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Results for “RNA Replication”

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PubMed1996

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants.

Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.

Animals↗

PubMed1986

Conservative replication and transcription of Saccharomyces cerevisiae viral double-stranded RNA in vitro.

All double-stranded RNA viruses have capsid-associated RNA polymerase activities. In the reoviruses, the transcriptase synthesizes the viral plus strand in a conservative mode and the replicase synthesizes the viral minus strand, again conservatively. In bacteriophage phi 6 and in some fungal viruses, the transcriptase activity is semiconservative, acting by displacement synthesis. In this work we demonstrate Saccharomyces cerevisiae viral RNA replication in vitro for the first time and, using more sensitive techniques than those previously used, show that both the transcriptase and the replicase appear to act conservatively, like those of reovirus. There is therefore clearly no universal life cycle for the double-stranded RNA viruses.

RNA Viruses↗

PubMed2004

C-terminal nsP1a protein of human astrovirus colocalizes with the endoplasmic reticulum and viral RNA.

Computational and biological approaches were undertaken to characterize the role of the human astrovirus nonstructural protein nsP1a/4, located at the C-terminal fragment of nsP1a. Computer analysis reveals sequence similarities to other nonstructural viral proteins involved in RNA replication and/or transcription and allows the identification of a glutamine- and proline-rich region, the prediction of many phosphorylation and O-glycosylation sites, and the occurrence of a KKXX-like endoplasmic reticulum retention signal. Immunoprecipitation analysis with an antibody against a synthetic peptide of the nsP1a/4 sequence detected polyprotein precursors of 160, 75, and 38 to 40 kDa as well as five smaller proteins in the range of 21 to 27 kDa. Immunofluorescence labeling showed that the nsP1a/4 protein is accumulated at the perinuclear region, in association with the endoplasmic reticulum and the viral RNA. These results suggest the involvement of nsP1a/4 protein in the RNA replication process in endoplasmic reticulum-derived intracellular membranes.

Caco-2 Cells↗

PubMed1992

Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome.

The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.

Base Sequence↗

PubMed1994

Biochemical studies on poliovirus polypeptide 2C: evidence for ATPase activity.

Poliovirus 2C is a nonstructural polypeptide proposed to function in viral RNA replication. Poliovirus 2C is a member of a rapidly expanding family of proteins containing a consensus for nucleotide binding (NTP-B). Site-directed mutagenesis of conserved residues in the consensus A and B sites have suggested a functional role for the NTP-B motif in viral RNA replication and proliferation of poliovirus. We have expressed wildtype 2C and a 2C mutant, carrying a single amino acid exchange in the NTP-B motif A (Lys135Gln) using the baculovirus system. Both wildtype and mutant proteins are membrane associated. Following membrane solubilization, we have purified wildtype and mutant proteins to near homogeneity using conventional chromatography. We present biochemical evidence that wildtype 2C copurifies with an ATPase activity that is absent in the mutant preparation.

Adenosine Triphosphatases↗

PubMed2005

Specific binding of tombusvirus replication protein p33 to an internal replication element in the viral RNA is essential for replication.

The mechanism of template selection for genome replication in plus-strand RNA viruses is poorly understood. Using the prototypical tombusvirus, Tomato bushy stunt virus (TBSV), we show that recombinant p33 replicase protein binds specifically to an internal replication element (IRE) located within the p92 RNA-dependent RNA polymerase coding region of the viral genome. Specific binding of p33 to the IRE in vitro depends on the presence of a C.C mismatch within a conserved RNA helix. Interestingly, the absence of the p33:p33/p92 interaction domain in p33 prevented specific but allowed nonspecific RNA binding, suggesting that a multimeric form of this protein is involved in the IRE-specific interaction. Further support for the selectivity of p33 binding in vitro was provided by the inability of the replicase proteins of the closely related Turnip crinkle virus and distantly related Hepatitis C virus to specifically recognize the TBSV IRE. Importantly, there was also a strong correlation between p33:IRE complex formation in vitro and viral replication in vivo, where mutations in the IRE that disrupted selective p33 binding in vitro also abolished TBSV RNA replication both in plant and in Saccharomyces cerevisiae cells. Based on these findings and the other known properties of p33 and the IRE, it is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes. Since all genera in Tombusviridae encode comparable replicase proteins, these results may be relevant to other members of this large virus family.

