scholarly journals Requirements for Assembly of Poliovirus Replication Complexes and Negative-Strand RNA Synthesis

2001 ◽  
Vol 75 (8) ◽  
pp. 3841-3850 ◽  
Author(s):  
Natalya L. Teterina ◽  
Denise Egger ◽  
Kurt Bienz ◽  
David M. Brown ◽  
Bert L. Semler ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Internal Ribosomal Entry Site ◽  
Rna Replication ◽  
Viral Proteins ◽  
Encephalomyocarditis Virus ◽  
Minus Strand ◽  
Entry Site ◽  
Sequence Production ◽  
Positive Strand Rna

ABSTRACT HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5′ noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5′-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5′-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3′ end.

scholarly journals Heat Shock Protein A6, a Novel HSP70, Is Induced During Enterovirus A71 Infection to Facilitate Internal Ribosomal Entry Site-Mediated Translation

2021 ◽  
Vol 12 ◽  
Author(s):  
Yu-Siang Su ◽  
Lih-Hwa Hwang ◽  
Chi-Ju Chen
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Neurological Diseases ◽  
Internal Ribosomal Entry Site ◽  
Viral Proteins ◽  
Cellular Protein ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Enterovirus A71 ◽  
Ribosomal Entry

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot, and mouth disease (HFMD) in children. Its infection can lead to severe neurological diseases or even death in some cases. While being produced in a large quantity during infection, viral proteins often require the assistance from cellular chaperones for proper folding. In this study, we found that heat shock protein A6 (HSPA6), whose function in viral life cycle is scarcely studied, was induced and functioned as a positive regulator for EV-A71 infection. Depletion of HSPA6 led to the reductions of EV-A71 viral proteins, viral RNA and virions as a result of the downregulation of internal ribosomal entry site (IRES)-mediated translation. Unlike other HSP70 isoforms such as HSPA1, HSPA8, and HSPA9, which regulate all phases of the EV-A71 life, HSPA6 was required for the IRES-mediated translation only. Unexpectedly, the importance of HSPA6 in the IRES activity could be observed in the absence of viral proteins, suggesting that HSPA6 facilitated IRES activity through cellular factor(s) instead of viral proteins. Intriguingly, the knockdown of HSPA6 also caused the reduction of luciferase activity driven by the IRES from coxsackievirus A16, echovirus 9, encephalomyocarditis virus, or hepatitis C virus, supporting that HSPA6 may assist the function of a cellular protein generally required for viral IRES activities.

scholarly journals Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

2004 ◽  
Vol 78 (23) ◽  
pp. 13007-13018 ◽  
Author(s):  
Christopher T. Cornell ◽  
Jo Ellen Brunner ◽  
Bert L. Semler
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Genetically Modified ◽  
Rna Replication ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Wild Type ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACT We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

scholarly journals Identification of an RNA Hairpin in Poliovirus RNA That Serves as the Primary Template in the In Vitro Uridylylation of VPg

2000 ◽  
Vol 74 (22) ◽  
pp. 10359-10370 ◽  
Author(s):  
Aniko V. Paul ◽  
Elizabeth Rieder ◽  
Dong Wook Kim ◽  
Jacques H. van Boom ◽  
Eckard Wimmer
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Covalent Attachment ◽  
Minus Strand ◽  
Coding Region ◽  
Rna Sequences ◽  
Cellular Factors ◽  
Rna Hairpin ◽  
Made In

ABSTRACT The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3Dpol, and Mg2+ (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280–284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CDpro. Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.

scholarly journals Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

1998 ◽  
Vol 72 (8) ◽  
pp. 6546-6553 ◽  
Author(s):  
Julie A. Lemm ◽  
Anders Bergqvist ◽  
Carol M. Read ◽  
Charles M. Rice
Keyword(s):  
Rna Polymerase ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Functional Assay ◽  
Minus Strand ◽  
Virus Rna ◽  
A Genome ◽  
Strand Initiation

ABSTRACT Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

scholarly journals Unr Is Required In Vivo for Efficient Initiation of Translation from the Internal Ribosome Entry Sites of both Rhinovirus and Poliovirus

2003 ◽  
Vol 77 (6) ◽  
pp. 3353-3359 ◽  
Author(s):  
Oréda Boussadia ◽  
Michael Niepmann ◽  
Laurent Créancier ◽  
Anne-Catherine Prats ◽  
François Dautry ◽  
...  
Keyword(s):  
Rna Binding ◽  
Es Cells ◽  
Internal Ribosomal Entry Site ◽  
Embryonic Stem ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Initiation Of Translation ◽  
Mouth Disease Virus

ABSTRACT Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (−/+) or both (−/−) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr +/+, unr +/−, and unr −/− cell lines. Translation directed by the HRV IRES was severely impaired in unr −/− cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr −/− cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.

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 Synthesis of Minus-Strand RNA of Bamboo Mosaic Potexvirus Initiates from Multiple Sites within the Poly(A) Tail

2002 ◽  
Vol 76 (12) ◽  
pp. 6114-6120 ◽  
Author(s):  
Jai-Hong Cheng ◽  
Chi-Weng Peng ◽  
Yau-Heiu Hsu ◽  
Ching-Hsiu Tsai
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Initiation Site ◽  
Minus Strand ◽  
Sequence Analyses ◽  
Mobility Shift ◽  
Rapid Amplification ◽  
Gel Mobility Shift ◽  
Rna Pseudoknot

ABSTRACT The 3′ terminus of the bamboo mosaic potexvirus (BaMV) contains a poly(A) tail, the 5′ portion of which participates in the formation of an RNA pseudoknot required for BaMV RNA replication. Recombinant RNA-dependent RNA polymerase (RdRp) of BaMV binds to the pseudoknot poly(A) tail in gel mobility shift assays (C.-Y. Huang, Y.-L. Huang, M. Meng, Y.-H. Hsu, and C.-H. Tsai, J. Virol. 75:2818-2824, 2001). Approximately 20 nucleotides of the poly(A) tail adjacent to the 3′ untranslated region (UTR) are protected from diethylpyrocarbonate modification, suggesting that this region may be used to initiate minus-strand RNA synthesis. The 5′ terminus of the minus-strand RNA synthesized by the RdRp in vitro was examined using 5′ rapid amplification of cDNA ends (RACE) and DNA sequencing. Minus-strand RNA synthesis was found to initiate from several positions within the poly(A) tail, with the highest frequency of initiation being from the 7th to the 10th adenylates counted from the 5′-most adenylate of the poly(A) tail. Sequence analyses of BaMV progeny RNAs recovered from Nicotiana benthamiana protoplasts which were inoculated with mutants containing a mutation at the 1st, 4th, 7th, or 16th position of the poly(A) tail suggested the existence of variable initiation sites, similar to those found in 5′ RACE experiments. We deduce that the initiation site for minus-strand RNA synthesis is not fixed at one position but resides opposite one of the 15 adenylates of the poly(A) tail immediately downstream of the 3′ UTR of BaMV genomic RNA.

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 Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Philipp Schult ◽  
Maren Nattermann ◽  
Chris Lauber ◽  
Stefan Seitz ◽  
Volker Lohmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Replication ◽  
Rna Dependent Rna Polymerase ◽  
Internal Initiation ◽  
Positive Strand Rna

ABSTRACT Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5′ untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or template-switching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps. IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNA-dependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo. Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.

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