scholarly journals Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication

2010 ◽  
Vol 84 (12) ◽  
pp. 6103-6118 ◽  
Author(s):  
Peter Friebe ◽  
Eva Harris
Keyword(s):  
Dengue Virus ◽  
Rna Structure ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Rna ◽  
Coding Region ◽  
Stem Loop ◽  
Complementary Sequences ◽  
Translation Start ◽  
Rna Elements

ABSTRACT Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV 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 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.

scholarly journals Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication

2008 ◽  
Vol 83 (2) ◽  
pp. 993-1008 ◽  
Author(s):  
María F. Lodeiro ◽  
Claudia V. Filomatori ◽  
Andrea V. Gamarnik
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Stem Loop ◽  
Denv Serotypes ◽  
Functional Studies ◽  
Promoter Recognition

ABSTRACT The 5′ untranslated region (5′UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication. At the 5′ end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as the 5′ upstream AUG region (5′UAR). Here, we analyzed the secondary structure of the SLA in solution and the structural requirements of this element for viral replication. Using infectious DENV clones, viral replicons, and in vitro polymerase assays, we defined two helical regions, a side stem-loop, a top loop, and a U bulge within SLA as crucial elements for viral replication. The determinants for SLA-polymerase recognition were found to be common in different DENV serotypes. In addition, structural elements within the SLA required for DENV RNA replication were also conserved among different mosquito- and tick-borne flavivirus genomes, suggesting possible common strategies for polymerase-promoter recognition in flaviviruses. Furthermore, a conserved oligo(U) track present downstream of the SLA was found to modulate RNA synthesis in transfected cells. In vitro polymerase assays indicated that a sequence of at least 10 residues following the SLA, upstream of the 5′UAR, was necessary for efficient RNA synthesis using the viral 3′UTR as template.

scholarly journals Structural Properties of a Multifunctional T-Shaped RNA Domain That Mediate Efficient Tomato Bushy Stunt Virus RNA Replication

2004 ◽  
Vol 78 (19) ◽  
pp. 10490-10500 ◽  
Author(s):  
Debashish Ray ◽  
Hong Na ◽  
K. Andrew White
Keyword(s):  
Rna Structure ◽  
Function Analysis ◽  
Rna Replication ◽  
Tomato Bushy Stunt Virus ◽  
Viral Rna ◽  
Genome Replication ◽  
Dependent Manner ◽  
Stem Loop ◽  
Virus Rna ◽  
Positive Strand Rna

ABSTRACT In positive-strand RNA viruses, 5′ untranslated regions (5′ UTRs) mediate many essential viral processes, including genome replication. Previously, we proposed that the 5′-terminal portion of the genomic leader sequence of Tomato bushy stunt virus (TBSV) forms an RNA structure containing a 3-helix junction, termed the T-shaped domain (TSD). In the present study, we have carried out structure-function analysis of the proposed TSD and have confirmed an important role for this domain in mediating efficient viral RNA amplification. Using a model TBSV defective interfering RNA replicon and a protoplast system, we demonstrated that various TSD subelements contribute to the efficiency of viral RNA replication. In particular, the stabilities of all three stems (S1, S2, and S4) forming the 3-helix junction are important, while stem-loop 3—a terminal extension of S2—is largely dispensable. Additionally, some of the sequences forming the 3-helix junction are required in an identity-dependent manner. Thus, both secondary structure and nucleotide identity are important for TSD-mediated viral RNA replication. Importantly, these results are fully consistent with the dual functions we defined previously for the sequences corresponding to loops 3 and 4, respectively, in facilitating 5′ cap- and 3′ poly(A) tail-independent translation of the genome by forming a loop-loop interaction with the 3′-proximal translational enhancer and in mediating viral RNA replication through formation of a pseudoknot with the adjacent downstream RNA domain. Also, since comparable TSDs and associated interactions are predicted in the 5′ UTRs of all sequenced Aureusvirus genomes, members of at least one other genus in the family Tombusviridae appear to utilize this type of multifunctional RNA domain.

scholarly journals Dengue virus 2 capsid protein chaperones strand displacement without altering the capsid-coding region hairpin element’s structural functionality

2021 ◽  
Author(s):  
Xin Ee Yong ◽  
Palur Venkata Raghuvamsi ◽  
Ganesh S. Anand ◽  
Thorsten Wohland ◽  
Kamal K. Sharma
Keyword(s):  
Dengue Virus ◽  
Capsid Protein ◽  
Rna Synthesis ◽  
Chaperone Activity ◽  
Start Codon ◽  
Strand Displacement ◽  
Coding Region ◽  
Conserved Sequence ◽  
Rna Elements ◽  
Codon Selection

ABSTRACTBy virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5’-3’ panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during RNA rearrangement of the 9-nt conserved sequence (5CS) to its complementary 3CS counterpart. The cHP element has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that the cHP hairpin impedes annealing between the 5CS and the 3CS elements. Although DENV2C does not modulate structural functionality of the cHP hairpin, it accelerates annealing and specifically promotes strand displacement of 3CS during 5’-3’ panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favoring one of the active conformations of the cHP element. Based on our results, we propose mechanisms for annealing and strand displacement involving the cHP element. Thus, our results provide mechanistic insights on how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.

scholarly journals Poliovirus CRE-Dependent VPg Uridylylation Is Required for Positive-Strand RNA Synthesis but Not for Negative-Strand RNA Synthesis

2003 ◽  
Vol 77 (8) ◽  
pp. 4739-4750 ◽  
Author(s):  
Kenneth E. Murray ◽  
David J. Barton
Keyword(s):  
Rna Structure ◽  
Rna Synthesis ◽  
Critical Role ◽  
Rna Replication ◽  
Stem Loop ◽  
Positive Strand ◽  
Negative Strand ◽  
Step Model ◽  
Positive Strand Rna

ABSTRACT The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpUOH, the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpUOH, however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpUOH synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpUOH were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpUOH in the asymmetric replication of poliovirus RNA.

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 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.

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