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The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase) domain of the vesicular stomatitis virus (VSV) L protein possesses a dual-functional "priming-capping loop" that governs terminal de novo initiation for leader RNA synthesis and capping of monocistronic mRNAs during the unique stop-start transcription cycle. Here, we investigated the roles of basic amino acid residues on a helix structure directly connected to the priming-capping loop in viral RNA synthesis and identified single point mutations that cause previously unreported defective phenotypes at different steps of stop-start transcription. Mutations of residue R1183 (R1183A and R1183K) dramatically reduced the leader RNA synthesis activity by hampering early elongation, but not terminal de novo initiation or productive elongation, suggesting that the mutations negatively affect escape from the leader promoter. On the other hand, mutations of residue R1178 (R1178A and R1178K) decreased the efficiency of polyadenylation-coupled termination of mRNA synthesis at the gene junctions, but not termination of leader RNA synthesis at the leader-to-N-gene junction, resulting in the generation of larger amounts of aberrant polycistronic mRNAs. In contrast, both the R1183 and R1178 residues are not essential for cap-forming activities. The R1183K mutation was lethal to VSV, whereas the R1178K mutation attenuated VSV and triggered the production of the polycistronic mRNAs in infected cells. These observations suggest that the PRNTase domain plays multiple roles in conducting accurate stop-start transcription beyond its known role in pre-mRNA capping.

The L proteins of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus (RABV), possess an unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase) domain with a loop structure protruding into an active site cavity of the RNA-dependent RNA polymerase (RdRp) domain. Here, using complementary VSV and RABV systems, we show that the loop governs RNA synthesis and capping during the dynamic stop-start transcription cycle. A conserved tryptophan residue in the loop was identified as critical for terminal de novo initiation from the genomic promoter to synthesize the leader RNA and virus replication in host cells, but not for internal de novo initiation or elongation from the gene-start sequence for mRNA synthesis or pre-mRNA capping. The co-factor P protein was found to be essential for both terminal and internal initiation. A conserved TxΨ motif adjacent the tryptophan residue in the loop was required for pre-mRNA capping in the step of the covalent enzyme-pRNA intermediate formation, but not for either terminal or internal transcription initiation. These results provide insights into the regulation of stop-start transcription by the interplay between the RdRp active site and the dual-functional priming-capping loop of the PRNTase domain in non-segmented negative strand RNA viruses.

Vesicular stomatitis virus (VSV) is a member of the order Mononegavirales, which consists of viruses with genomes of nonsegmented negative-sense (NNS) RNA. Many insights into the molecular biology of NNS viruses were first made in VSV, which is often studied as a prototype for members of this order. Like those of other NNS viruses, the VSV RNA polymerase consists of a complex of the large protein (L) and the phosphoprotein (P). Recent discoveries have produced a model in which the N-terminal disordered segment of P (P(NTD)) coordinates the C-terminal accessory domains to produce a “compacted” L conformation. Despite this advance, the role of the three phosphorylation sites in P(NTD) has remained unknown. Using nuclear magnetic resonance spectroscopy to analyze the interactions between P(NTD) and the L protein C-terminal domain (L(CTD)), we demonstrated our ability to test sensitively for changes in the interface between the two proteins. This method showed that the binding site for P(NTD) on L(CTD) is longer than was previously appreciated. We demonstrated that phosphorylation of P(NTD) modulates its interaction with L(CTD), and we used a minigenome reporter system to validate the functional significance of the P(NTD)-L(CTD) interaction. Using an electron microscopy approach, we showed that L bound to phosphorylated P(NTD) displays increased conformational heterogeneity in solution. Taken as a whole, our studies suggest a model in which phosphorylation of P(NTD) modulates its cofactor and conformational regulatory activities with L. IMPORTANCE Polymerase-cofactor interactions such as those addressed in this study are absolute requirements for mononegavirus RNA synthesis. Although cofactor phosphorylation is present in most of these interactions, its effect, if any, on this protein-protein interaction had not been addressed. Our study is the first to address the effects of phosphorylation on P during its interactions with L in residue-by-residue detail. Since phosphorylation is the biologically relevant state of the cofactor, our demonstration of its effects on L conformation suggests that the structural picture of L during infection might be more complex than previously appreciated.

