Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription.

Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.

Evidence for ADAR-induced hypermutation of the Drosophila sigma virus (Rhabdoviridae).

BACKGROUND: ADARs are RNA editing enzymes that target double stranded RNA and convert adenosine to inosine, which is read by translation machinery as if it were guanosine. Aside from their role in generating protein diversity in the central nervous system, ADARs have been implicated in the hypermutation of some RNA viruses, although why this hypermutation occurs is not well understood. RESULTS: Here we describe the hypermutation of adenosines to guanosines in the genome of the sigma virus--a negative sense RNA virus that infects Drosophila melanogaster. The clustering of these mutations and the context in which they occur indicates that they have been caused by ADARs. However, ADAR-editing of viral RNA is either rare or edited viral RNA are rapidly degraded, as we only detected evidence for editing in two of the 104 viral isolates we studied. CONCLUSION: This is the first evidence for ADARs targeting viruses outside of mammals, and it raises the possibility that ADARs could play a role in the antiviral defences of insects.

[Physico-chemical properties and functional peculiarities of mRNP of different subcellular location of Nicotiana rustica plants affected by potato curly dwarf virus]. / Fiziko-khimicheskie svoistva i funktsional'nye osobennosti mRNP razlichnoi subkletochnoi lokalizatsii rastenii Nicotiana rustica, porazhennykh virusom kurchavoi karlikovosti kartofelia.

mRNP of different subcellular location of N. rustica plants affected by PCDV contain virus-specific proteins. RNA-replicase, poly (A)-polymerase and protein kinase have been found in mRNP under study. The most expressed replicase, poly(A)-polymerase and protein kinase activity has been noticed in the systems containing membrane-bound non-polysomal mRNP.

[Enzyme activity of nucleocapsid proteins of the curly potato dwarf virus]. / Fermentativnaia aktivnost' nukleokapsidnykh belkov fitorabdovirusa kurchavoi karlikovosti kartofelia.

The individual nucleocapsid proteins of phytorhabdovirus of curly potato dwarf were isolated: N-protein (m. w. 56 kDa), NS-protein (m. w. 49 kDa), L-protein (m. w. 128 kDa). It was establish that L and NS proteins displayed enzyme activity in the replication, phosphorylation and adenylation systems in vitro. Major N protein (m. w. 56 kDa) did not show the enzyme activity in these systems. The revelation of RNA-polymerase, poly(A)-polymerase and proteinkinase activities of nucleocapsid proteins are characteristic of the viruses belonging to the Rhabdoviridae family.

Role of cellular kinases in the gene expression of nonsegmented negative strand RNA viruses.

Nonsegmented negative strand RNA viruses package an RNA-dependent RNA polymerase composed of two subunits, a large protein L and a phosphoprotein P, for transcription and replication of their genome RNAs. The RNA polymerase activity resides within the L protein, while the P protein acts as a transcription factor or transactivator of the polymerase. Since P protein is heavily phosphorylated and phosphorylation is known to regulate function of many viral as well as cellular proteins, the role of phosphorylation of P protein in the gene expression of this group of RNA viruses has recently been investigated. Through expression in bacteria the P protein was produced in large quantity in the nonphosphorylated form and involvement of cellular kinase(s) in its phosphorylation was studied. Casein kinase II and/or protein kinase C have been shown to play a critical role in the activation of P protein in transcription. These findings have opened up a new avenue for studying an important regulatory step in virus gene expression that may lead to the development of an effective antiviral agent.

Characterization of the components and activity of Sonchus yellow net rhabdovirus polymerase.