Binding Sites↗

PubMed2003

Manganese-dependent polioviruses caused by mutations within the viral polymerase.

Viral RNA-dependent RNA polymerases exhibit great sequence diversity. Only six core amino acids are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. While exploring the function of one of these completely conserved residues, asparagine 297 in the prototypic poliovirus polymerase 3D(pol), we identified three viable mutants with noncanonical amino acids at this conserved position. Although asparagine 297 could be replaced by glycine or alanine in these mutants, the viruses exhibited Mn(2+)-dependent RNA replication and viral growth. All known RNA polymerases and replicative polymerases of bacterial, eukaryotic, and viral organisms are thought to be magnesium dependent in vivo, and therefore these mutant polioviruses may represent the first viruses with a requirement for an alternative polymerase cation. These results demonstrate the extreme functional flexibility of viral RNA-dependent RNA polymerases. Furthermore, the finding that strictly conserved residues in the nucleotide binding pocket of the polymerase can be altered in a manner that supports virus production suggests that drugs targeting this region of the enzyme will still be susceptible to the problem of drug-resistant escape mutants.

Amino Acid Sequence↗

PubMed1998

The activity of Sendai virus genomic and antigenomic promoters requires a second element past the leader template regions: a motif (GNNNNN)3 is essential for replication.

The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of approximately 15 kb, contains six transcription units flanked at the 3' and 5' ends by a short (approximately 50- to 60-nucleotide) extracistronic sequence, dubbed the positive and negative leader regions. These leader template regions, present at the 3' end of the genome and the antigenome, have been shown to contain essential signals governing RNA replication activity. Whether they are sufficient to promote replication is still open to question. By using a series of Sendai virus defective interfering RNAs carrying a nested set of deletions in the promoter regions, it is shown here that for both the genomic and antigenomic promoters, a 3'-end RNA sequence of 96 nucleotides is required to allow replication. Sequence comparison of active and inactive promoters led to the identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5'-3' positive-strand]. Sequential mutation of each hexamer into its complementary sequence confirmed their essential role. The three hexamers are required, and their relative positioning is important, since displacing them by 6 nucleotides destroyed promoter function. RNAs carrying degenerate nucleotides in the three hexamers were used as replication templates. They led to the selection of actively replicating RNA species exclusively carrying the basic motif (GNNNNN)3 from nucleotides 79 to 96. These results clearly show that, apart from the region from nucleotides 1 to 31, previously identified as governing Sendai virus replication activity, a second element, spanning at the most nucleotides 79 to 96, appears essential. Thus, the paramyxovirus replication promoters are not confined to the leader template regions, as seems to be the case for the rhabdoviruses.

Binding Sites↗

PubMed1999

Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a.

Brome mosaic virus (BMV) RNA replication is directed by two virus-encoded proteins, 1a and 2a. The amino-terminal half of 1a is a distant homolog of alphavirus nonstructural protein nsP1, which has been implicated in capping viral RNAs. In this study, we examined the enzymatic activities of BMV 1a expressed in yeast, where the protein is fully functional in RNA replication. 1a methylated GTP, dGTP, and the cap analogs GpppG and GpppA, using S-adenosylmethionine (AdoMet) as the methyl donor. Product analysis by nuclear magnetic resonance spectroscopy showed that 1a methylation was specific for guanine position 7. Additionally, 1a interacted with GTP to form a covalent 1a-m(7)GMP complex. This reaction was specific for GTP, required AdoMet, and was accompanied by transfer of (3)H-methyl from AdoMet to the covalent 1a-guanylate complex. The covalent complex could be immunoprecipitated by 1a antibodies. The 1a-m(7)GMP complex was inhibited in catalyzing further methyltransferase reactions. Mutation of conserved amino acids in the N-terminal half of 1a reduced both methyltransferase and covalent complex formation activities to very low or undetectable levels. Covalent 1a-guanylate complex formation took place in similar, AdoMet-dependent fashion in extracts of BMV-infected barley protoplasts. These results show that BMV 1a has activities similar to those of alphavirus nsP1, demonstrating conservation of these putative capping functions across a wide span of sequence divergence within the alphavirus-like superfamily. Conservation of this unusual combination of functions also supports the inference that the superfamily caps viral RNAs by an unusual pathway proceeding via a m(7)GMP intermediate.