Vesicular stomatitis virus (VSV) is an archetypical member of Mononegavirales, viruses with a genome of negative-sense single-stranded RNA (−ssRNA). Like other viruses of this order, VSV encodes a unique polymerase, a complex of viral L (large, the enzymatic component) protein and P (phosphoprotein, a cofactor component). The L protein has a modular layout consisting of a ring-shaped core trailed by three accessory domains and requires an N-terminal segment of P (P N-terminal disordered [P(NTD)]) to perform polymerase activity. To date, a binding site for P on L had not been described. In this report, we show that the connector domain of the L protein, which previously had no assigned function, binds a component of P(NTD). We further show that this interaction is a positive regulator of viral RNA synthesis, and that the interfaces mediating it are conserved in other members of Mononegavirales. Finally, we show that the connector-P interaction fits well into the existing structural information of VSV L. IMPORTANCE This study represents the first functional assignment of the connector domain of a Mononegavirales L protein. Furthermore, this study localizes P polymerase cofactor activity to specific amino acids. The functional necessity of this interaction, combined with the uniqueness of L and P proteins to the order Mononegavirales, makes disruption of the P-connector site a potential target for developing antivirals against other negative-strand RNA viruses. Furthermore, the connector domain as an acceptor site for the P protein represents a new understanding of Mononegavirales L protein biology.

Vesicular stomatitis virus (VSV) represents a promising platform for developing oncolytic viruses, as well as vaccines against significant human pathogens. To safely control VSV infection in humans, small-molecule drugs that selectively inhibit VSV infection may be needed. Here, using a cell-based high-throughput screening assay followed by an in vitro transcription assay, compounds with a 7-hydroxy-6-methyl-3,4-dihydroquinolin-2(1H)-one structure and an aromatic group at position 4 (named vesiculopolins, VPIs) were identified as VSV RNA polymerase inhibitors. The most effective compound, VPI A, inhibited VSV-induced cytopathic effects and in vitro mRNA synthesis with micromolar to submicromolar 50% inhibitory concentrations. VPI A was found to inhibit terminal de novo initiation rather than elongation for leader RNA synthesis, but not mRNA capping, with the VSV L protein, suggesting that VPI A is targeted to the polymerase domain in the L protein. VPI A inhibited transcription of Chandipura virus, but not of human parainfluenza virus 3, suggesting that it specifically acts on vesiculoviral L proteins. These results suggest that VPIs may serve not only as molecular probes to elucidate the mechanisms of transcription of vesiculoviruses, but also as lead compounds to develop antiviral drugs against vesiculoviruses and other related rhabdoviruses.

The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase; block V) domain in RNA polymerase L proteins of non-segmented negative strand (NNS) RNA viruses (e.g. rabies, measles, Ebola) contains five collinear sequence elements, Rx(3)Wx(3-8)ΦxGxζx(P/A) (motif A; Φ, hydrophobic; ζ, hydrophilic), (Y/W)ΦGSxT (motif B), W (motif C), HR (motif D) and ζxxΦx(F/Y)QxxΦ (motif E). We performed site-directed mutagenesis of the L protein of vesicular stomatitis virus (VSV, a prototypic NNS RNA virus) to examine participation of these motifs in mRNA capping. Similar to the catalytic residues in motif D, G1100 in motif A, T1157 in motif B, W1188 in motif C, and F1269 and Q1270 in motif E were found to be essential or important for the PRNTase activity in the step of the covalent L-pRNA intermediate formation, but not for the GTPase activity that generates GDP (pRNA acceptor). Cap defective mutations in these residues induced termination of mRNA synthesis at position +40 followed by aberrant stop-start transcription, and abolished virus gene expression in host cells. These results suggest that the conserved motifs constitute the active site of the PRNTase domain and the L-pRNA intermediate formation followed by the cap formation is essential for successful synthesis of full-length mRNAs.

The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5′-phospho-RNA (pRNA) from 5′-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5′-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m 7 G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m 7 GpppA (cap 0), respectively. Furthermore, either the 2′- or 3′-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5′-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential for the substrate activity. The findings presented here are useful not only for understanding the mechanism of the substrate recognition with PRNTase but also for designing antiviral agents targeting this enzyme.

