Structure of a rabies virus polymerase complex from electron cryo-microscopy.

Nonsegmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS RNA viruses carry all of the enzymatic functions required for viral messenger RNA (mRNA) transcription and replication: RNA polymerization, mRNA capping, and cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 Å resolution. The complex closely resembles the vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L-protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a preinitiation conformation. Comparison with the likely elongation state, seen in two structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.

Molecular Function Analysis of Rabies Virus RNA Polymerase L Protein by Using an L Gene-Deficient Virus.

While the RNA-dependent RNA polymerase L protein of rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, has potential to be a therapeutic target for rabies, the molecular functions of this protein have remained largely unknown. In this study, to obtain a novel experimental tool for molecular function analysis of the RABV L protein, we established by using a reverse genetics approach an L gene-deficient RABV (Nishi-ΔL/Nluc), which infects, propagates, and correspondingly produces NanoLuc luciferase in cultured neuroblastoma cells transfected to express the L protein. trans-Complementation with wild-type L protein, but not that with a functionally defective L protein mutant, efficiently supported luciferase production by Nishi-ΔL/Nluc, confirming its potential for function analysis of the L protein. Based on the findings obtained from comprehensive genetic analyses of L genes from various RABV and other lyssavirus species, we examined the functional importance of a highly conserved L protein region at positions 1914 to 1933 by a trans-complementation assay with Nishi-ΔL/Nluc and a series of L protein mutants. The results revealed that the amino acid sequence at positions 1929 to 1933 (NPYNE) is functionally important, and this was supported by other findings that this sequence is critical for binding of the L protein with its essential cofactor, P protein, and thus also for L protein's RNA polymerase activity. Our findings provide useful information for the development of an anti-RABV drug targeting the L-P protein interaction.IMPORTANCE To the best of our knowledge, this is the first report on the establishment of an L gene-deficient, reporter gene-expressing virus in all species of the order Mononegavirales, also highlighting its applicability to a trans-complementation assay, which is useful for molecular function analyses of their L proteins. Moreover, this study revealed for the first time that the NPYNE sequence at positions 1929 to 1933 in the RABV L protein is important for L protein's interaction with the P protein, consistent with and extending the results of a previous study showing that the P protein-binding domain in the L protein is located in its C-terminal region, at positions 1562 to 2127. This study indicates that the NPYNE sequence is a promising target for the development of an inhibitor of viral RNA synthesis, which has high potential as a therapeutic drug for rabies.

Phylogenetic analysis of partial RNA-polymerase blocks II and III of Rabies virus isolated from the main rabies reservoirs in Brazil.

This study describes the results of the sequencing and analysis of segments of Blocks II and III of the RNA polymerase L gene of Rabies virus isolates from different reservoir species of Brazil. The phylogenetic relations of the virus were determined and a variety of species-specific nucleotides were found in the analyzed areas, but the majority of these mutations were found to be synonymous. However, an analysis of the putative amino acid sequences were shown to have some characteristic mutations between some reservoir species of Brazil, indicating that there was positive selection in the RNA polymerase L gene of Rabies virus. On comparing the putative viral sequences obtained from the Brazilian isolates and other Lyssavirus, it was determined that amino acid mutations occurred in low-restriction areas. This study of the L gene of Rabies virus is the first to be conducted with samples of virus isolates from Brazil, and the results obtained will help in the determination of the phylogenetic relations of the virus.

Molecular basis of neurovirulence of flury rabies virus vaccine strains: importance of the polymerase and the glycoprotein R333Q mutation.

The molecular mechanisms associated with rabies virus (RV) virulence are not fully understood. In this study, the RV Flury low-egg-passage (LEP) and high-egg-passage (HEP) strains were used as models to explore the attenuation mechanism of RV. The results of our studies confirmed that the R333Q mutation in the glycoprotein (G(R333Q)) is crucial for the attenuation of Flury RV in mice. The R333Q mutation is stably maintained in the HEP genome background but not in the LEP genome background during replication in mouse brain tissue or cell culture. Further investigation using chimeric viruses revealed that the polymerase L gene determines the genetic stability of the G(R333Q) mutation during replication. Moreover, a recombinant RV containing the LEP G protein with the R333Q mutation and the HEP L gene showed significant attenuation, genetic stability, enhancement of apoptosis, and immunogenicity. These results indicate that attenuation of the RV Flury strain results from the coevolution of G and L elements and provide important information for the generation of safer and more effective modified live rabies vaccine.