Amino Acid Sequence↗

PubMed2005

Function follows form: the structure of the N-terminal domain of HCV NS5A.

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The nonstructural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kd) with an amphipathic -helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into 3 domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif, and a disulfide bond. We use molecular surface analysis to suggest the location of protein-, RNA-, and membrane-interaction sites.

Binding Sites↗

PubMed2005

Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase.

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic alpha-helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.

Amino Acid Motifs↗

PubMed1993

Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA.

RNA 3 of alfalfa mosaic virus (AIMV) encodes the movement protein P3 and the viral coat protein which is translated from the subgenomic RNA 4. The 5'-leader sequences of RNA 3 of AIMV strains S, A, and Y differ in length from 314 to 392 nucleotides and contain a variable number of internal control regions of type 2 (ICR2 motifs) each located in a 27 nt repeat. Infectious cDNA clones were used to exchange the leader sequences of the three strains. This revealed that the leader sequence controls the specific ratio in which RNAs 3 and 4 are synthesized for each strain. In addition, it specifies strain specific differences in the kinetics of P3 accumulation in plants. Subsequent deletion analysis revealed that a 5'-sequence of 112 nt containing one ICR2 motif was sufficient for a 10 to 20% level of RNA 3 accumulation in protoplasts and a delayed accumulation in plants. An additional leader sequence of maximally 114 nt, containing two ICR2 motifs, was required to permit wildtype levels of RNA 3 accumulation. The effect of deletions in the leader sequence on P3 synthesis in vitro and in vivo was investigated.

Base Sequence↗

PubMed2006

Intramolecular RNA replicase: possibly the first self-replicating molecule in the RNA world.

Although there is more and more evidence suggested the existence of an RNA World during the origin of life, the scenario concerning the origin of the RNA World remains blurry. Usually it is speculated that it originated from a prebiotic nucleotide pool, during which a self-replicating RNA synthesis ribozyme may have emerged as the first ribozyme - the RNA replicase. However, there is yet no ersuasive supposition for the mechanism for the self-favouring feature of the replicase, thus the speculation remains unconvincing. Here we suggest that intramolecular catalysis is a possible solution. Two RNA synthesis ribozymes may be integrated into one RNA molecule, as two functional domains which could catalyze the copy of each other. Thus the RNA molecule could self-replicate and be referred to as "intramolecular replicase" here. Computational simulation to get insight into the dynamic mechanism of emergence of the intramolecular replicase from a nucleotide pool is valuable and would be included in a following work of our group.

Catalysis↗

PubMed2000

Effects of palmitoylation of replicase protein nsP1 on alphavirus infection.

The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.

Alphavirus Infections↗

PubMed2004

Evidence of viral infection in the myocardium of American and Japanese patients with idiopathic dilated cardiomyopathy.

Enteroviruses have been implicated in the pathogenesis of idiopathic dilated cardiomyopathy (IDC). Recently, the association of adenovirus or parvovirus with IDC has been reported. Viral infection in the myocardium of American and Japanese patients with end-stage IDC was evaluated. Myocardial specimens from 30 American patients with IDC and 47 Japanese patients with IDC were analyzed for the presence of cardiotropic viruses. The strand-specific detection of enteroviral ribonucleic acid (RNA) was performed to determine viral activity in hearts with IDC. Established reverse transcription-polymerase chain reaction (PCR) or PCR techniques were used to detect genomic sequences of influenza viruses, mumps virus, adenovirus, parvovirus, herpes simplex viruses, varicella-zoster virus, and Epstein-Barr virus. Enteroviral RNA was detected in 7 of the 30 American patients (23%) and in 15 of the 47 Japanese patients (32%). Minus-strand enteroviral RNA, an indicator of active enteroviral RNA replication, was demonstrated in 5 of 7 plus-strand-positive American patients (71%) and in 12 of 15 plus-strand-positive Japanese patients (80%). Sequence analysis revealed that the viruses detected were Coxsackie B viruses. No genomic sequences of other viruses were detected in the myocardium of either American or Japanese patients with IDC. Therefore, active group B Coxsackie virus RNA replication in the myocardium was demonstrated in a significant proportion of American and Japanese patients with end-stage IDC. There was no evidence of persistent infection by other viruses in hearts with IDC. Specific therapy should be designed for Coxsackie virus positive patients with IDC.