Rabies virus (RABV) is a causative agent of a fatal neurological disease in humans and animals. The large (L) protein of RABV is a multifunctional RNA-dependent RNA polymerase, which is one of the most attractive targets for developing antiviral agents. A remarkable homology of the RABV L protein to a counterpart in vesicular stomatitis virus, a well-characterized rhabdovirus, suggests that it catalyzes mRNA processing reactions, such as 5′-capping, cap methylation, and 3′-polyadenylation, in addition to RNA synthesis. Recent breakthroughs in developing in vitro RNA synthesis and capping systems with a recombinant form of the RABV L protein have led to significant progress in our understanding of the molecular mechanisms of RABV RNA biogenesis. This review summarizes functions of RABV replication proteins in transcription and replication, and highlights new insights into roles of an unconventional mRNA capping enzyme, namely GDP polyribonucleotidyltransferase, domain of the RABV L protein in mRNA capping and transcription initiation.

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5′-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5′-monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and Borna disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents.

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV) elicits GTPase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities to produce a 5′-cap core structure, guanosine(5′)triphospho(5′)adenosine (GpppA), on viral mRNAs. Here, we report that the L protein produces an unusual cap structure, guanosine(5′)tetraphospho(5′)adenosine (GppppA), that is formed by the transfer of the 5′-monophosphorylated viral mRNA start sequence to GTP by the PRNTase activity before the removal of the γ-phosphate from GTP by GTPase. Interestingly, GppppA-capped and polyadenylated full-length mRNAs were also found to be synthesized by an in vitro transcription system with the native VSV RNP.

The nucleocapsid (N) protein of nonsegmented negative-strand (NNS) RNA viruses, when expressed in eukaryotic cells, aggregates and forms nucleocapsid-like complexes with cellular RNAs. The phosphoprotein (P) has been shown to prevent such aggregation by forming a soluble complex with the N protein free from cellular RNAs (designated N 0 ). The N 0 -P complex presumably mediates specific encapsidation of the viral genome RNA. The precise mechanism by which the P protein carries out this function remains unclear. Here, by using a series of deleted and truncated mutant forms of the P protein of vesicular stomatitis virus (VSV), Indiana serotype, we present evidence that the N-terminal 11 to 30 amino acids (aa) of the P protein are essential in keeping the N protein soluble. Furthermore, glutathione S-transferase fused to the N-terminal 40 aa by itself is able to form the N 0 -P complex. Interestingly, the N-terminal 40-aa stretch failed to interact with the viral genome N-RNA template whereas the C-terminal 72 aa of the P protein interacted specifically with the latter. With an in vivo VSV minigenome transcription system, we further show that a deletion mutant form of P( P1-10) lacking the N-terminal 10 aa which is capable of forming the N 0 -P complex was unable to support VSV minigenome transcription, although it efficiently supported transcription in vitro in a transcriptionreconstitution reaction when used as purified protein. However, the same mutant protein complemented minigenome transcription when expressed together with a transcription-defective P deletion mutant protein containing N-terminal aa 1 to 210 (PIIIII). Since the minigenome RNA needs to be encapsidated before transcription ensues, it seems that the entire N-terminal 210 aa are required for efficient genome RNA encapsidation. Taking these results together, we conclude that the N-terminal 11 to 30 aa are required for N 0 -P complex formation but the N-terminal 210 aa are required for genome RNA encapsidation. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus belonging to the family Rhabdoviridae. Its genome RNA is enwrapped in a helical form with the nucleocapsid (N) protein and packaged within the virion together with the RNAdependent RNA polymerase large protein (L) and the phosphoprotein (P). During cell infection, the associated RNA polymerase transcribes the genome RNA into five mRNAs that are subsequently translated into the glycoprotein (G), the matrix (M) protein, and the N, P, and L proteins. The N protein is an RNA-binding protein since it forms a high-mo

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

Non-segmented negative strand RNA viruses belonging to the Mononegavirales order possess RNA-dependent RNA polymerase L proteins within viral particles. The L protein is a multifunctional enzyme catalyzing viral RNA synthesis and processing (i.e., mRNA capping, cap methylation, and polyadenylation). Using vesicular stomatitis virus (VSV) as a prototypic model virus, we have shown that the L protein catalyzes the unconventional mRNA capping reaction, which is strikingly different from the eukaryotic reaction. Furthermore, co-transcriptional pre-mRNA capping with the VSV L protein was found to be required for accurate stop?start transcription to synthesize full-length mRNAs in vitro and virus propagation in host cells. This article provides a review of historical and present studies leading to the elucidation of the molecular mechanism of VSV mRNA capping.

Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae , Paramyxoviridae , and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. IMPORTANCE Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the design of new therapeutics against negative-strand RNA viruses.

The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase complex and plays a central role in viral transcription and replication. Using both the yeast two-hybrid system and coimmunoprecipitation assays, we confirmed the self-association of the P protein of Indiana serotype (Pind) and heterotypic interaction between Pind and the P protein of New Jersey serotype (Pnj). Furthermore, by using various truncation and deletion mutants of Pind, the self-association domain of the Pind protein was mapped to amino acids 161 to 210 within the hinge region. The self-association domain of Pind protein is not required for its binding to nucleocapsid and large proteins. We further demonstrated that the self-association domain of Pind protein is essential for VSV transcription in a minireplicon system and that a synthetic peptide spanning amino acids 191 to 210 in the self-association domain of Pind protein strongly inhibited the transcription of the VSV genome in vitro in a dose-dependent manner. These results indicated that the self-association domain of Pind protein plays a critical role in VSV transcription.

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in young children (12). This pathogen induces a characteristic histopathological lesion referred to as an attaching/effacing lesion, which is defined by the intimate attachment of bacteria to the epithelial surface and the effacement of host cell microvilli (25). Factors responsible for the formation of attaching/effacing lesions are encoded by a 35-kbp locus designated LEE (24), which encodes the following components: (i) the type III secretion (TTS) apparatus (15), (ii) E. coli-secreted proteins (EspA, EspB, and EspD) and several effectors, including Tir, (iii) type III-specific chaperones, and (iv) regulators (9). LEE is highly conserved among enterohemorrhagic E. coli, rabbit EPEC, and Citrobacter rodentium. TTS systems are found in many other gram-negative bacterial species, and they constitute a strategy for the delivery of effectors into host cells, a process which in turn disrupts host physiology and thereby contributes to the disease process (14). Unlike the sec-dependent secreted proteins, the effector does not have a typical signal sequence at its N terminus, and it is directly delivered via the TTS system without N-terminal processing in the bacterial periplasm. Analysis by transmission electron microscopy (TEM) revealed that the supermolecular structures of the TTS apparatus in EPEC (8, 31), Salmonella enterica serovar Typhimurium (18, 20-22), and Shigella flexneri (2, 32) are highly conserved with respect to each other and that their shapes are similar to that of the flagellar basal body complex. The core TTS apparatus, which is referred to as a needle complex (NC), is composed of two distinct portions: (i) a needle structure that extrudes from the bacterial outer membrane and functions as an injector of effectors into host cells and (ii) a cylindrical basal body that is similar to the flagellar basal body and functions as a channel that spans the outer and inner membranes of the bacterium as well as the periplasmic region. The basal body is further divided into three major portions: (i) an outer ring, (ii) an inner ring, and (iii) a presumed central rod (22) that can connect the outer and inner rings to build a channel. Although the supermolecular structure of the EPEC NC is similar in shape to that of the NCs of both Salmonella and Shigella, a unique extracellular appendage in the needle structure of the EPEC NC was discovered previously (31). The EPEC needle structure is composed of a thin needle (neck portion) and an expandable sheath-like structure (31). EPEC EscF is predicted to be an 8-kDa protein and may polymerize to form the thin needle in the needle structure. EscF shows homology to PrgI (24% identity) in the Salmonella pathogenicity island 1, Shigella MxiH (25% identity), and Yersinia enterocolitica YscF (20% identity), which are major components of the thin and stiff needle structures of the Salmonella, Shigella, and Yersinia NCs, respectively (21, 26, 32). EscF is required for NC formation and the secretion of the Esp proteins (31); this observation agrees with findings of secretion-defective phenotypes of Salmonella prgI and