scholarly journals The 5′-End Sequence of the Genome of Aichi Virus, a Picornavirus, Contains an Element Critical for Viral RNA Encapsidation

2003 ◽  
Vol 77 (6) ◽  
pp. 3542-3548 ◽  
Author(s):  
Jun Sasaki ◽  
Koki Taniguchi
Keyword(s):  
Mutational Analysis ◽  
Rna Replication ◽  
Virus Genome ◽  
Middle Part ◽  
Viral Rna ◽  
Aichi Virus ◽  
Stem Loop ◽  
Positive Strand ◽  
The Family ◽  
Infected Cells

ABSTRACT Picornavirus positive-strand RNAs are selectively encapsidated despite the coexistence of viral negative-strand RNAs and cellular RNAs in infected cells. However, the precise mechanism of the RNA encapsidation process in picornaviruses remains unclear. Here we report the first identification of an RNA element critical for encapsidation in picornaviruses. The 5′ end of the genome of Aichi virus, a member of the family Picornaviridae, folds into three stem-loop structures (SL-A, SL-B, and SL-C, from the most 5′ end). In the previous study, we constructed a mutant, termed mut6, by exchanging the seven-nucleotide stretches of the middle part of the stem in SL-A with each other to maintain the base pairings of the stem. mut6 exhibited efficient RNA replication and translation but formed no plaques. The present study showed that in cells transfected with mut6 RNA, empty capsids were accumulated, but few virions containing RNA were formed. This means that mut6 has a severe defect in RNA encapsidation. Site-directed mutational analysis indicated that as the mutated region was narrowed, the encapsidation was improved. As a result, the mutation of the 7 bp of the middle part of the stem in SL-A was required for abolishing the plaque-forming ability. Thus, the 5′-end sequence of the Aichi virus genome was shown to play an important role in encapsidation.

scholarly journals 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

2001 ◽  
Vol 75 (17) ◽  
pp. 8021-8030 ◽  
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.

scholarly journals The 5′-Terminal Region of the Aichi Virus Genome Encodes cis-Acting Replication Elements Required for Positive- and Negative-Strand RNA Synthesis

2005 ◽  
Vol 79 (11) ◽  
pp. 6918-6931 ◽  
Author(s):  
Shigeo Nagashima ◽  
Jun Sasaki ◽  
Koki Taniguchi
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Virus Genome ◽  
Encephalomyocarditis Virus ◽  
Viral Rna ◽  
Aichi Virus ◽  
Pseudoknot Structure ◽  
Positive Strand ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5′ end) formed at the 5′ end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5′-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5′-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5′-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5′-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5′ end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3′ end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5′ end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.

scholarly journals 5′ cis Elements Direct Nodavirus RNA1 Recruitment to Mitochondrial Sites of Replication Complex Formation

2009 ◽  
Vol 83 (7) ◽  
pp. 2976-2988 ◽  
Author(s):  
Priscilla M. Van Wynsberghe ◽  
Paul Ahlquist
Keyword(s):  
Complex Formation ◽  
Mutational Analysis ◽  
Protein A ◽  
Rna Replication ◽  
Viral Rna ◽  
Yeast Cells ◽  
Positive Strand ◽  
Replication Complex ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Positive-strand RNA viruses replicate their genomes on intracellular membranes, usually in conjunction with virus-induced membrane rearrangements. For the nodavirus flock house virus (FHV), we recently showed that multifunctional FHV replicase protein A induces viral RNA template recruitment to a membrane-associated state, but the site(s) and function of this recruitment were not determined. By tagging viral RNA with green fluorescent protein, we show here in Drosophila cells that protein A recruits FHV RNA specifically to the outer mitochondrial membrane sites of RNA replication complex formation. Using Drosophila cells and yeast cells, which also support FHV replication, we also defined the cis-acting regions that direct replication and template recruitment for FHV genomic RNA1. RNA1 nucleotides 68 to 205 were required for RNA replication and directed efficient protein A-mediated RNA recruitment in both cell types. RNA secondary structure prediction, structure probing, and phylogenetic comparisons in this region identified two stable, conserved stem-loops with nearly identical loop sequences. Further mutational analysis showed that both stem-loops and certain flanking sequences were required for RNA1 recruitment, negative-strand synthesis, and subsequent positive-strand amplification in yeast and Drosophila cells. Thus, we have shown that protein A recruits RNA1 templates to mitochondria, as expected for RNA replication, and identified a new RNA1 cis element that is necessary and sufficient for RNA1 template recognition and recruitment to these mitochondrial membranes for negative-strand RNA1 synthesis. These results establish RNA recruitment to the sites of replication complex formation as an essential, distinct, and selective early step in nodavirus replication.

scholarly journals Stem-Loop III in the 5′ Untranslated Region Is a cis-Acting Element in Bovine Coronavirus Defective Interfering RNA Replication

