Replicase Proteins Research Articles

Using the TIR Domain to Recognize Pathogens

Plants respond to pathogens with a response called the hypersensitive response that involves necrotic cell death of the infected tissue to prevent further infection. Mounting this response involves the recognition of pathogen elicitor proteins and the plant products of the Resistance ( R ) genes, but the exact mechanism remains unclear. Burch-Smith et al . used the p50 helicase domain of the Tobacco mosaic virus replicase protein to study in plants the interaction between this p50 protein elicitor and the product of the N gene, which encodes an R protein with a leucine-rich repeat (LRR), a nucleotide-binding (NB) site, and a Toll-interleukin-1 receptor (TIR) homolog domain. Plant tissue in which a tagged version of p50 and a tagged version of N were coexpressed exhibited the hypersensitive response. Biochemical fractionation indicated that both proteins were soluble, and fluorescence analysis indicated that both proteins could be found in the cytoplasm and the nucleus of infected leaf cells. Inhibition of the nuclear localization of the N protein through the addition of a nuclear export sequence prevented the hypersensitive response, whereas blocking nuclear localization of p50 did not. This suggests that N and p50 interact in the cytoplasm but that N has nuclear functions that are required for activation of the immune response. Coimmunoprecipitation studies with the tagged proteins, as well as bimolecular fluorescence complementation studies, indicated that the two proteins interacted. However, purified proteins did not interact in vitro, which suggests that the interaction is indirect and that there is another molecule required for p50 and N interaction. Although the LRR domain is best known for recognition of pathogen sequences, the TIR domain of N appeared to be necessary and sufficient for the interaction of p50 with N. Deletion or point mutation of the TIR domain blocked the interaction between the two proteins. The isolated TIR domain of N, but not that of other R proteins, interacted with p50. The authors propose that the TIR domain of N (through an unknown adaptor) recognizes p50, which leads to a change in the N protein, and that the "activated" N protein then engages the defense response through a process that requires the nuclear localization or activation of a nuclear pool of N. T. M. Burch-Smith, M. Schiff, J. F. Caplan, J. Tsao, K. Czymmek, S. P. Dinesh-Kumar, A novel role for the TIR domain in association with pathogen-derived elicitors. PLoS Biol. 5 , e68 (2007). [PubMed]

Enzymatic Defects of the nsP2 Proteins of Semliki Forest Virus Temperature-Sensitive Mutants

We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24 degrees C and 35 degrees C. The protease domain mutants (ts4, ts6, and ts11) had reduced protease activity at 35 degrees C but displayed normal NTPase and RTPase. The helicase domain mutation ts1 did not have enzymatic consequences, whereas ts13a and ts9 reduced both NTPase and protease activities but in different and mutant-specific ways. The effects of these helicase domain mutants on protease function suggest interdomain interactions within nsP2. NTPase activity was not directly required for protease activity. The similarities of the NTPase and RTPase results, as well as competition experiments, suggest that these two reactions utilize the same active site. The mutations were also studied in recombinant viruses first cultivated at the permissive temperature and then shifted up to the restrictive temperature. Processing of the nonstructural polyprotein was generally retarded in cells infected with viruses carrying the ts4, ts6, ts11, and ts13a mutations, and a specific defect appeared in ts9. All mutations except ts13a were associated with a large reduction in the production of the subgenomic 26S mRNA, indicating that both protease and helicase domains influence the recognition of the subgenomic promoter during virus replication.

Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 Replication

Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 Replication

Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

Role of the Amphipathic Peptide of Semliki Forest Virus Replicase Protein nsP1 in Membrane Association and Virus Replication

Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G245STLYTESRKLLRSWHLPSV264, has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A+L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A+L256A only yielded virus encoding L255A+L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein.

Competition of bacteriophage polypeptides with native replicase proteins for binding to the DNA sliding clamp reveals a novel mechanism for DNA replication arrest in Staphylococcus aureus

Competition of bacteriophage polypeptides with native replicase proteins for binding to the DNA sliding clamp reveals a novel mechanism for DNA replication arrest in <i>Staphylococcus aureus</i>

