SEARCH · PubMed Health

Results for “RNA Replication”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,387 records · Page 77Linked to original sources

PubMed2002

Mutational analysis of the Uukuniemi virus (Bunyaviridae family) promoter reveals two elements of functional importance.

We have performed an extensive mutational analysis of the proposed promoter region of the phlebovirus Uukuniemi (UUK), a member of the Bunyaviridae family. This was achieved by using a recently developed RNA polymerase I (Pol I)-driven reverse genetics system (R. Flick and R. F. Pettersson, J. Virol. 75:1643-1655, 2001). Chimeric cDNAs containing the coding region for the reporter chloramphenicol acetyltransferase (CAT) in an antisense orientation were flanked by the 5'- and 3'-terminal nontranslated regions of the UUK virus-sense RNA (vRNA) derived from the medium-sized (M) RNA segment. The chimeric cDNAs (Pol I expression cassettes) were cloned between the murine Pol I promoter and terminator, and the plasmids were transfected into BHK-21 cells. CAT activity was determined after cotransfection with viral expression plasmids encoding the RNA-dependent RNA polymerase (L) and the nucleoprotein (N) or, alternatively, after superinfection with UUK virus helper virus. Using oligonucleotide-directed mutagenesis, single point mutations (substitutions, deletions, and insertions) were introduced into the viral promoter region. Differences in CAT activities were interpreted to reflect the efficiency of mRNA transcription from the mutated promoter and the influence on RNA replication. Analysis of 109 mutants allowed us to define two important regulatory regions within the proximal promoter region (site A, positions 3 to 5 and 2 to 4; site B, positions 8 and 8, where underlined nucleotides refer to positions in the vRNA 3' end). Complementary double nucleotide exchanges in the proximal promoter region, which maintained the possibility for base pairing between the 5' and 3' ends, demonstrated that nucleotides in the two described regions are essential for viral polymerase recognition in a base-specific manner. Thus, mere preservation of panhandle base pairing between the 5' and 3' ends is not sufficient for promoter activity. In conclusion, we have been able to demonstrate that both ends of the M RNA segment build up the promoter region and are involved in the specific recognition by the viral polymerase.

Animals↗

PubMed1993

A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo.

Mutants of foot-and-mouth disease virus (FMDV) with altered biological properties can be selected during the course of persistent infection of BHK-21 cells with FMDV C-S8c1 (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). Two nucleotide substitutions, U to C at position -376 and A to G at position -15, (counting as +1 the A of the first functional AUG), were fixed within the internal ribosome entry site (IRES) of R100, the virus rescued after 100 passages of the carrier BHK-21 cells. IRES-directed cap-independent protein synthesis was quantitated by using bicistronic constructs of the form chloramphenicol acetyltransferase gene-IRES-luciferase gene. The IRES from R100 was 1.5- to 5-fold more active than that of C-S8c1 in directing cap-independent luciferase synthesis. This enhanced translational activity was observed when the RNAs were transcribed either in the nucleus or in the cytoplasm by a weak or a strong promoter, respectively. C-S8c1 and R100 IRES elements were functional in both FMDV-sensitive and FMDV-resistant cells (including persistently infected R cells), indicating that factors mediating cap-independent protein synthesis are not limited in any of the analyzed cell lines. Constructs in which each of the two mutations in the R100 IRES were analyzed separately indicate that the transition at position -376 is responsible for the enhanced activity of the R100 IRES. By estimating the effect that an increase in the initial translation efficiency may have on subsequent RNA replication steps, we suggest that the modifications in the IRES elements can account for the previously described hypervirulence of FMDV R100 for BHK-21 cells. The results show that a single point mutation in an IRES element of a picornavirus can cause an increase in translation efficiency.

Animals↗

PubMed2002

Hyper-phosphorylation of the adeno-associated virus Rep78 protein inhibits terminal repeat binding and helicase activity.