Sonchus yellow net virus (SYNV) is the best-characterized member of a group of plant rhabdoviruses that replicate in the host cell nucleus. Using a recently developed method for partial purification of active SYNV polymerase by salt extraction of nuclei from infected plant tissue (J. D. O. Wagner et al, J. Virol. 70:468-477, 1996), we have identified the nucleocapsid (N), M2, and L proteins as polymerase complex components (based on copurification with the polymerase activity and by coimmunoprecipitation assays). Furthermore, the L protein was shown by antibody inhibition analysis to be a functional component of the polymerase. A second complex of M2 and L proteins, thought to be a precursor to the polymerase complex, was also identified. In addition, we conducted a detailed characterization of SYNV RNA synthesis in vitro. The results demonstrate that the RNAs are transcribed sequentially, beginning with the N mRNA and followed successively by the remaining five mRNAs in the order of their genome organization. Gene expression conforms to a cascade pattern, with synthesis of the 3'-proximal N mRNA occurring at the highest level, followed by consecutively lower levels of transcription from each subsequent gene. The reaction conditions favor transcription over minus-sense RNA replication, which, we posit, is inhibited near specific signal sequences located on the antigenomic template. The results support the concept that the mechanism of transcription is highly conserved among diverse rhabdoviruses and are compatible with a unified model for the regulation of genomic and antigenomic RNA synthesis.

Extraction of nuclei from sonchus yellow net rhabdovirus-infected plants yields a polymerase that synthesizes viral mRNAs and polyadenylated plus-strand leader RNA.

Although the primary sequence of the genome of the plant rhabdovirus sonchus yellow net virus (SYNV) has been determined, little is known about the composition of the viral polymerase or the mechanics of viral transcription and replication. In this paper, we report the partial isolation and characterization of an active SYNV polymerase from nuclei of SYNV-infected leaf tissue. A salt extraction procedure is shown to be an effective purification step for recovery of the polymerase from the nuclei. Full-length, polyadenylated SYNV N and M2 mRNAs and plus-strand leader RNA are among the products of the in vitro polymerase reactions. Polyadenylation of the plus-strand leader RNA in vitro is shown with RNase H and specific oligonucleotides. This is the first report of a polyadenylated plus-strand leader RNA for a minus-strand RNA virus, a feature that may reflect adaptation of SYNV to replication in the nucleus. Analysis of the SYNV proteins present in the polymerase extract suggests that the N, M2, and L proteins are components of the transcription complex. Overall, the system we developed promises to be useful for an in-depth characterization of the mechanics of SYNV RNA synthesis.

Distant strains of the fish rhabdovirus VHSV maintain a sixth functional cistron which codes for a nonstructural protein of unknown function.

We used direct RNA sequencing to determine the genomic organization of the region downstream from the G gene of viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus. This region contains a gene coding for a protein, identified as nonvirion protein (NV), and the gene coding for the RNA polymerase (L). Thus, VHSV genome organization was confirmed to be 3'-N-P-M-G-NV-L-5'. In both a virulent European (07-71) and an avirulent North American (Makah) strain, the NV gene is transcribed into a small mRNA that codes for a protein of 122 amino acids. It has no significant sequence similarity with the infectious hematopoietic necrosis virus NV protein nor with any other known protein. We expressed the NV protein as a fusion protein with the glutathione S-transferase of Schistosoma japonicum and used the purified fusion protein to immunize rabbits. The rabbit antiserum precipitated from infected cell extracts--and not from noninfected cells or purified virions--a protein of 14 kDa, well in accordance with the expected NV gene product size. The prediction that the NV protein is a nonstructural protein is supported by its absence from mature virions although it is present in infected cells.

Comparison of the polymerases (L genes) of spring viremia of carp virus and infectious hematopoietic necrosis virus.

We have determined the partial nucleotide sequences of the polymerase genes of the fish rhabdoviruses, spring viremia of carp virus (SVCV) and infectious hematopoietic necrosis virus (IHNV). At this point we have deduced the amino acid sequences and analysed the first 1,400 amino acids comprising two thirds of the polymerase genes of SVCV and IHNV. We have compared sequence similarities of SVCV and IHNV polymerases with other rhabdovirus and paramyxovirus polymerases. The SVCV polymerase showed the closest relationship with the vesicular stomatitis virus polymerases and also shared significant sequence identity with the polymerase of rabies virus. Other rhabdovirus and paramyxovirus polymerases showed lower sequences identities with the SVCV polymerase. The IHNV polymerase shared a relatively low amino acid sequence identity with the rabies virus polymerase, and similar low identities with other rhabdovirus and paramyxovirus polymerases. Several domains of various lengths were conserved in the virus polymerases included in this study. These domains were less conserved in the IHNV polymerase than in the SVCV polymerase, and some of the domains present in the other polymerases were not identified in the IHNV. These preliminary results indicate that SVCV is closely related to mammalian vesiculoviruses and that IHNV may be only distantly related to mammalian lyssa and vesiculotype rhabdoviruses.