Novel replication complex architecture in rubella replicon-transfected cells

SummaryRubella virus (RUB) assembles its replication complexes (RCs) in modified organelles of endo‐lysosomal origin, known as cytopathic vacuoles (CPVs). These peculiar structures are key elements of RUB factories, where rough endoplasmic reticulum, mitochondria, and Golgi are recruited. Bicistronic RUB replicons expressing an antibiotic resistance gene either in the presence or the absence of the RUB capsid (C) gene were used to study the structure of RCs in transfected cells. Confocal microscopy showed that the RUB replicase components P90 and P150 localized to CPVs, as did double‐stranded RNA (dsRNA), a marker for RNA synthesis. Electron microscopy (EM) showed that replicons generated CPVs containing small vesicles and large vacuoles, similar to CPVs from RUB‐infected cells and that the replicase proteins were sufficient for organelle recruitment. Some of these CPVs contained straight membranes. When cross‐sectioned, these rigid membranes appeared to be sheets of closely packed proteins. Immuno‐EM revealed that these sheets, apparently in contact with the cytosol, contained both P150 and P90, as well as dsRNA, and thus could be two‐dimensional arrays of functional viral replicases. Labelling of dsRNA after streptolysin‐O permeabilization showed that replication of viral genome takes place on the cytoplasmic side of CPVs. When present, C accumulated around CPVs. Mitochondrial protein P32 was detected within modified CPVs, the first demonstration of involvement of this protein, which interacts with C, with RCs.

Competition of bacteriophage polypeptides with native replicase proteins for binding to the DNA sliding clamp reveals a novel mechanism for DNA replication arrest in Staphylococcus aureus

Bacteriophages have evolved specific mechanisms that redirect bacterial metabolic pathways to the bacteriophage reproduction cycle. In this study, we characterized the bactericidal mechanism of two polypeptides from bacteriophages Twort and G1 that target the DNA sliding clamp of Staphylococcus aureus. The DNA sliding clamp, which tethers DNA polymerase to its template and thereby confers processivity upon the enzyme, was found to be essential for the viability of S. aureus. Expression of polypeptides TwortORF168 and G1ORF240 in S. aureus selectively inhibited DNA replication which in turn resulted in cell death. Both polypeptides specifically inhibited the S. aureus DNA replicase that was reconstituted in vitro but not the corresponding replicase of Streptococcus pyogenes. We demonstrated that inhibition of DNA synthesis is multifaceted and occurs via binding the DNA sliding clamp: TwortORF168 and G1ORF240 bound tightly to the DNA sliding clamp and prevented both its loading onto DNA and its interaction with DNA polymerase C. These results elucidate the impact of bacteriophage polypeptide expression upon DNA replication in the growing cell.

Genome Packaging by Spherical Plant RNA Viruses

The majority of positive-strand RNA viruses of plants replicate and selectively encapsidate their progeny genomes into stable virions in cytoplasmic compartments of the cell where the opportunity to copackage cellular RNA also exists. Remarkably, highly purified infectious virions contain almost exclusively viral RNA, suggesting that mechanisms exist to regulate preferential packaging of viral genomes. The general principle that governs RNA packaging is an interaction between the structural CP and a specific RNA signal. Mechanisms that enhance selective packaging of viral genomes and formation of infectious virions may involve factors other than CP and nucleic acid sequences. The possible involvement of replicase proteins is an example. Our knowledge concerning genome packaging among spherical plant RNA viruses is still maturing. The main focus of this review is to discuss factors that have limited progress and to evaluate recent technical breakthroughs likely to help unravel the mechanism of RNA packaging among viruses of agronomic importance. A key breakthrough is the development of in vivo systems and comparisons with results obtained in vitro.

C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage

Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5′ end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5′ end of the C gene and the 3′ end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles.

Complete Genome Sequence of the Broad Host Range Single-Stranded RNA Phage PRR1 Places It in the Levivirus Genus with Characteristics Shared with Alloleviviruses

Single-stranded RNA (ssRNA) bacteriophages of the family Leviviridae infect gram-negative bacteria. They are restricted to a single host genus. Phage PRR1 is an exception, having a broad host range due to the promiscuity of the receptor encoded by the IncP plasmid. Here we report the complete genome sequence of PRR1. Three proteins homologous with those of other ssRNA phages, i.e., maturation, coat, and replicase proteins, were identified. A fourth protein has a lysis function. Comparison of PRR1 with other members of the Leviviridae family places PRR1 in the genus Levivirus with some characteristics more similar to those of members of the genus Allolevivirus.