The replication (Rep) proteins of adeno-associated virus (AAV) play prominent roles in regulation of viral DNA replication, RNA transcription, assembly of an infectious virion and establishment of the provirus. We have previously demonstrated that all four Rep proteins are phosphorylated on serine residues [Virology 23 (1997) 332-336]. Reversible phosphorylation may provide a mechanism for regulating Rep protein function. To test this hypothesis, we used the phosphatase inhibitor okadaic acid (OA) to obtain hyper-phosphorylated Rep proteins. OA treatment of AAV- and adenovirus (Ad)-infected cells and baculovirus-infected insect cells at a concentration of 100 nM resulted in a significant increase in Rep protein phosphorylation. This concentration suggests that protein phosphatase 2A (PP2A) is one of the enzymes involved in regulation of Rep phosphorylation. The increased phosphorylation occurred primarily on serine residues with a detectable amount of phosphate on threonine. Hyper-phosphorylation of Rep78 resulted in reduced binding to the AAV origin of DNA replication. Hyper-phosphorylated Rep78 also had diminished helicase activity. These results suggest that regulated phosphorylation of Rep78 plays a role in controlling Rep functions in the virus replication cycle.

Cell Line↗

PubMed1983

The 5'-terminal sequences of the genomic RNAs of several alphaviruses.

The 5'-terminal sequences of the genomic RNAs of several alphaviruses have been determined. The nucleotide sequences at the extreme 5' termini are not highly conserved among the alphaviruses, but a similar stem and loop structure, which begins at the 5' end and utilizes about the first 40 nucleotides, can be formed in each case. Downstream from this structure, beginning about 150 nucleotides from the 5' end, a conserved sequence of 51 nucleotides is found which can form two stable hairpin structures. Examination of the 5'-terminal and 3'-terminal sequences suggests that part of this conserved nucleotide sequence may be involved in cyclization of the RNA. A model is proposed for the function of the 5'-terminal sequences in RNA replication. In addition, sequence homologies among these RNAs strongly support the hypothesis that an AUG codon, which occurs at 60 to 80 nucleotides from the 5' end, depending on the virus, and which may or may not be the first AUG codon, is used for initiation of translation of the non-structural proteins and allows a comparison of the deduced amino acid sequences in the NH2-terminal regions.

Alphavirus↗

PubMed2002

Identification of the lymphocytic choriomeningitis virus (LCMV) proteins required to rescue LCMV RNA analogs into LCMV-like particles.

We have used a reverse genetic approach to identify the viral proteins required for packaging and assembly of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). Plasmids encoding individual LCMV proteins under the control of an RNA polymerase II promoter were cotransfected with a plasmid containing an LCMV minigenome (MG). Intracellular synthesis of the LCMV MG was driven by T7 RNA polymerase whose expression was also mediated by a Pol II promoter. The supernatant from transfected cells was passaged onto fresh cells that were subsequently infected with LCMV to provide the minimal viral trans-acting factors, NP and L, that are required for LCMV MG RNA replication and expression. Reconstitution of LCMV-specific packaging and passage was detected by expression of the chloramphenicol acetyl transferase (CAT) reporter gene present in the MG. NP and L did not direct detectable levels of MG passage. Addition of Z and GP resulted in high levels of passage of CAT activity, which could be prevented by LCMV neutralizing antibodies. Passage of LCMV MG was inhibited by omission of either GP or Z.

Animals↗

PubMed1992

Utilization of heterologous alphavirus junction sequences as promoters by Sindbis virus.