Shigella mxiH mutant strains (21, 32). EspA is predicted to be a 20-kDa protein and is secreted via the EPEC TTS system (17). We previously demonstrated that EspA is directly associated with the tip of the putative EscF needle and polymerizes into an expandable filamentous structure, referred to as the sheath-like structure (31). The EPEC needle structure, including the EspA sheath-like structure, extended to a length of more than 600 nm and was 10 times longer than the Shigella needle (45 nm) (31). Three-dimensional structure analysis of the EspA filament revealed that the structure consists of a helical tube with a diameter of 12 nm enclosing a central channel with a diameter of 2.5 nm (7). Recent TEM analysis of purified EspA revealed that EspA alone is able to polymerize into irregular short filaments (33). On the other hand, the widths of the outer and inner membrane rings in the basal body of the EPEC NC are estimated to be 17 and 18 nm, respectively, and the height of the basal body is 31 nm (31). However, the molecular composition of the EPEC NC basal body remains unclear. From the results of a membrane fractionation study (13), yeast two-hybrid analysis (5), whole mutation analyses of LEE (10), and computer modeling predictions, several proteins are thought to be components for the EPEC TTS apparatus. The outer ring protein of the basal body has been suggested to be EscC, according to a membrane fractionation study (13), and this hypothesis received further support from a demonstration of its similarity to the YscC protein (31.1% identity) in the Yersinia TTS system, which forms a ring-shaped oligomeric complex with a diameter of 20 nm in the outer membrane (4, 19). EscC belongs to a member of the secretin superfamily, which participates in the delivery of large molecules through the outer membrane by the formation of a channel. EscC is predicted to be synthesized as a 56-kDa preprotein possessing a signal sequence that is cleavable with type I signal peptidase after amino acid residue 19. Then, the EscC preprotein most likely undergoes signal peptide cleavage after its export across the inner membrane by a sec-dependent secretion pathway, thus generating a mature 54-kDa protein. Indeed, a mature form of Salmonella InvG, an EscC orthologue, starts at amino acid residue 25, suggesting that its preprotein is cleaved by type I signal peptidase in order to reach maturation (20). Recently, the association of EscC with EscD was suggested by use of a yeast two-hybrid system (5). EscD is predicted to be a 45-kDa protein and shows amino acid sequence similarity to Yersinia YscD, a bacterial inner membrane protein (27). In Salmonella, PrgK and PrgH together form the inner membrane ring of the TTS apparatus (18). Although a PrgH orthologue has not been found in EPEC, a PrgK orthologue, EscJ, is thought to form a portion of the inner ring. Molecular modeling using the 1.8-A crystal structure of EscJ suggests that EscJ oligomerizes to form a large 24-subunit ring structure (34). Furthermore, a membrane fraction study indicates that EscJ localizes mainly to the inner membrane (34). These findings suggest that the inner ring of the EPEC TTS apparatus contains the EscJ multimeric complex. EscJ is predicted to be produced as a 21-kDa preprotein with a lipoprotein signal sequence that can be cleaved after amino acid residue 19 by type II signal peptidase. After sec-dependent translocation, the EscJ preprotein may undergo lipid modification and signal peptide cleavage to form a mature 19-kDa lipoprotein, which may be anchored to the inner membrane via its N-terminal lipid moiety (34). Although several Esc proteins, namely, EscR, EscS, EscT, and EscU, are predicted to be components of the basal body, their precise functions and localizations in the EPEC TTS apparatus remain unclear. In this study, we analyzed the structures of NCs isolated from EPEC by using centrifugation techniques, including CsCl density gradient centrifugation. Furthermore, protein components of NCs were examined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), Western blotting, and immunoelectron microscopy. In addition, the protein-protein interactions required for assembly of the EPEC TTS apparatus were analyzed using a glutathione S-transferase (GST) pulldown assay, and confirmation of the putative components of the TTS apparatus needed for NC assembly, as well as those needed for the secretion of Esp proteins and the Tir effector, was carried out by the construction of deletion mutants.