Adult↗

PubMed2006

The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site similar to that of the hepatitis C virus NS5A protein.

The recent demonstration that the NS5A protein of hepatitis C virus (HCV) contains an unconventional zinc-binding site with the format Cx(17)CxCx(20)C and the presence of a similar sequence element in the NS5A proteins of members of the Pestivirus genus has led to the hypothesis that the NS5A protein of the pestivirus bovine viral diarrhea virus (BVDV) is a zinc-binding protein. A method for the expression and partial purification of BVDV NS5A was developed, and the partially purified protein was analyzed for zinc content by atomic absorption spectroscopy. BVDV NS5A was found to coordinate a single zinc atom per protein molecule. Mutation of any of the four cysteines of the predicted zinc-binding motif eliminated zinc coordination. Furthermore, analysis of mutations at these cysteine residues in the context of a BVDV replicon system indicated that these residues were absolutely essential for RNA replication. The recently determined crystal structure of the N-terminal zinc-binding domain of the HCV NS5A protein, combined with secondary structure predictions of the region surrounding the mapped BVDV zinc-binding region, indicates that the BVDV zinc-binding motif fits the general template Cx(22)CxCx(24)C and likely comprises a three-stranded antiparallel beta-sheet fold. These data highlight the similarities between the Hepacivirus and Pestivirus NS5A proteins and suggest that both proteins perform a not-yet-defined function in RNA replication that requires coordination of a single zinc atom.

Amino Acid Sequence↗

PubMed1995

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.

Cell Line↗

PubMed1992

Synthesis of the complete 200K polyprotein encoded by cowpea mosaic virus B-RNA in insect cells.

The coding sequence for the entire 200K polyprotein of cowpea mosaic virus (CPMV) B-RNA was expressed in insect cells by using baculovirus expression vectors. The 200K polyprotein, which harbours all virus functions required for RNA replication, is completely cleaved into 170K and 32K products by the 24K protease activity contained within the polyprotein. Further processing of the 170K protein into CPMV-specific products of 60K, 84K, 87K, 110K and 112K occurred to a limited extent, similar to that observed in cowpea cells. Electron microscopy of insect cells in which the 200K protein was produced revealed the presence of membranous vesicles and electron-dense structures which were not seen in cells infected with wild-type baculovirus. Similar cytopathic structures develop in the cytoplasm of CPMV-infected cowpea cells and are thought to be the site of membrane-bound viral RNA replication. The electron-dense structures in insect cells could be preferentially labelled with several CPMV-specific antisera and Protein A-gold. Since electron-dense structures were not observed in cells in which the 170K protein only was produced, it seems that the 32K protein has a role in keeping the B-RNA-encoded proteins in these structures together. Membranous vesicles were also observed in insect cells in which the 60K protein only was produced. Use of specific antibodies and Protein A-gold showed that the 60K protein is associated with these vesicles, indicating that the 60K protein may induce the formation of vesicles. Although proteolytic processing of the 200K polyprotein and the induction of cytopathic structures indicate that the CPMV proteins produced in insect cells are functional, it has not been possible to demonstrate RNA polymerase activity in extracts of these cells using an oligo(U)-primed assay. The results indicate that in the assay an additional component is lacking and/or that the CPMV polymerase is not able to start RNA synthesis on an exogenous template.

Animals↗