In silico structural elucidation of the rabies RNA-dependent RNA polymerase (RdRp) toward the identification of potential rabies virus inhibitors.

The rabies virus (RABV) is a non-segmented, negative single-stranded RNA virus which causes acute infection of the central nervous system in humans. Once symptoms appear, the result is nearly always death, and to date, post-exposure prophylaxis (PEP) is the only treatment applicable only immediately after an exposure. Previous studies have identified viral RNA-dependent RNA polymerase (RdRp) as a potential drug target due to its significant role in viral replication and transcription. Herein we generated an energy-minimized homology model of RABIES-RdRp and used it for virtual screening against 2045 NCI Diversity Set III library. The best five ligand-RdRp complexes were picked for further energy minimization via molecular dynamics (MDs) where the complex with ligand Z01690699 shows a minimum score characterized with stable hydrogen bonds and hydrophobic interactions with the catalytic site residues. Our study identified an important ligand for development of remedial approach for treatment of rabies infection.

Segmentation of the rabies virus genome.

We established a system for the recovery of a segmented recombinant rabies virus, the virus genome RNA of which was divided into two parts: segment 1 encoding the nucleoprotein, phosphoprotein, matrix protein, and glycoprotein genes, and segment 2 encoding the large RNA-dependent RNA polymerase gene. The morphology of the segmented recombinant rabies virus was bullet-like in shape with a length of approximately 130 nm, which is shorter than the 200-nm long non-segmented recombinant rabies virus. The segmented recombinant rabies virus was maintained for at least 18 passages. The virus multiplication rate of the segmented recombinant rabies virus was lower than that of the non-segmented recombinant rabies virus during the passages, and the relative amounts of virus genome RNAs for segment 1 and segment 2 differed in the supernatant of the segmented recombinant rabies virus infected cells. These results suggest that the segmented recombinant rabies virus packages either segment 1 or segment 2 into each virus particle. Thus, co-infection with segmented recombinant rabies virus particles packaging segment 1 or segment 2 may be necessary for the production of progeny virus.

The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.

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.

Development of a mouse monoclonal antibody cocktail for post-exposure rabies prophylaxis in humans.

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans.

Rabies virus P protein interacts with STAT1 and inhibits interferon signal transduction pathways.

Rabies virus P protein is a cofactor of RNA polymerase. We investigated other potential roles of P (CVS strain) by searching for cellular partners using two-hybrid screening. We isolated a cDNA encoding the signal transducer and activator of transcription 1 (STAT1) that is a critical component of interferon type I (IFN-alpha/beta) and type II (IFN-gamma) signaling. We confirmed this interaction by glutathione S-transferase-pull-down assay. Deletion mutant analysis indicated that the carboxy-terminal part of P interacted with a region containing the DNA-binding domain and the coiled-coil domain of STAT1. The expression of P protein inhibits IFN-alpha- and IFN-gamma-induced transcriptional responses, thus impairing the IFN-induced antiviral state. Mechanistic studies indicate that P protein does not induce STAT1 degradation and does not interfere with STAT1 phosphorylation but prevents IFN-induced STAT1 nuclear accumulation. These results indicate that rabies P protein overcomes the antiviral response of the infected cells.

An RNA polymerase activity in purified rabies virions.

An RNA polymerase activity has been demonstrated in purified rabies virions. Efficiency of the reaction is low since the rate of incorporation was equal to 3 to 5 pmol of uridine per hour, per mg of protein. As with other mammalian rhabdoviruses the optimal temperature was 31 degrees C. Unlike vesicular stomatitis virus, manganese could be substituted for magnesium as a divalent cation, at an optimum concentration of 10 to 20 mM.