The Sendai virus (SeV) RNA-dependent RNA polymerase complex, which consists of L and P proteins, participates in the synthesis of viral mRNAs that possess a methylated cap structure. To identify the SeV protein(s) involved in mRNA cap methylation, we developed an in vitro assay system to detect mRNA (guanine-7-)methyltransferase (G-7-MTase) activity. Viral ribonucleoprotein complexes and purified recombinant L protein but not P protein exhibited G-7-MTase activity. On the other hand, mRNA synthesis in a reconstituted transcription system using purified N-RNA (N protein-genomic RNA) complex as a template required both the L and P proteins. The enzymatic properties of SeV G-7-MTase were different from those of cellular G-7-MTase. In particular, unlike cellular G-7-MTase, the SeV enzyme preferentially methylated capped RNA containing the viral mRNA 5'-end sequences (GpppApGpG-). The C-terminal part (amino acid residues 1,756-2,228) of the L protein catalyzed cap methylation, whereas the N-terminal half (residues 1-1,120) containing putative RNA polymerase subdomains did not. This is to our knowledge the first direct biochemical evidence that supports the idea that mononegavirus L protein catalyzes cap methylation as well as RNA synthesis.

The immature core protein (p23, residues 1 to 191) of hepatitis C virus undergoes posttranslational modifications including intramembranous proteolysis within its C-terminal signal sequence by signal peptide peptidase to generate the mature form (p21). In this study, we analyzed the cleavage site and other amino acid modifications that occur on the core protein. To produce the posttranslationally modified core protein, we used a baculovirus-insect cell expression model system. As previously reported, p23 is processed to form p21 in insect as well as in mammalian cells. p21 was found to be associated with the cytoplasmic membrane, and its significant portion behaved as an integral membrane protein. The protein was purified from the membrane by a simple and unique procedure on the basis of its membrane-binding properties and solubility in detergents. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of purified p21 showed that the average molecular mass ( m/z 19,307) of its single-charged ion differs by m/z 1,457 from that calculated for p23. To determine the posttranslational modifications, tryptic p21 peptides were analyzed by MALDI-TOF MS. We found three peptides that did not match the theoretically derived peptides of p23. Analysis of these peptides by MALDI-TOF tandem MS revealed that they correspond to N-terminal peptides (residues 2 to 9 and 2 to 10) starting with α- N -acetylserine and C-terminal peptide (residues 150 to 177) ending with phenylalanine. These results suggest that the mature core protein (molecular mass of 19,306 Da) includes residues 2 to 177 and that its N terminus is blocked with an acetyl group.

Cellular tubulin has been shown to activate in vitro transcription with Sendai virus (SeV) particles. In this study, the molecular basis for the transcriptional activation by tubulin was investigated. We showed that tubulin dissociates viral matrix (M) protein, which acts as a negative regulator for transcription, from viral ribonucleoprotein (RNP) consisting of L, P, N proteins, and the genome RNA. Both alpha and beta subunits of human tubulin, which were expressed as GST fusion proteins, were found to stimulate viral mRNA synthesis similar to native alpha/beta-heterodimer tubulin. Pull-down assay using GST-tubulin subunits demonstrated that M protein is released from the RNP as a complex with each tubulin subunit. In vitro-binding analyses revealed that M protein directly interacts with tubulin as well as microtubules. These findings suggest that interaction of M protein with tubulin may have an important role in the regulation of SeV transcription.

The multifunctional RNA-dependent RNA polymerase L protein of vesicular stomatitis virus catalyzes unconventional pre-mRNA capping via the covalent enzyme-pRNA intermediate formation, which requires the histidine–arginine (HR) motif in the polyribonucleotidyltransferase domain. Here, the effects of cap-defective mutations in the HR motif on transcription were analyzed using an in vitro reconstituted transcription system. The wild-type L protein synthesized the leader RNA from the 3′-end of the genome followed by 5′-capped and 3′-polyadenylated mRNAs from internal genes by a stop–start transcription mechanism. Cap-defective mutants efficiently produced the leader RNA, but displayed aberrant stop–start transcription using cryptic termination and initiation signals within the first gene, resulting in sequential generation of ∼40-nucleotide transcripts with 5′-ATP from a correct mRNA-start site followed by a 28-nucleotide transcript and long 3′-polyadenylated transcript initiated with non-canonical GTP from atypical start sites. Frequent transcription termination and re-initiation within the first gene significantly attenuated the production of downstream mRNAs. Consistent with the inability of these mutants in in vitro mRNA synthesis and capping, these mutations were lethal to virus replication in cultured cells. These findings indicate that viral mRNA capping is required for accurate stop–start transcription as well as mRNA stability and translation and, therefore, for virus replication in host cells.

Enteropathogenic Escherichia coli (EPEC) secretes several Esp proteins via the type III secretion system (secreton). EspA, EspB, and EspD are required for translocation of the effector proteins into host cells, in which EspB and EspD are thought to form a pore in the host membrane. Recent study has shown that EspA forms a filamentous structure that assembles as a physical bridge between bacteria and host cell surfaces, which then functions as a conduit for the translocation of bacterial effectors into host cells. To investigate the supermolecular structure of the type III secreton in EPEC, we partially purified it from the bacteria membrane and observed it via transmission electron microscopy. The EPEC type III secreton was composed of a basal body and a needle part and was similar to those of Salmonella and Shigella , except for a sheath-like structure at the tip of the needle. The length of sheath-like structures varied; it extended more than 600 nm and was 10 times longer than the Shigella needle part. The putative major needle component, EscF, was required for both secretion of Esp proteins and needle complex formation. Interestingly, elongation of the sheath-like structure was observed under constitutive expression of EspA but not of EscF. Furthermore, the transmission electron microscopy view with immunogold labeled anti-EspA antibodies clearly showed that EspA is a component of the sheath-like structure. This study revealed, to our knowledge for the first time, the supermolecular structure of the EPEC type III secreton and its direct association with the EspA-sheath-like structure.

