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 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 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 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 and Functional Analysis of the cis-Acting Elements Required for Plus-Strand RNA Synthesis of Bamboo Mosaic Virus

2005 ◽  
Vol 79 (14) ◽  
pp. 9046-9053 ◽  
Author(s):  
Jen-Wen Lin ◽  
Hsiao-Ning Chiu ◽  
I-Hsuan Chen ◽  
Tzu-Chi Chen ◽  
Yau-Heiu Hsu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Minus Strand ◽  
Internal Deletion ◽  
Stem Loop ◽  
Cis Acting ◽  
Rna Accumulation

ABSTRACT Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3′-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3′ terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/Δ5, with a deletion of the 3′-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/Δ16 and Ba-77/Δ31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three cis-acting elements in the 3′ end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.

scholarly journals Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro

2003 ◽  
Vol 77 (21) ◽  
pp. 11408-11416 ◽  
Author(s):  
Mark H. Fogg ◽  
Natalya L. Teterina ◽  
Ellie Ehrenfeld
Keyword(s):  
Membrane Trafficking ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Rna ◽  
Chain Elongation ◽  
Electron Microscopic ◽  
Ribonucleoprotein Complexes ◽  
Cell Extracts ◽  
Infected Cells

ABSTRACT Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.

scholarly journals Restriction of poliovirus RNA replication in persistently infected nerve cells

2002 ◽  
Vol 83 (5) ◽  
pp. 1087-1093 ◽  
Author(s):  
Sophie Girard ◽  
Anne-Sophie Gosselin ◽  
Isabelle Pelletier ◽  
Florence Colbère-Garapin ◽  
Thérèse Couderc ◽  
...  
Keyword(s):  
Acute Phase ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Rna ◽  
Paralytic Poliomyelitis ◽  
Minus Strand ◽  
Relative Level ◽  
Persistently Infected ◽  
Cellular Factors

The aetiology of post-polio syndrome may involve persistence of poliovirus (PV) in the CNS. PV persists in the CNS of infected paralysed mice for over a year after the acute phase of paralytic poliomyelitis. However, infectious PV particles cannot be recovered from homogenates of CNS from paralysed mice after the acute phase of disease, indicating that PV replication is restricted. To identify the molecular mechanism by which PV replication is limited, PV RNA synthesis was analysed by estimating the relative level of genomic (plus-strand) and complementary (minus-strand) PV RNA in the CNS of persistently infected mice. PV RNA replication decreased during the 6 months following onset of paralysis, due mainly to inhibition of plus-strand RNA synthesis. Thus, restriction of PV RNA synthesis may contribute to persistence by limiting virus replication in the mouse CNS. Interestingly, viral RNA replication was similarly inhibited in neuroblastoma IMR-32 cell cultures persistently infected with PV. This in vitro model thus shows that cellular factors play a role in the inhibition of viral RNA synthesis.

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 Structural and functional characterization of the coxsackievirus B3 CRE(2C): role of CRE(2C) in negative- and positive-strand RNA synthesis

2006 ◽  
Vol 87 (1) ◽  
pp. 103-113 ◽  
Author(s):  
Mark J. M. van Ooij ◽  
Dorothee A. Vogt ◽  
Aniko Paul ◽  
Christian Castro ◽  
Judith Kuijpers ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Coxsackievirus B3 ◽  
Viral Rna ◽  
Free System ◽  
Positive Strand ◽  
Negative Strand ◽  
Cell Extracts ◽  
Positive Strand Rna

A stem–loop element located within the 2C-coding region of the coxsackievirus B3 (CVB3) genome has been proposed to function as a cis-acting replication element (CRE). It is shown here that disruption of this structure indeed interfered with viral RNA replication in vivo and abolished uridylylation of VPg in vitro. Site-directed mutagenesis demonstrated that the previously proposed enteroviral CRE consensus loop sequence, R1NNNAAR2NNNNNNR3, is also applicable to CVB3 CRE(2C) and that a positive correlation exists between the ability of CRE(2C) mutants to serve as template in the uridylylation reaction and the capacity of these mutants to support viral RNA replication. To further investigate the effects of the mutations on negative-strand RNA synthesis, an in vitro translation/replication system containing HeLa S10 cell extracts was used. Similar to the results observed for poliovirus and rhinovirus, it was found that a complete disruption of the CRE(2C) structure interfered with positive-strand RNA synthesis, but not with negative-strand synthesis. All CRE(2C) point mutants affecting the enteroviral CRE consensus loop, however, showed a marked decrease in efficiency to induce negative-strand synthesis. Moreover, a transition (A5G) regarding the first templating adenosine residue in the loop was even unable to initiate complementary negative-strand synthesis above detectable levels. Taken together, these results indicate that the CVB3 CRE(2C) is not only required for the initiation of positive-strand RNA synthesis, but also plays an essential role in the efficient initiation of negative-strand RNA synthesis, a conclusion that has not been reached previously by using the cell-free system.

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 Human Rhinovirus Type 14 Gain-of-Function Mutants for oriI Utilization Define Residues of 3C(D) and 3Dpol That Contribute to Assembly and Stability of the Picornavirus VPg Uridylylation Complex

2007 ◽  
Vol 81 (22) ◽  
pp. 12485-12495 ◽  
Author(s):  
Miaoqing Shen ◽  
Qixin Wang ◽  
Yan Yang ◽  
Harsh B. Pathak ◽  
Jamie J. Arnold ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Human Rhinovirus ◽  
Adaptive Mutation ◽  
Sequence Specificity ◽  
Wild Type ◽  
Stem Loop ◽  
Additional Support ◽  
Type 3 ◽  
Enhanced Stability

ABSTRACT VPg linkage to the 5′ ends of picornavirus RNAs requires production of VPg-pUpU. VPg-pUpU is templated by an RNA stem-loop (the cre or oriI) found at different locations in picornavirus genomes. At least one adaptive mutation is required for human rhinovirus type 14 (HRV-14) to use poliovirus type 3 (PV-3) or PV-1 oriI efficiently. One mutation changes Leu-94 of 3C to Pro; the other changes Asp-406 of 3Dpol to Asn. By using an in vitro VPg uridylylation system for HRV-14 that recapitulates biological phenotypes, we show that the 3C adaptive mutation functions at the level of 3C(D) and the 3D adaptive mutation functions at the level of 3Dpol. Pro-94 3C(D) has an expanded specificity and enhanced stability relative to wild-type 3C(D) that leads to production of more processive uridylylation complexes. PV-1/HRV-14 oriI chimeras reveal sequence specificity in 3C(D) recognition of oriI that resides in the upper stem. Asn-406 3Dpol is as active as wild-type 3Dpol in RNA-primed reactions but exhibits greater VPg uridylylation activity due to more efficient recruitment to and retention in the VPg uridylylation complex. Asn-406 3Dpol from PV-1 exhibits identical behavior. These studies suggest a two-step binding mechanism in the assembly of the 3C(D)-oriI complex that leads to unwinding of at least the upper stem of oriI and provide additional support for a direct interaction between the back of the thumb of 3Dpol and 3C that is required for 3Dpol recruitment to and retention in the uridylylation complex.

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