The Tobacco mosaic virus Replicase Protein Disrupts the Localization and Function of Interacting Aux/IAA Proteins

Previously, we identified a correlation between the interaction of the Tobacco mosaic virus (TMV) 126/183-kDa replicase with the auxin response regulator indole acetic acid (IAA)26/PAP1 and the development of disease symptoms. In this study, the TMV replicase protein is shown to colocalize with IAA26 in the cytoplasm and prevent its accumulation within the nucleus. Furthermore, two additional auxin (Aux)/IAA family members, IAA27 and IAA18, were found to interact with the TMV replicase and displayed alterations in their cellular localization or accumulation that corresponded with their ability to interact with the TMV replicase. In contrast, the localization and accumulation of noninteracting Aux/IAA proteins were unaffected by the presence of the viral replicase. To investigate the effects of the replicase interaction on Aux/IAA function, transgenic plants expressing a proteolysis-resistant IAA26-P108L-green fluorescent protein (GFP) protein were created. Transgenic plants accumulating IAA26-P108L-GFP displayed an abnormal developmental phenotype that included severe stunting and leaf epinasty. However, TMV infection blocked the nuclear localization of IAA26-P108L-GFP and attenuated the developmental phenotype displayed by the transgenic plants. Combined, these findings suggest that TMV-induced disease symptoms can be attributed, in part, to the ability of the viral replicase protein to disrupt the localization and subsequent function of interacting Aux/IAA proteins.

Plasmid Diversity in Neisseriae

Horizontal gene transfer constitutes an important force in prokaryotic genome evolution, and it is well-known that plasmids are vehicles for DNA transfer. Chromosomal DNA is frequently exchanged between pathogenic and commensal neisseriae, but relatively little is known about plasmid diversity and prevalence among these nasopharyngeal inhabitants. We investigated the plasmid contents of 18 Neisseria lactamica isolates and 20 nasopharyngeal Neisseria meningitidis isolates. Of 18 N. lactamica strains, 9 harbored one or more plasmids, whereas only one N. meningitidis isolate contained a plasmid. Twelve plasmids were completely sequenced, while five plasmid sequences from the public databases were also included in the analyses. On the basis of nucleic acid sequences, mobilization, and replicase protein alignments, we distinguish six different plasmid groups (I to VI). Three plasmids from N. lactamica appeared to be highly similar on the nucleotide level to the meningococcal plasmids pJS-A (>99%) and pJS-B (>75%). The genetic organizations of two plasmids show a striking resemblance with that of the recently identified meningococcal disease-associated (MDA) phage, while four putative proteins encoded by these plasmids show 25% to 39% protein identity to those encoded by the MDA phage. The putative promoter of the gene encoding the replicase on these plasmids contains a polycytidine tract, suggesting that replication is subjected to phase variation. In conclusion, extensive plasmid diversity is encountered among commensal neisseriae. Members of three plasmid groups are found in both pathogenic and commensal neisseriae, indicating plasmid exchange between these species. Resemblance between plasmids and MDA phage may be indicative of dissemination of phage-related sequences among pathogenic and commensal neisseriae.

Ribosomal Protein S6 Associates with Alphavirus Nonstructural Protein 2 and Mediates Expression from Alphavirus Messages

Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

The Crystal Structure of ORF-9b, a Lipid Binding Protein from the SARS Coronavirus

SummaryTo achieve the greatest output from their limited genomes, viruses frequently make use of alternative open reading frames, in which translation is initiated from a start codon within an existing gene and, being out of frame, gives rise to a distinct protein product. These alternative protein products are, as yet, poorly characterized structurally. Here we report the crystal structure of ORF-9b, an alternative open reading frame within the nucleocapsid (N) gene from the SARS coronavirus. The protein has a novel fold, a dimeric tent-like β structure with an amphipathic surface, and a central hydrophobic cavity that binds lipid molecules. This cavity is likely to be involved in membrane attachment and, in mammalian cells, ORF-9b associates with intracellular vesicles, consistent with a role in the assembly of the virion. Analysis of ORF-9b and other overlapping genes suggests that they provide snapshots of the early evolution of novel protein folds.

Nuclear localization of flavivirus RNA synthesis in infected cells.

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.

Phosphatidylserine is involved in gene expression from Sindbis virus subgenomic promoter

Sindbis virus replication is mediated by an RNA replicase translated from viral RNA genome. The replicase synthesizes progeny genomic RNA and shorter RNA (subgenomic RNA) carrying viral structural genes in association with cytoplasmic membranes. Here we examined the involvement of a membrane lipid, phosphatidylserine (PS), in Sindbis virus gene expression using Chinese hamster ovary cell mutants. When the mutant cells were transfected with viral replicon RNA, in which the structural genes downstream of the subgenomic promoter were replaced by a reporter gene, reporter expression was inhibited under PS-deficient conditions. In contrast, reporter expression from an SV40 promoter-driven construct was normal under similar conditions. Furthermore, expression of a viral replicase protein from the genomic RNA and accumulation of the subgenomic RNA were not inhibited by PS deficiency. These findings indicate that reduced cellular PS level impairs a posttranscriptional event of Sindbis virus subgenomic promoter-driven gene expression.