Sequence similarity between Borna disease virus p40 and a duplicated domain within the paramyxovirus and rhabdovirus polymerase proteins.

We report the sequence of a Borna disease virus clone (pBDV-40) that encodes a 40-kDa protein (p40) found in the nuclei of infected cells. Comparative sequence analysis indicates that p40 is distantly similar to two different regions in the L-polymerase proteins encoded by paramyxoviruses and rhabdoviruses. The p40 sequence similarity indicates a previously undetected duplication in these viral polymerases. Phylogenetic reconstruction suggests that the gene that encodes p40 last shared a common ancestor with these viral polymerase genes prior to the duplication event. These findings support the hypothesis that Borna disease virus is a negative-strand RNA virus and suggest that p40 is involved in transcription and/or replication. The discovery of a duplication within the polymerase proteins of paramyxoviruses and rhabdoviruses has profound implications for the mapping of enzymatic activities within these multifunctional proteins.

Sequence comparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains.

The large (L) protein subunit of unsegmented negative-strand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagreement with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis.

Effect of melittin on transcription by vesiculovirus mutant and wild-type viruses.

The bee venom peptide melittin activated the virion transcriptase activity of three vesiculoviruses with preservation of virion structure. The kinetics of RNA synthesis were similar to those observed with purified transcribing nucleoprotein (TNP) preparations. Six temperature-sensitive host range (tdCE) mutants of Chandipura virus displayed 1.7- to 5.5-fold greater efficiencies of transcription at 39 degrees with melittin-permeabilized virions in comparison with TNP preparations. Comparative study of other host range mutants (tdCE3) and tsB1) of vesicular stomatitis virus (VSV) New Jersey and a thermosensitive polymerase mutant (tsG114) of VSV Indiana suggested that the enhanced transcription at 39 degrees associated with melittin-activated tdCE mutants was due to the retention of host factors in the virions.

In vitro transcriptase deficiency of temperature-dependent host range mutants of Chandipura virus.

Ten of 12 Chandipura virus tdCE mutants, which exhibit temperature-dependent restriction of growth in chick embryo (CE) cells but not in BS-C-1 cells, showed deficient transcriptase activity in vitro at 39 degrees C relative to wild-type virus. A gradation in transcriptional activity at 39 degrees C in vitro was observed. Reversion of the tdCE phenotype to unrestricted growth in CE cells at 39 degrees C was accompanied by partial restoration of normal transcriptase activity at 39 degrees C, suggesting that reversion was mediated by either extragenic or intragenic suppression. Viral protein synthesis was reduced or absent in CE cells at 39 degrees C indicating that transcription was also defective in vivo under these conditions. Induction of heat-shock proteins in CE cells at 39 degrees C occurred normally in tdCE mutant-infected cells and RNA methylation in vitro was unaffected.

Biological characteristics of rhabdoviruses in different host cells.

Rhabdoviruses are a group of viruses capable of infecting vertebrates, invertebrates and plants and showing a wide range of host-parasite relationships. Biological characteristics of viruses can vary according to the type of cells in which they grow. The Authors analyze differences in viral structure and multiplication related to the host. Some final considerations on the evolution of this group of viruses are also reported.

Spring viremia of carp virus RNA and virion-associated transcriptase activity.

An RNA-dependent RNA polymerase activity has been demonstrated for spring viremia of carp virus (SVCV). The optimal temperature for in vitro synthesis of RNA was 20 to 25 degrees C. The SVCV enzyme activity was stimulated when the methyl donor S-adenosyl-L-methionine was included in the reaction mixture. S-adenosyl-L-methionine was not particularly effective in stimulating the virion RNA polymerase activity of vesicular stomatitis virus or pike fry rhabdovirus. The 5' nucleotide of the SVCV viral RNA is pppAp.