2003 ◽  
Vol 77 (12) ◽  
pp. 6720-6730 ◽  
Author(s):  
Sharmila Raman ◽  
Peter Bouma ◽  
Gwyn D. Williams ◽  
David A. Brian
Keyword(s):  
Untranslated Region ◽  
Rna Replication ◽  
Virus Genome ◽  
Higher Order ◽  
Bovine Coronavirus ◽  
Stem Loop ◽  
Positive Strand ◽  
Amino Acid Peptide ◽  
Rna Accumulation ◽  
Order Structures

ABSTRACT Higher-order structures in the 5′ untranslated region (UTR) of plus-strand RNA viruses are known in many cases to function as cis-acting elements in RNA translation, replication, or transcription. Here we describe evidence supporting the structure and a cis-acting function in defective interfering (DI) RNA replication of stem-loop III, the third of four predicted higher-order structures mapping within the 210-nucleotide (nt) 5′ UTR of the 32-kb bovine coronavirus (BCoV) genome. Stem-loop III maps at nt 97 through 116, has a calculated free energy of −9.1 kcal/mol in the positive strand and −3.0 kcal/mol in the negative strand, and has associated with it beginning at nt 100 an open reading frame (ORF) potentially encoding an 8-amino-acid peptide. Stem-loop III is presumed to function in the positive strand, but its strand of action has not been established. Stem-loop III (i) shows phylogenetic conservation among group 2 coronaviruses and appears to have a homolog in coronavirus groups 1 and 3, (ii) has in all coronaviruses for which sequence is known a closely associated short, AUG-initiated intra-5′ UTR ORF, (iii) is supported by enzyme structure-probing evidence in BCoV RNA, (iv) must maintain stem integrity for DI RNA replication in BCoV DI RNA, and (v) shows a positive correlation between maintenance of the short ORF and maximal DI RNA accumulation in BCoV DI RNA. These results indicate that stem-loop III in the BCoV 5′ UTR is a cis-acting element for DI RNA replication and that its associated intra-5′ UTR ORF may function to enhance replication. It is postulated that these two elements function similarly in the virus genome.

scholarly journals Sequence Requirements for Viral RNA Replication and VPg Uridylylation Directed by the Internal cis-Acting Replication Element (cre) of Human Rhinovirus Type 14

2002 ◽  
Vol 76 (15) ◽  
pp. 7485-7494 ◽  
Author(s):  
Yan Yang ◽  
Rene Rijnbrand ◽  
Kevin L. McKnight ◽  
Eckard Wimmer ◽  
Aniko Paul ◽  
...  
Keyword(s):  
Rna Structure ◽  
Rna Synthesis ◽  
Mutational Analysis ◽  
Rna Replication ◽  
Human Rhinovirus ◽  
Viral Rna ◽  
Base Substitution ◽  
Stem Loop ◽  
Additional Support

ABSTRACT Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5′ and 3′ noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5′ half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA replication is to act as template for VPg uridylylation.

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 Sequence and Structural Elements at the 3′ Terminus of Bovine Viral Diarrhea Virus Genomic RNA: Functional Role during RNA Replication

1999 ◽  
Vol 73 (5) ◽  
pp. 3638-3648 ◽  
Author(s):  
Haiying Yu ◽  
Claus W. Grassmann ◽  
Sven-Erik Behrens
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Bovine Viral Diarrhea ◽  
Rna Motifs ◽  
Stem Loop ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Interaction Sites ◽  
Viral Diarrhea Virus

ABSTRACT Bovine viral diarrhea virus (BVDV), a member of the genusPestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5′ and 3′ ends. Computer modeling suggested the 3′ UTR comprised single-stranded regions as well as stem-loop structures—features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364–2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3′ UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3′C) of the DI9c 3′ UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3′ terminus and a considerably less stable stem-loop, SL II, that forms the 5′ portion of 3′C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3′-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3′C region of the BVDV 3′ UTR create a common cis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.

scholarly journals Long-Range RNA-RNA Interactions Circularize the Dengue Virus Genome

2005 ◽  
Vol 79 (11) ◽  
pp. 6631-6643 ◽  
Author(s):  
Diego E. Alvarez ◽  
María F. Lodeiro ◽  
Silvio J. Ludueña ◽  
Lía I. Pietrasanta ◽  
Andrea V. Gamarnik
Keyword(s):  
Dengue Virus ◽  
Rna Binding ◽  
Rna Replication ◽  
Virus Genome ◽  
Viral Rna ◽  
Physical Interaction ◽  
Virus Rna ◽  
Rna Elements ◽  
Rna Interaction

ABSTRACT Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5′ and 3′ ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5′ and 3′ CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5′ and 3′ untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5′ and 3′ UAR (upstream AUG region). In order to investigate the functional role of 5′-3′ UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5′ or 3′ UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

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