Analysis of Rubella Virus Capsid Protein-Mediated Enhancement of Replicon Replication and Mutant Rescue

The rubella virus capsid protein (C) has been shown to complement a lethal deletion (termed deltaNotI) in P150 replicase protein. To investigate this phenomenon, we generated two lines of Vero cells that stably expressed either C (C-Vero cells) or C lacking the eight N-terminal residues (Cdelta8-Vero cells), a construct previously shown to be unable to complement DeltaNotI. In C-Vero cells but not Vero or Cdelta8-Vero cells, replication of a wild-type (wt) replicon expressing the green fluorescent protein (GFP) reporter gene (RUBrep/GFP) was enhanced, and replication of a replicon with deltaNotI (RUBrep/GFP-deltaNotI) was rescued. Surprisingly, replicons with deleterious mutations in the 5' and 3' cis-acting elements were also rescued in C-Vero cells. Interestingly, the Cdelta8 construct localized to the nucleus while the C construct localized in the cytoplasm, explaining the lack of enhancement and rescue in Cdelta8-Vero cells since rubella virus replication occurs in the cytoplasm. Enhancement and rescue in C-Vero cells were at a basic step in the replication cycle, resulting in a substantial increase in the accumulation of replicon-specific RNAs. There was no difference in translation of the nonstructural proteins in C-Vero and Vero cells transfected with the wt and mutant replicons, demonstrating that enhancement and rescue were not due to an increase in the efficiency of translation of the transfected replicon transcripts. In replicon-transfected C-Vero cells, C and the P150 replicase protein associated by coimmunoprecipitation, suggesting that C might play a role in RNA replication, which could explain the enhancement and rescue phenomena. A unifying model that accounts for enhancement of wt replicon replication and rescue of diverse mutations by the rubella virus C protein is proposed.

Mutagenesis analysis of the nsp4 main proteinase reveals determinants of arterivirus replicase polyprotein autoprocessing.

Nonstructural protein 4 (nsp4; 204 amino acids) is the chymotrypsin-like serine main proteinase of the arterivirus Equine arteritis virus (order Nidovirales), which controls the maturation of the replicase complex. nsp4 includes a unique C-terminal domain (CTD) connected to the catalytic two-beta-barrel structure by the poorly conserved residues 155 and 156. This dipeptide might be part of a hinge region (HR) that facilitates interdomain movements and thereby regulates (in time and space) autoprocessing of replicase polyproteins pp1a and pp1ab at eight sites that are conserved in arteriviruses. To test this hypothesis, we characterized nsp4 proteinase mutants carrying either point mutations in the putative HR domain or a large deletion in the CTD. When tested in a reverse genetics system, three groups of mutants were recognized (wild-type-like, debilitated, and dead), which was in line with the expected impact of mutations on HR flexibility. When tested in a transient expression system, the effects of the mutations on the production and turnover of replicase proteins varied widely. They were cleavage product specific and revealed a pronounced modulating effect of moieties derived from the nsp1-3 region of pp1a. Mutations that were lethal affected the efficiency of polyprotein autoprocessing most strongly. These mutants may be impaired in the accumulation of nsp5-7 and/or suffer from delayed or otherwise perturbed processing at the nsp5/6 and nsp6/7 junctions. On average, the production of nsp7-8 seems to be the most resistant to debilitating nsp4 mutations. Our results further prove that the CTD is essential for a vital nsp4 property other than catalysis.

Cytological analysis of Saccharomyces cerevisiae cells supporting cymbidium ringspot virus defective interfering RNA replication

The replicase proteins p33 and p92 of Cymbidium ringspot virus (CymRSV) were found to support the replication of defective interfering (DI) RNA in Saccharomyces cerevisiae cells. Two yeast strains were used, differing in the biogenesis of peroxisomes, the organelles supplying the membranous vesicular environment in which CymRSV RNA replication takes place in infected plant cells. Double-labelled immunofluorescence showed that both p33 and p92 replicase proteins localized to peroxisomes, independently of one another and of the presence of the replication template. It is suggested that these proteins are sorted initially from the cytosol to the endoplasmic reticulum and then to peroxisomes. However, only the expression of p33, but not p92, increased the number of peroxisomes and induced membrane proliferation. DI RNA replication occurred in yeast cells, as demonstrated by the presence of monomers and dimers of positive and negative polarities. Labelling with BrUTP showed that peroxisomes were the sites of nascent viral synthesis, whereas in situ hybridization indicated that DI RNA progeny were diffused throughout the cytoplasm. DI RNA replication also took place in yeast cells devoid of peroxisomes. It is suggested that replication in these cells was targeted to the endoplasmic reticulum.