Overexpression of cytochrome C by a recombinant rabies virus attenuates pathogenicity and enhances antiviral immunity.

The pathogenicity of individual rabies virus strains appears to correlate inversely with the extent of apoptotic cell death they induce and with the expression of rabies virus glycoprotein, a major inducer of an antiviral immune response. To determine whether the induction of apoptosis by rabies virus contributes to a decreased pathogenicity by stimulating antiviral immunity, we have analyzed these parameters in tissue cultures and in mice infected with a recombinant rabies virus construct that expresses the proapoptotic protein cytochrome c. The extent of apoptosis was strongly increased in primary neuron cultures infected with the recombinant virus carrying the active cytochrome c gene [SPBN-Cyto c(+)], compared with cells infected with the recombinant virus containing the inactive cytochrome c gene [SPBN-Cyto c(-)]. Mortality in mice infected intranasally with SPBN-Cyto c(+) was substantially lower than in SPBN-Cyto c(-)-infected mice. Furthermore, virus-neutralizing antibody (VNA) titers were significantly higher in mice immunized with SPBN-Cyto c(+) at the same dose. The VNA titers induced by these recombinant viruses paralleled their protective activities against a lethal rabies virus challenge infection, with SPBN-Cyto c(+) revealing an effective dose 20 times lower than that of SPBN-Cyto c(-). The strong increase in immunogenicity, coupled with the marked reduction in pathogenicity, identifies the SPBN-Cyto c(+) construct as a candidate for a live rabies virus vaccine.

Transcriptase activity associated with rabies virion.

Rabies virion-associated transcriptase activity was investigated in vitro and compared with that of the New Jersey serotype of vesicular stomatitis virus. The concentration of detergent that affected [3H]GMP incoporation into acid-insoluble material was significantly different for both viruses. Vesicular stomatitis virus New Jersey required 0.05 to 0.1% nonionic detergent, whereas rabies virion could not be fully activated unless 4 to 5% detergent was used. Other optimal conditions were as follows: 40 mM NaCl, 5 mM Mg2+, 40 mM Tris-hydrochloride (pH 7.4), 5 mM dithiothreitol, and 30 degrees C. The reaction required four nucleoside triphosphates. The initial rate of RNA synthesis by rabies virion enzyme was 140 pmol of GMP incorporated/mg of viral protein per h and linearly increased until about 8 h, with a slight initial lag phase. The enzyme activity that correlated with the content of L protein was highest when rabies virions were grown at 33 degrees C. The product was single-stranded RNA, which was complementary in base sequences to rabies viral RNA. Most of the RNA synthesized sedimented at 6-16S.

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.

Studies on the rabies virus RNA polymerase: 2. Possible relationships between the two forms of the non-catalytic subunit (P protein).

We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [3H]leucine and [32P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s).

Transcribing complexes in cells infected by vesicular stomatitis virus and rabies virus.

We analyzed cell extracts from BHK(21) cells infected with vesicular stomatitis virus (VSV) and rabies virus for in vitro RNA polymerase activity. Cells infected with VSV B virions exhibited several complexes with in vitro RNA polymerase activity in sucrose gradients. These complexes synthesize VSV transcriptase product (4 to 18S) polyadenylated in RNA complementary to virion RNA. Cells infected with a high multiplicity of B virions and T particles show only one RNA polymerase complex active in vitro. This complex sediments at 110S and makes only small (2S) RNA. Carrier BHK(21) cells persistently noncytopathically infected with VSV contain several complexes active in RNA polymerase, but both exhibit very low activity. Cytoplasms of cells noncytopathically infected with rabies virus also show very low levels of a complex containing RNA polymerase activity. No transcriptase nor any other in vitro polymerase activity could be found associated with purified rabies virions, although they do carry out primary transcription in cells treated with actinomycin D and cycloheximide before infection.

Phosphorylation of the N and M1 proteins of rabies virus.