The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV), a prototypic nonsegmented negative strand (NNS) RNA virus classified into the Rhabdoviridae family, has been used to investigate the fundamental molecular mechanisms of NNS RNA viral mRNA synthesis and processing. In vitro studies on mRNA cap formation with the VSV L protein eventually led to the discovery of the unconventional mRNA capping pathway catalyzed by the guanosine 5'-triphosphatase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities. The PRNTase activity is a novel enzymatic activity, which transfers 5'-monophosphorylated (p-) RNA from 5'-triphosphorylated (ppp-) RNA to GDP to form 5'-capped RNA (GpppRNA) in a viral mRNA-start sequence-dependent manner. This unconventional capping (pRNA transfer) reaction with PRNTase can be experimentally distinguished from the conventional capping (GMP transfer) reaction with eukaryotic GTP:RNA guanylyltransferase (GTase) on the basis of the following differences in their substrate specificity for the cap formation: PRNTase uses GDP and pppRNA, but not ppRNA, whereas GTase employs GTP, but not GDP, and ppRNA. The pRNA transfer reaction with PRNTase proceeds through a covalent enzyme-pRNA intermediate with a phosphoamide bond. Hence, to prove the PRNTase activity, it is necessary to demonstrate the following consecutive steps separately: (1) the enzyme forms a covalent enzyme-pRNA intermediate, and (2) the intermediate transfers pRNA to GDP. This article describes the methods for in vitro transcription and capping with the recombinant VSV L protein, which permit detailed characterization of its enzymatic reactions and mapping of active sites of its enzymatic domains. It is expected that these systems are adaptable to rhabdoviruses and, by extension, other NNS RNA viruses belonging to different families.

Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2 −/−) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1 −/− mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2−/− mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2 −/− mice and induced interferon-β. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2 −/− mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2 −/− mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2−/− mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2 −/− mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon., Author Summary In mammals, the first line of defense against virus infection is the interferon system. Viruses induce synthesis of interferon in the infected cells and its secretion to circulation. Interferon acts upon the as yet uninfected cells and protects them from oncoming infection by inducing the synthesis of hundreds of new proteins, many of which interfere with virus replication. Vesicular stomatitis virus (VSV), a virus similar to rabies virus, is very sensitive to interferon but it is not known which interferon-induced protein inhibits its replication. Here, we have identified a single interferon-induced protein as the protector of mice from death by VSV infection. Knocking out the gene encoding this protein, Ifit2, made mice very vulnerable to neuropathogenesis caused by VSV infection; a related protein, Ifit1, did not share this property. Moreover, Ifit2 failed to protect mice from another neurotropic virus, encephalomyocarditis virus, nor was it necessary for protecting organs other than brain from infection by VSV. Our observation that a single IFN-induced protein protects a specific organ from infection by a specific virus revealed an unexpected degree of specificity of the antiviral action of IFN.

mRNAs of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative strand (NNS) RNA viruses (e.g., rabies, measles, mumps, Ebola, and Borna disease viruses), possess the 5′-terminal cap structure identical to that of eukaryotic mRNAs, but the mechanism of mRNA cap formation is distinctly different from the latter. The elucidation of the unconventional capping of VSV mRNA remained elusive for three decades since the discovery of the cap structure in some viral and eukaryotic mRNAs in 1975. Only recently our biochemical studies revealed an unexpected strategy employed by vesiculoviruses (VSV and Chandipura virus, an emerging arbovirus) to generate the cap structure. This article summarizes the historical and current research that led to the discovery of the novel vesiculoviral mRNA capping reaction.