We used Sindbis virus, an alphavirus, as a model to study the evolution of the recognition of viral cis-acting sequences. During the life cycle of alphaviruses, a full-length minus-strand RNA is made and serves as a template for both genomic RNA replication and subgenomic mRNA transcription. Transcription initiates at an internal promoter site, the junction sequence, to produce a subgenomic mRNA. The junction sequences of alphaviruses are highly conserved, but they do contain a number of base differences. These could have been essentially neutral mutations during evolution, such that any of the contemporary sequences can be recognized efficiently by any of the alphaviruses. Alternately, the changes could have resulted in significant functional divergence, such that the contemporary viruses can no longer recognize heterologous junction sequences as promoters. To distinguish between these possibilities, we constructed Sindbis virus derivatives with two subgenomic mRNA promoters. One is the wild-type Sindbis virus promoter used for expression of the structural proteins. The other is either the minimal Sindbis virus promoter or the corresponding junction sequences from other alphaviruses, which are placed upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene. RNA analyses were used to determine the relative promoter strengths of the various junction sequences. The results showed that all but two were recognized as promoters by Sindbis virus. CAT enzyme assays were used to measure the accumulation of CAT protein made from mRNAs transcribed by using the heterologous junction sequences as promoters. Most of the viruses expressed amounts of CAT enzyme within 10-fold of each other. The two viruses with junction sequences that were not recognized as promoters did not give significant CAT expression. We conclude that, with respect to Sindbis virus, the junction sequences are functionally conserved; i.e., most of the contemporary nucleotide differences in the junction sequences are neutral or near-neutral mutations. The functional conservation suggests that neither the cis-acting sequence nor the cognate binding site of the transcription factor can change independently. This type of coupled evolution between cis-acting sequences and their cognate viral protein binding sites may be a general phenomenon. For example, it explains the ubiquitous presence of conserved cis-acting sequences in each of the families of RNA viruses. There are implications of this hypothesis for the design of antiviral drugs.

Alphavirus↗

PubMed2005

Specific anti-viral effects of RNA interference on replication and expression of hepatitis B virus in mice.

BACKGROUND: RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs (siRNAs) have sufficiently inhibited hepatitis B virus (HBV) replication and expression in vitro. In this study we observed the RNAi-mediated inhibitory effects on HBV replication in mice models and accessed the specificity of these effects. METHODS: A mutant RNAi vector (pSI-C mut) with two base pairs different from the original target gene sequence at the RNAi vector (pSI-C) was constructed according to the method described in this study. A mouse model of acute hepatitis B virus infection was established by injecting naked plasmid pHBV1.3 via the tail vein with acute circulatory overload. pSI-C, pSI-C mut and the irrelevant RNAi control plasmid for green fluorescent protein (GFP) gene, pSIGFP were respectively delivered with pHBV1.3 by tail vein injection method. Six days post injection, enzyme-linked immunosorbent assay (ELISA) assay was used to measure the concentration of HBV surface antigen (HBsAg) in mouse serum, immunohistochemical straining method was used to visualize the expression of HBV core protein (HBcAg) in liver tissues, and the transcriptional level of HBV C mRNA in liver tissues was detected by reverse transcriptase PCR (RT-PCR) analysis. RESULTS: Injection of pSI-C exerted magnificent and specific inhibitory effects on the replication and expression of HBV in the murine model. After 6-day post-injection (p.i.), the OD values were shown to be 5.07 +/- 1.07 in infecting group and 0.62 +/- 0.59 in pSI-C group. The concentration of HBsAg in pSI-C group was significantly lower than that in infecting group (P < 0.01). Liver intracellular synthesis of viral core protein was sharply reduced to 0.9% +/- 0.1%, compared with 5.4% +/- 1.2% of positive hepatocytes in infecting group (P < 0.01), and the transcriptional level of HBV C mRNA was greatly reduced by 84.7%. However, the irrelevant RNAi control plasmid (pSIGFP), and the pSI-C mut did not show the same robust inhibitory effects as pSI-C. CONCLUSION: pSI-C exert efficient and specific inhibitory effects on HBV replication and expression in mice models.

Animals↗

PubMed1999

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus.

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-A resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the "fingertips," that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669-1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.

Amino Acid Sequence↗

PubMed1996

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

Animals↗

PubMed1990

Phosphorylation of Sindbis virus nsP3 in vivo and in vitro.