Phosphorylation of rabies virus proteins was followed in vivo and in vitro. The N and M1 proteins were both found to be phosphorylated. The M1 protein was present in the virion in two phosphorylated states, but only the hypophosphorylated form of M1 was found in infected cells. The hypothesis that some of the M1 molecules become hyperphosphorylated during the maturation process by a membrane-bound kinase was examined. The phosphorylation of the viral proteins by the kinase present in purified rabies virions was studied using an in vitro transcriptase assay: under the conditions of the assay, additional phosphate groups were rapidly attached to the N protein. The M1 protein was similarly hyperphosphorylated although more slowly. Whether the hyperphosphorylation of the N protein is responsible for the poor efficiency of the in vitro transcriptase reaction is not clear. No detectable change in the phosphorylation of cellular proteins was observed in the course of rabies virus infection.

Differential transcription attenuation of rabies virus genes by intergenic regions: generation of recombinant viruses overexpressing the polymerase gene.

Gene expression of nonsegmented negative-sense RNA viruses involves sequential synthesis of monocistronic mRNAs and transcriptional attenuation at gene borders resulting in a transcript gradient. To address the role of the heterogeneous rabies virus (RV) intergenic regions (IGRs) in transcription attenuation, we constructed bicistronic model RNAs in which two reporter genes are separated by the RV N/P gene border. Replacement of the 2-nucleotide (nt) N/P IGR with the 5-nt IGRs from the P/M or M/G border resulted in attenuation of downstream gene transcription to 78 or 81%, respectively. A severe attenuation to 11% was observed for the 24-nt G/L border. This indicated that attenuation in RV is correlated with the length of the IGR, and, in particular, severe downregulation of the L (polymerase) gene by the 24 nt IGR. By reverse genetics, we recovered viable RVs in which the strongly attenuating G/L gene border of wild-type (wt) RV (SAD L16) was replaced with N/P-derived gene borders (SAD T and SAD T2). In these viruses, transcription of L mRNA was enhanced by factors of 1.8 and 5.1, respectively, resulting in exaggerated general gene expression, faster growth, higher virus titers, and induction of cytopathic effects in cell culture. The major role of the IGR in attenuation was further confirmed by reintroduction of the wt 24-nt IGR into SAD T, resulting in a ninefold drop of L mRNA. The ability to modulate RV gene expression by altering transcriptional attenuation is an advantage in the study of virus protein functions and in the development of gene delivery vectors.

An L (polymerase)-deficient rabies virus defective interfering particle RNA is replicated and transcribed by heterologous helper virus L proteins.

A rabies virus-derived defective interfering particle (DI) was isolated and characterized. The DI genome contained an internal deletion of 6.4 kb spanning the 3' moiety of the pseudogene region (psi) and most of the L gene. DI-specific monocistronic N, NS, and M mRNAs as well as a G/L fusion mRNA were transcribed in cells coinfected with DI and helper virus. In addition, polycistronic DI RNAs and standard virus RNAs with internal A stretches and intergenic regions were found. Superinfection experiments showed that heterologous rabies-related viruses (Lyssavirus serotypes 2, 3, and 4) can complement the L deficiency of the DI genome. The heterologous polymerase proteins recognize correctly the replicational and transcriptional signal sequences of the Lyssavirus serotype 1-derived DI.

Effect of neurotropic virus infection on neuronal and inducible nitric oxide synthase activity in rat brain.

To elucidate the potential role of inducible nitric oxide synthase (iNOS) and neuronal constitutive nitric oxide synthase (cNOS) in the pathogenesis of virus-induced encephalopathy, the activities of both NOS isoforms were determined in the brains of rats infected with Borna disease virus (BDV) or rabies virus. iNOS activity strongly increased, whereas neuronal cNOS activity significantly decreased in a time-dependent manner after either BDV or rabies virus infection. Choline acetyltransferase activity in the brain remained unchanged during both virus infections, suggesting that the decrease in cNOS activity does not reflect a generalized neuronal loss. Immunohistochemistry and Northern blot analyses indicate that the decrease in neuronal cNOS activity is due to a decrease in cNOS protein and mRNA synthesis. These results suggest that both an excessive generation of NO by activated macrophages or microglia, as well as a decrease of NO production in neurons may contribute to the neuropathogenesis of neurotropic virus infections.