Chandipura virus (CHPV) is an emerging human pathogen associated with acute encephalitis and is related closely to vesicular stomatitis virus (VSV), a prototype rhabdovirus. Here, we demonstrate that the RNA polymerase L protein of CHPV exhibits a VSV-like RNA : GDP polyribonucleotidyltransferase (PRNTase) activity, which transfers the 5′-monophosphorylated (p-) viral mRNA start sequence to GDP to produce a capped RNA, and that the conserved HR motif in the CHPV L protein is essential for the PRNTase activity. Interestingly, the CHPV L protein was found to form two distinct SDS-resistant complexes with the CHPV mRNA and leader RNA start sequences; mutations in the HR motif significantly reduced the formation of the former complex (a putative covalent enzyme–pRNA intermediate in the PRNTase reaction), but not the latter complex. These results suggest that the rhabdoviral L proteins universally use the active-site HR motif for the PRNTase reaction at the step of the enzyme–pRNA intermediate formation.

In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors). Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin. In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain. This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparent M(r) of 46,000 (p46). From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK). Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46. Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation. On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated. The enzymatic activity per se of PGK did not seem to be required for its activity. West-Western blot analysis showed that PGK could directly interact with tubulin. These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex.

The Escherichia coli sensory kinase, ArcB, possesses a histidine-containing phosphotransfer (HPt) domain, which is implicated in the His-Asp multistep phosphorelay. We searched for a presumed phosphohistidine phosphatase, if present, which affects the function of the HPt domain through its dephosphorylation activity. Using in vivo screening, we first identified a gene that appeared to interfere with the His-Asp phosphorelay between the HPt domain and the receiver domain of OmpR, provided that the gene product was expressed through a multicopy plasmid. The cloned gene (named sixA) was found to encode a protein consisting of 161 amino acids, which has a noticeable sequence motif, an arginine-histidine-glycine (RHG) signature, at its N-terminus. Such an RHG signature, which presumably functions as a nucleophilic phosphoacceptor, was previously found in a set of divergent enzymes, including eukaryotic fructose-2,6-bisphosphatase, E. coli periplasmic phosphatase and E. coli glucose-1-phosphate phosphatase, and ubiquitous phosphoglycerate mutase. Otherwise, the entire amino acid sequences of none of these enzymes resembles that of SixA. It was demonstrated in vitro that the purified SixA protein exhibited the ability to release the phosphoryl group from the HPt domain of ArcB, but the mutant protein lacking the crucial histidine residue in the RHG signature did not. Evidence was also provided that a deletion mutation of sixA on the chromosome affected the in vivo phosphotransfer signalling. These results support the view that SixA is capable of functioning as a phosphohistidine phosphatase that may be implicated in the His-Asp phosphorelay through regulating the phosphorylation state of the HPt domain.

The structure of chloropeptin I, a gp120-CD4 binding inhibitor having a potent anti-HIV activity, was elucidated by 1H and 13C NMR experiments and chemical degradation. It is a peptide antibiotic consisting of six aryl amino acids residues and an alpha-oxo aryl acid some of which have chlorine atoms.

In Escherichia coli, expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity and other environmental stimuli. A two-component signal transduction system, mediated by EnvZ and OmpR, is crucially responsible for this osmotic regulation of the ompC and ompF genes. In this study, an E. coli gene was cloned, which interferes with expression of both the ompC and ompF genes at the level of transcription, provided that the cloned gene was introduced in E. coli cells by a multicopy plasmid. The gene product was identified as F107, which was previously characterized as a hypothetical protein in E. coli genome databases. F107 containing 107 amino acids appears to be highly hydrophobic, and has a sequence similarity to the eukaryotic type of cytochrome-c oxidase subunit III. The mechanism by which F107 inhibits transcription of ompC and ompF was examined extensively, mainly by using a set of envZ and ompR mutants. These results suggested that F107 interferes specifically with a function of the EnvZ osmosensory kinase. Possible mechanisms by which F107 affects the EnvZ function are discussed.

Hashimoto's encephalopathy (HE) is a rare autoimmune disease associated with Hashimoto's thyroiditis (HT). To identify the HE-related autoantigens, we developed a human brain proteome map using two-dimensional electrophoresis and applied it to the immuno-screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI-TOF-MASS analysis, immuno-positive spots of 48 kDa (pI 7.3-7.8) detected from HE patient sera were identified as a novel autoimmuno-antigen, alpha-enolase, harboring several modifications. Specific high reactivities against human alpha-enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut-off level. Although a few HT patients showed faint reactions to alpha-enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti-alpha-enolase antibody is useful for defining HE-related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies.