

Exon 1 ◽

Long Stem

The first intron of the early region 3 from adenovirus type 2 contains a cryptic 5' splice site, Dcr1, 74 nucleotides downstream from the natural site D1. The cryptic site can be activated when the natural site is inactivated by mutagenesis. To investigate the basis for selection between a natural and a cryptic 5' splice site, we searched for cis-acting elements responsible for the exclusive selection of the natural site. We show that both the relative intrinsic strength of the sites and the sequence context affect the selection. A 120-nucleotide segment located at the 3' end of exon 1 enhances splicing at the proximal site D1; in its absence the two sites are used according to their strength. Thus, three cis-acting elements are involved in the silencing of the cryptic site: the sequence of D1, the sequence of Dcr1, and an upstream exonic sequence. We show that the exonic element folds, in solution, into a 113-nucleotide-long stem-loop structure. We propose that this potential stem-loop structure which is located 6 nucleotides upstream of the exon 1-intron junction is responsible for the preferential use of the natural 5' splice site.

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Analysis of secondary structure within sgm and kgmB mRNA

Archives of Biological Sciences ◽

10.2298/abs1003515v ◽

2010 ◽

Vol 62 (3) ◽

pp. 515-524 ◽

Cited By ~ 1

Author(s):

Sandra Vojnovic ◽

Tatjana Ilic-Tomic ◽

Ivana Moric ◽

Branka Vasiljevic

Keyword(s):

Secondary Structure ◽

Regulatory Sequence ◽

Loop Structure ◽

In Silico Prediction ◽

Stable Secondary Structure ◽

Stem Loop ◽

Cis Acting ◽

Stem Loop Structure ◽

Structure Probing ◽

A Site

Sgm methyltransferase from Micromonospora zionensis and KgmB methyltransferase from Streptoalloteichus tenebrarius are resistant to aminoglycoside antibiotics as a result of their ability to specifically methylate G1405 within the bacterial 16S rRNA A-site. The (C)CGCCC motif, assumed to be a regulatory sequence responsible for the autoregulation of the sgm gene, could most likely also be responsible for the autoregulation of the kgmB gene. This sequence, found within the 5' untranslated region of both sgm and kgmB mRNAs, as indicated by in silico prediction, may be involved in the formation of a specific stem-loop structure. Sgm and KgmB are mutually down-regulated and it is likely that they share the same cis-acting elements. Structure probing experiments confirmed the existence of a stable secondary structure within the 5' UTR of the sgm mRNA, while the analysis of kgmB mRNA failed to confirm the predicted structure. .

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Identification of a Conserved RNA Replication Element (cre) within the 3Dpol-Coding Sequence of Hepatoviruses

Journal of Virology ◽

10.1128/jvi.00787-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 10118-10128 ◽

Cited By ~ 32

Author(s):

Yan Yang ◽

MinKyung Yi ◽

David J. Evans ◽

Peter Simmonds ◽

Stanley M. Lemon

Keyword(s):

Rna Structure ◽

Hepatitis A Virus ◽

Synonymous Codon ◽

Phylogenetic Analyses ◽

Rna Replication ◽

Loop Structure ◽

Human Hepatoma Cells ◽

Stem Loop ◽

Coding Sequence ◽

Stem Loop Structure

ABSTRACT Internally located, cis-acting RNA replication elements (cre) have been identified within the genomes of viruses representing each of the major picornavirus genera (Enterovirus, Rhinovirus, Aphthovirus, and Cardiovirus) except Hepatovirus. Previous efforts to identify a stem-loop structure with cre function in hepatitis A virus (HAV), the type species of this genus, by phylogenetic analyses or thermodynamic predictions have not succeeded. However, a region of markedly suppressed synonymous codon variability was identified in alignments of HAV sequences near the 5′ end of the 3Dpol-coding sequence of HAV, consistent with noncoding constraints imposed by an underlying RNA secondary structure. Subsequent MFOLD predictions identified a 110-nucleotide (nt) complex stem-loop in this region with a typical AAACA/G cre motif in its top loop. A potentially homologous RNA structure was identified in this region of the avian encephalitis virus genome, despite little nucleotide sequence relatedness between it and HAV. Mutations that disrupted secondary RNA structure or the AAACA/G motif, without altering the amino acid sequence of 3Dpol, ablated replication of a subgenomic HAV replicon in transfected human hepatoma cells. Replication competence could be rescued by reinsertion of the native 110-nt stem-loop structure (but not an abbreviated 45-nt stem-loop) upstream of the HAV coding sequence in the replicon. These results suggest that this stem-loop is functionally similar to cre elements of other picornaviruses and likely involved in templating VPg uridylylation as in other picornaviruses, despite its significantly larger size and lower free folding energy.