nsP3 is one of four viral nonstructural proteins required for RNA replication of Sindbis virus. In this report, post-translational modifications of nsP3 which occur in both vertebrate and mosquito cell cultures have been examined. In pulse-chase experiments, analyzed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nsP3 was initially observed as a single species (termed nsP3a, approximately 76 kDa) which was gradually converted to slower mobility forms ranging from 78 kDa (termed nsP3b) to 106 kDa (termed nsP3c). The slower mobility forms, but not nsP3a or the other nonstructural proteins, could be labeled in vivo with [32P]orthophosphate. Treatment of nsP3 immunoprecipitates with calf intestinal alkaline phosphatase converted the slower mobility forms to nsP3a. Phosphoamino acid analysis of nsP3b and nsP3c demonstrated that both contained phosphoserine and phosphothreonine but not phosphotyrosine, nsP34, a polyprotein produced by readthrough of the in-frame opal codon preceding nsP4, was also phosphorylated on serine and threonine residues. nsP3 phosphorylation did not require ongoing RNA synthesis since phosphorylated forms were also observed in the absence of Sindbis-specific RNA synthesis. Furthermore, when immunoprecipitates of nsP3 were incubated with [gamma-32P]ATP in the presence of Mg2+ or Mn2+, a kinase activity which was able to phosphorylate nsP3 on serine and threonine residues in vitro was detected. This kinase activity was inhibited by heparin, was activated by spermidine, and could utilize GTP and ATP as the phosphate donor. These latter properties are similar to those of cellular casein kinase II. Although it is possible that this nsP3-associated kinase is of cellular origin, autophosphorylation of nsP3 has not been excluded.

Animals↗

PubMed1991

Expression and characterization of poliovirus proteins 3BVPg, 3Cpro, and 3Dpol in recombinant baculovirus-infected Spodoptera frugiperda cells.

As an initial step toward investigating the roles of poliovirus proteins in viral RNA replication, a baculovirus expression system was used to produce poliovirus proteins from the P3 region. Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus, vETL-PoV3A*BCD, which contains cDNA coding for poliovirus proteins 3D, 3C, 3B, and a portion of 3A protein sequence. Immunofluorescence microscopy revealed that the majority of 3D (polymerase) was in the cytoplasm of recombinant baculovirus-infected Sf9 cells. In the same cells, the 3C (protease) and 3B (VPg) proteins appeared to be located in distinct subcellular regions, possibly membrane structures, suggesting that the expressed polyprotein was cleaved to generate mature proteins. Processing of the polypeptide was confirmed by immunoblot analysis which demonstrated that 3Cpro sequences were active in cleavage of the polyproteins 3A*BCD and 3CD. Over 95% of the 3D sequences accumulated in the form of mature 3Dpol, with only low levels of 3CD remaining. The majority of 3Dpol remained in the supernatant after low speed centrifugation of sonicated cells. The 3Dpol had RNA-dependent RNA polymerase activity as measured by elongation of an oligo(U) primer using a poly(A) template. The protein 3CDpro was active in cleaving P1 protein. The yield and activities of the poliovirus proteins expressed will facilitate future biochemical studies.

Animals↗

PubMed2005

Viral and cellular proteins involved in coronavirus replication.

As the largest RNA virus, coronavirus replication employs complex mechanisms and involves various viral and cellular proteins. The first open reading frame of the coronavirus genome encodes a large polyprotein, which is processed into a number of viral proteins required for viral replication directly or indirectly. These proteins include the RNA-dependent RNA polymerase (RdRp), RNA helicase, proteases, metal-binding proteins, and a number of other proteins of unknown function. Genetic studies suggest that most of these proteins are involved in viral RNA replication. In addition to viral proteins, several cellular proteins, such as heterogeneous nuclear ribonucleoprotein (hnRNP) A1, polypyrimidine-tract-binding (PTB) protein, poly(A)-binding protein (PABP), and mitochondrial aconitase (m-aconitase), have been identified to interact with the critical cis-acting elements of coronavirus replication. Like many other RNA viruses, coronavirus may subvert these cellular proteins from cellular RNA processing or translation machineries to play a role in viral replication.

Aconitate Hydratase↗

PubMed1999

RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein.

Repeated, specific interactions between capsid protein (CP) subunits direct virus capsid assembly and exemplify regulated protein-protein interactions. The results presented here reveal a striking in vivo switch in CP assembly. Using cryoelectron microscopy, three-dimensional image reconstruction, and molecular modeling, we show that brome mosaic virus (BMV) CP can assemble in vivo two remarkably distinct capsids that selectively package BMV-derived RNAs in the absence of BMV RNA replication: a 180-subunit capsid indistinguishable from virions produced in natural infections and a previously unobserved BMV capsid type with 120 subunits arranged as 60 CP dimers. Each such dimer contains two CPs in distinct, nonequivalent environments, in contrast to the quasi-equivalent CP environments throughout the 180-subunit capsid. This 120-subunit capsid utilizes most of the CP interactions of the 180-subunit capsid plus nonequivalent CP-CP interactions. Thus, the CP of BMV, and perhaps other viruses, can encode CP-CP interactions that are not apparent from mature virions and may function in assembly or disassembly. Shared structural features suggest that the 120- and 180-subunit capsids share assembly steps and that a common pentamer of CP dimers may be an important assembly intermediate. The ability of a single CP to switch between distinct capsids by means of alternate interactions also implies reduced evolutionary barriers between different capsid structures. The in vivo switch between alternate BMV capsids is controlled by the RNA packaged: a natural BMV genomic RNA was packaged in 180-subunit capsids, whereas an engineered mRNA containing only the BMV CP gene was packaged in 120-subunit capsids. RNA features can thus direct the assembly of a ribonucleoprotein complex between alternate structural pathways.

Bromovirus↗

PubMed1987

Transcriptionally defective nucleocapsids of vesicular stomatitis virus from cells treated with indomethacin.

Indomethacin blocks the biosynthesis of vesicular stomatitis virus (VSV) at the level of primary transcription, RNA replication, and protein synthesis (P. K. Mukherjee and R. W. Simpson (1985), Virology 140, 188-191). Nucleocapsids of infecting virus particles recovered from indomethacin-treated cells were analyzed for in vitro transcriptase activity. Incorporation of [3H]UTP in mixtures containing nucleocapsids from HEp-2 cells pretreated with 10(-3) M indomethacin was inhibited approximately 80% compared to control reactions containing nucleocapsids from untreated infected cells. The level of inhibition of in vitro transcriptase activity of viral nucleocapsids from drug-treated cultures varied according to the cell line used for infection. After indomethacin removal, cells regained their ability to produce enzymatically competent viral-transcribing complexes unless they were subsequently exposed to metabolic inhibitors such as actinomycin D or alpha-amanitin. Enzymatically defective nucleocapsids from indomethacin-treated cells showed enhanced in vitro transcriptase activity in the presence of modulators of prostaglandins and cyclic nucleotides. Electrophoretic analysis of product from in vitro transcriptase reactions revealed that these defective nucleocapsids are unable to synthesize VSV messenger RNA or normal size leader RNA species but only smaller transcripts of undetermined identity.

Animals↗

PubMed2006

A second RGD motif in the 1D capsid protein of a SAT1 type foot-and-mouth disease virus field isolate is not essential for attachment to target cells.

The amino acid sequence motif Arg-Gly-Asp (RGD), located in the surface-exposed betaG-betaH loop of the 1D protein of different serotypes and subtypes of foot-and-mouth disease virus (FMDV), is highly conserved and participates in binding of FMDV to susceptible cells. Previous sequence analyses of the 1D-encoding region of a FMDV serotype SAT1 field isolate from Namibia (NAM/307/98) indicated the presence of a second RGD motif upstream of the conserved betaG-betaH loop RGD. The role of these RGD sequences in virus infection was investigated by mutating the betaG-betaH loop RGD to a KGE tripeptide, using a genome-length infectious chimeric cDNA clone. Although the infectivity of the derived mutant viruses for baby hamster kidney cells (BHK-21) was lost, subsequent replacement of the KGE sequence with RGD in the mutant cDNA clone led to recovery of infectious viruses. Furthermore, viral RNA replication could be demonstrated with the genetically engineered mutant and non-mutant viruses. The presence of virus particles in the transfected cells could be also demonstrated by electron microscopy. These results demonstrate that, in contrast to the betaG-betaH loop RGD motif, the second RGD sequence in the capsid protein 1D of NAM/307/98 does not function as a ligand for receptor binding in BHK-21 cells.

Amino Acid Motifs↗

PubMed1999

Mutational analysis, using a full-length rubella virus cDNA clone, of rubella virus E1 transmembrane and cytoplasmic domains required for virus release.

We report on the construction of a full-length cDNA clone, pBRM33, derived from wild-type rubella virus M33 strain. The RNA transcripts synthesized in vitro from pBRM33 are highly infectious, and the viruses produced retain the phenotypic characteristics of the parental M33 virus in growth rate and plaque size. This cDNA clone was used to study the role of E1 transmembrane and cytoplasmic domains in virus assembly by site-directed mutagenesis. Three different alanine substitutions were introduced in the transmembrane domain of E1. These included substitution of leucine 464, cysteine 466, cysteine 467, and both cysteines 466 and 467 to alanine. In the E1 cytoplasmic domain, cysteine 470 and leucine 471 were altered to alanine. We found that these mutations did not significantly affect viral RNA replication, viral structural protein synthesis and transport, or E2/E1 heterodimer formation. Except for the substitution of cysteine 470, these mutations did, however, lead to a reduction in virus release. Substitution of cysteine 467 in the transmembrane region and of leucine 471 in the cytoplasmic domain dramatically reduced virus yield, resulting in the production of only 1 and 10% of the parental virus yield, respectively, in a parallel infection. These data show that E1 transmembrane and cytoplasmic domains play an important role in late stages of virus assembly, possibly during virus budding, consistent with earlier studies indicating that the E1 cytoplasmic domain may interact with nucleocapsids and that this interaction drives virus budding.

Animals↗

PubMed2006

Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus.

There is little understanding of mechanisms underlying the assembly and release of infectious hepatitis C virus (HCV) from cultured cells. Cells transfected with synthetic genomic RNA from a unique genotype 2a virus (JFH1) produce high titers of virus, while virus yields are much lower with a prototype genotype 1a RNA containing multiple cell culture-adaptive mutations (H77S). To characterize the basis for this difference in infectious particle production, we constructed chimeric genomes encoding the structural proteins of H77S within the background of JFH1. RNAs encoding polyproteins fused at the NS2/NS3 junction ("H-NS2/NS3-J") and at a site of natural, intergenotypic recombination within NS2 ["H-(NS2)-J"] produced infectious virus. In contrast, no virus was produced by a chimera fused at the p7-NS2 junction. Chimera H-NS2/NS3-J virus (vH-NS2/NS3-J) recovered from transfected cultures contained compensatory mutations in E1 and NS3 that were essential for the production of infectious virus, while yields of infectious vH-(NS2)-J were enhanced by mutations within p7 and NS2. These compensatory mutations were chimera specific and did not enhance viral RNA replication or polyprotein processing; thus, they likely compensate for incompatibilities between proteins of different genotypes at sites of interactions essential for virus assembly and/or release. Mutations in p7 and NS2 acted additively and increased the specific infectivity of vH-(NS2)-J particles, while having less impact on the numbers of particles released. We conclude that interactions between NS2 and E1 and p7 as well as between NS2 and NS3 are essential for virus assembly and/or release and that each of these viral proteins plays an important role in this process.

Cell Line↗

PubMed1995

Amino terminal regions of poliovirus 2C protein mediate membrane binding.

The poliovirus-encoded, membrane-associated polypeptide 2C is required for viral replication. We have expressed 2C, its precursor 2BC, and a number of 2C deletion mutants in eukaryotic (HeLa) cells and examined their localization using indirect immunofluorescence. Results presented here demonstrate that proteins 2C and 2BC are capable of localizing to the endoplasmic reticulum area in transfected cells in the absence of other poliovirus proteins. Additionally, 2C binds tightly to microsomal membranes in a direct in vitro membrane binding assay. Although the 2C protein lacks a defined membrane binding domain, we demonstrate that the N-terminal region encompassing amino acids 21-54 and containing a putative amphipathic helix plays an important role in membrane binding both in vivo and in vitro. In contrast, most of the C-terminus portion of the protein appears to be unnecessary for membrane association. The susceptibility of membrane-associated 2C to proteinase K digestion in vitro suggests that all or most of 2C is exposed to the cytoplasmic face of the membrane. Furthermore, unlike most proteins targeted to the endoplasmic reticulum, 2C does not appear to be glycosylated or cleaved by signal peptidases. The implication of the membrane binding domain of 2C in viral RNA replication is discussed.

Amino Acid Sequence↗