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Construction of an Infectious cDNA Clone of Aichi Virus (a New Member of the Family Picornaviridae) and Mutational Analysis of a Stem-Loop Structure at the 5′ End of the Genome

Journal of Virology ◽

10.1128/jvi.75.17.8021-8030.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 8021-8030 ◽

Cited By ~ 55

Author(s):

Jun Sasaki ◽

Yasuhiro Kusuhara ◽

Yoshimasa Maeno ◽

Nobumichi Kobayashi ◽

Teruo Yamashita ◽

...

Keyword(s):

Virus Replication ◽

Cdna Clone ◽

Rna Replication ◽

Infectious Cdna Clone ◽

Viral Rna ◽

Aichi Virus ◽

Loop Structure ◽

Stem Loop ◽

Stem Loop Structure ◽

The Family

ABSTRACT Aichi virus is the type species of a new genus,Kobuvirus, of the family Picornaviridae. In this study, we constructed a full-length cDNA clone of Aichi virus whose in vitro transcripts were infectious to Vero cells. During construction of the infectious cDNA clone, a novel sequence of 32 nucleotides was identified at the 5′ end of the genome. Computer-assisted prediction of the secondary structure of the 5′ end of the genome, including the novel sequence, suggested the formation of a stable stem-loop structure consisting of 42 nucleotides. The function of this stem-loop in virus replication was investigated using various site-directed mutants derived from the infectious cDNA clone. Our data indicated that correct folding of the stem-loop at the 5′ end of the positive strand, but not at the 3′ end of the negative strand, is critical for viral RNA replication. The primary sequence in the lower part of the stem was also suggested to be crucial for RNA replication. In contrast, nucleotide changes in the loop segment did not so severely reduce the efficiency of virus replication. A double mutant, in which both nucleotide stretches of the middle part of the stem were replaced by their complementary nucleotides, had efficient RNA replication and translation abilities but was unable to produce viruses. These results indicate that the stem-loop at the 5′ end of the Aichi virus genome is an element involved in both viral RNA replication and production of infectious virus particles.

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An Internally Located RNA Hairpin Enhances Replication of Tomato Bushy Stunt Virus RNAs

Journal of Virology ◽

10.1128/jvi.77.1.245-257.2003 ◽

2003 ◽

Vol 77 (1) ◽

pp. 245-257 ◽

Cited By ~ 43

Author(s):

Debashish Ray ◽

K. Andrew White

Keyword(s):

Rna Virus ◽

Function Analysis ◽

Rna Replication ◽

Tomato Bushy Stunt Virus ◽

Loop Structure ◽

Minus Strand ◽

Independent Manner ◽

Stem Loop ◽

Stem Loop Structure ◽

Rna Hairpin

ABSTRACT Defective interfering (DI) RNAs of Tomato bushy stunt virus (TBSV), a plus-sense RNA virus, comprise four conserved noncontiguous regions (I through IV) derived from the viral genome. Region III, a 70-nucleotide-long sequence corresponding to a genomic segment located 378 nucleotides upstream of the 3′ terminus of the genome, has been found to enhance DI RNA accumulation by approximately 10-fold in an orientation-independent manner (D. Ray and K. A. White, Virology 256:162-171, 1999). In this study, a more detailed structure-function analysis of region III was conducted. RNA secondary-structure analyses indicated that region III contains stem-loop structures in both plus and minus strands. Through deletion analyses of a DI RNA, a primary determinant of region III activity was mapped to the 5′-proximal 35-nucleotide segment. Compensatory-type mutational analyses showed that a stem-loop structure in the minus strand of this subregion was required for enhanced DI RNA replication. The same stem-loop structure was also found to function in a position-independent manner in a DI RNA (albeit at reduced levels) and to be important for efficient accumulation within the context of the TBSV genome. Taken together, these observations suggest that the 5′-proximal segment of region III is a modular RNA replication element that functions primarily through the formation of an RNA hairpin structure in the minus strand.

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