Protein Of Rabies Virus Research Articles

The roles of rabies virus structural proteins in immune evasion and implications for vaccine development.

Rabies is a zoonotic infectious disease that targets the nervous system of human and animals and has about 100% fatality rate without treatment. Rabies virus is a bullet-like viral particle composed of five structural proteins, including nucleoprotein (N), phosphorylated protein (P), matrix protein (M), glycoprotein (G), and large subunit (L) of RNA-dependent RNA polymerase. These multifunctional viral proteins also play critical roles in the immune escape by inhibiting specific immune responses in the host, resulting in massive replication of the virus in the nervous system and abnormal behaviors of patients such as brain dysfunction and hydrophobia, which ultimately lead to the death of patients. Herein, the role of five structural proteins of rabies virus in the viral replication and immune escape and its implication for the development of vaccines were systemically reviewed, so as to shed light on the understanding of pathogenic mechanism of rabies virus.

T-Cell Epitope Vaccine Prediction Analysis Targeting Phosphoprotein (P) Rabies Virus Based on the Presence of HLA-I Alleles A, B, and C Loci Throughout Southeast Asia: An Immunoinformatics Study

T cell immunity, like responses of CD4+and CD8+ T-cell, plays an important role to fight against viral infections and pathological harm. Several previous studies have shown the results that rabies virus (RABV) protein can act as an ideal receptor for rabies neuroseptic vaccine by inducing a response of T-cell. In this research, we evaluated possible vaccine epitopes based on the Rabies virus sequence and human lymphocyte antigen (HLA) distribution. First, this study used the rabies virus protein P sequence obtained from the NCBI database. Next, we predicted rabies CTL protein epitopes based on the frequency of HLA-I distribution allele locus A, B, and C in Southeast Asia region (> 1%) using Immune Epitope Database and Analysis Resource (iedb.org). Our results predict the presence of 12 epitopes of the protein P RABV. A cluster analysis of epitopes shows that seven P-protein clusters cover 97.47% of the Southeast Asian population. After a conservative epitope analysis, 8 epitopes of protein P showed protection in 11 different types of isolated Rabies virus. Finally, 4 epitopes (SQTVEEIISY119-128; RSSGIFLYNF218-227, ASGPPALEW178-186, IISYVTVNF125-133) were used to vaccinate 97.47% of people in Southeast Asia. Our results suggest that both single and combined CTL epitopes which were predicted in this study can be used as a more effective alternative vaccine against rabies virus’ infections and development

Single Amino Acid Substitution in the Matrix Protein of Rabies Virus Is Associated with Neurovirulence in Mice.

Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines in vitro. The RABV fixed strain, HEP-Flury, was produced via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain-based on the sequence of the HEP (HEP-Flury) strain-with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP. In vivo, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.

Expression of nucleocapsid protein of rabies virus and its diagnostic potential

Expression of nucleocapsid protein of rabies virus and its diagnostic potential

Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12mmol cell/h) in SF and for glutamine (30.8 × 10-12mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis.

The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.

Structure and Dynamics of the Unassembled Nucleoprotein of Rabies Virus in Complex with Its Phosphoprotein Chaperone Module.

As for all non-segmented negative RNA viruses, rabies virus has its genome packaged in a linear assembly of nucleoprotein (N), named nucleocapsid. The formation of new nucleocapsids during virus replication in cells requires the production of soluble N protein in complex with its phosphoprotein (P) chaperone. In this study, we reconstituted a soluble heterodimeric complex between an armless N protein of rabies virus (RABV), lacking its N-terminal subdomain (NNT-ARM), and a peptide encompassing the N0 chaperon module of the P protein. We showed that the chaperone module undergoes a disordered-order transition when it assembles with N0 and measured an affinity in the low nanomolar range using a competition assay. We solved the crystal structure of the complex at a resolution of 2.3 Å, unveiling the details of the conserved interfaces. MD simulations showed that both the chaperon module of P and RNA-mediated polymerization reduced the ability of the RNA binding cavity to open and close. Finally, by reconstituting a complex with full-length P protein, we demonstrated that each P dimer could independently chaperon two N0 molecules.

Lyssavirus P Protein Isoforms Diverge Significantly in Subcellular Interactions Underlying Mechanisms of Interferon Antagonism.

Viral hijacking of microtubule (MT)-dependent transport is well understood, but several viruses also express discrete MT-associated proteins (vMAPs), potentially to modulate MT-dependent processes in the host cell. Specific roles for vMAP-MT interactions include subversion of antiviral responses by P3, an isoform of the P protein of rabies virus (RABV; genus Lyssavirus), which mediates MT-dependent antagonism of interferon (IFN)-dependent signal transducers and activators of transcription 1 (STAT1) signaling. P3 also undergoes nucleocytoplasmic trafficking and inhibits STAT1-DNA binding, indicative of intranuclear roles in a multipronged antagonistic strategy. MT association/STAT1 antagonist functions of P3 correlate with pathogenesis, indicating potential as therapeutic targets. However, key questions remain, including whether other P protein isoforms interact with MTs, the relationship of these interactions with pathogenesis, and the extent of conservation of P3-MT interactions between diverse pathogenic lyssaviruses. Using super-resolution microscopy, live-cell imaging, and immune signaling analyses, we find that multiple P protein isoforms associate with MTs and that association correlates with pathogenesis. Furthermore, P3 proteins from different lyssaviruses exhibit variation in intracellular localization phenotypes that are associated with STAT1 antagonist function, whereby P3-MT association is conserved among lyssaviruses of phylogroup I but not phylogroup II, while nucleocytoplasmic localization varies between P3 proteins of the same phylogroup within both phylogroup I and II. Nevertheless, the divergent P3 proteins retain significant IFN antagonist function, indicative of adaptation to favor different inhibitory mechanisms, with MT interaction important to phylogroup I viruses. IMPORTANCE Lyssaviruses, including rabies virus, cause rabies, a progressive encephalomyelitis that is almost invariably fatal. There are no effective antivirals for symptomatic infection, and effective application of current vaccines is limited in areas of endemicity, such that rabies causes ~59,000 deaths per year. Viral subversion of host cell functions, including antiviral immunity, is critical to disease, and isoforms of the lyssavirus P protein are central to the virus-host interface underpinning immune evasion. Here, we show that specific cellular interactions of P protein isoforms involved in immune evasion vary significantly between different lyssaviruses, indicative of distinct strategies to evade immune responses. These findings highlight the diversity of the virus-host interface, an important consideration in the development of pan-lyssavirus therapeutic approaches.

The Amino Acid at Position 95 in the Matrix Protein of Rabies Virus Is Involved in Antiviral Stress Granule Formation in Infected Cells.

Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the α subunit of eukaryotic initiation factor 2α (eIF2α). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host. IMPORTANCE Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses. However, the relationship between SG formation capacity and pathogenicity of RABV has remained unclear. In this study, by comparing two RABV strains with completely different levels of virulence, we found that the amino acid mutation from valine to alanine at position 95 of matrix protein (M95), which is known to be one of the amino acid mutations that determine the difference in virulence between the strains, plays a major role in SG formation. Importantly, M95 was involved in the accumulation of RIG-I in SGs and in promoting interferon induction. These findings are the first report of the effect of a single amino acid substitution associated with SGs on viral virulence.

Targeted brain delivery of RVG29-modified rifampicin-loaded nanoparticles for Alzheimer's disease treatment and diagnosis.

Alzheimer's disease (AD) is an aging‐related neurodegenerative disease. The main pathological features of AD are β‐amyloid protein (Aβ) deposition and tau protein hyperphosphorylation. Currently, there are no effective drugs for the etiological treatment of AD. Rifampicin (RIF) is a semi‐synthetic broad‐spectrum antibiotic with anti‐β‐amyloid deposition, anti‐inflammatory, anti‐apoptosis, and neuroprotective effects, but its application in AD treatment has been limited for its strong hydrophobicity, high toxicity, short half‐life, low bioavailability, and blood–brain barrier hindrance. We designed a novel brain‐targeted and MRI‐characteristic nanomedicine via loading rabies virus protein 29 (RVG29), rifampicin, and Gd on poly (l‐lactide) nanoparticles (RIF@PLA‐PEG‐Gd/Mal‐RVG29). The cytotoxicity assay demonstrated that RIF@PLA‐PEG‐Gd/Mal‐RVG29 had favorable biocompatibility and security. Fluorescence imaging in vivo showed that PLA‐PEG‐Gd/Mal‐RVG29 could deliver rifampicin into the brain by enhancing cellular uptake and brain targeting performance, leading to improvement of the bioavailability of rifampicin. In in vivo study, RIF@PLA‐PEG‐Gd/Mal‐RVG29 improved the spatial learning and memory capability of APP/PS1 mice in the Morris water maze, as compared to rifampicin. Immunofluorescence, TEM, immunoblotting, and H&E staining revealed that RIF@PLA‐PEG‐Gd/Mal‐RVG29 reduced Aβ deposition in hippocampal and cortex of APP/PS1 mice, improved the damage of synaptic ultrastructure, increased the expression level of PSD95 and SYP, as well as reduced the necrosis of neurons. These findings suggest that RIF@PLA‐PEG‐Gd/Mal‐RVG29 may be an effective strategy for the treatment of AD.

Rabies Virus Exploits Cytoskeleton Network to Cause Early Disease Progression and Cellular Dysfunction.

Rabies virus (RABV) is a cunning neurotropic pathogen and causes top priority neglected tropical diseases in the developing world. The genome of RABV consists of nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase L protein (L), respectively. The virus causes neuronal dysfunction instead of neuronal cell death by deregulating the polymerization of the actin and microtubule cytoskeleton and subverts the associated binding and motor proteins for efficient viral progression. These binding proteins mainly maintain neuronal structure, morphology, synaptic integrity, and complex neurophysiological pathways. However, much of the exact mechanism of the viral-cytoskeleton interaction is yet unclear because several binding proteins of the actin-microtubule cytoskeleton are involved in multifaceted pathways to influence the retrograde and anterograde axonal transport of RABV. In this review, all the available scientific results regarding cytoskeleton elements and their possible interactions with RABV have been collected through systematic methodology, and thereby interpreted to explain sneaky features of RABV. The aim is to envisage the pathogenesis of RABV to understand further steps of RABV progression inside the cells. RABV interacts in a number of ways with the cell cytoskeleton to produce degenerative changes in the biochemical and neuropathological trails of neurons and other cell types. Briefly, RABV changes the gene expression of essential cytoskeleton related proteins, depolymerizes actin and microtubules, coordinates the synthesis of inclusion bodies, manipulates microtubules and associated motors proteins, and uses actin for clathrin-mediated entry in different cells. Most importantly, the P is the most intricate protein of RABV that performs complex functions. It artfully operates the dynein motor protein along the tracks of microtubules to assist the replication, transcription, and transport of RABV until its egress from the cell. New remedial insights at subcellular levels are needed to counteract the destabilization of the cytoskeleton under RABV infection to stop its life cycle.

Research Advances on the Interactions between Rabies Virus Structural Proteins and Host Target Cells: Accrued Knowledge from the Application of Reverse Genetics Systems.

Rabies is a lethal zoonotic disease caused by lyssaviruses, such as rabies virus (RABV), that results in nearly 100% mortality once clinical symptoms appear. There are no curable drugs available yet. RABV contains five structural proteins that play an important role in viral replication, transcription, infection, and immune escape mechanisms. In the past decade, progress has been made in research on the pathogenicity of RABV, which plays an important role in the creation of new recombinant RABV vaccines by reverse genetic manipulation. Here, we review the latest advances on the interaction between RABV proteins in the infected host and the applied development of rabies vaccines by using a fully operational RABV reverse genetics system. This article provides a background for more in-depth research on the pathogenic mechanism of RABV and the development of therapeutic drugs and new biologics.

Implication of the nuclear trafficking of rabies virus P3 protein in viral pathogenicity.

Although the majority of viruses of the family Mononegvirales replicate exclusively in the host cell cytoplasm, many of these viruses encode proteins that traffic between the nucleus and cytoplasm, which is believed to enable accessory functions in modulating the biology of the infected host cell. Among these, the P3 protein of rabies virus localizes to the nucleus through the activity of several specific nuclear localization and nuclear export signals. The major defined functions of P3 are in evasion of interferon (IFN)-mediated antiviral responses, including through inhibition of DNA-binding by IFN-activated STAT1. P3 also localizes to nucleoli and promyelocytic leukemia (PML) nuclear bodies, and interacts with nucleolin and PML protein, indicative of several intranuclear roles. The relationship of P3 nuclear localization with pathogenicity, however, is unresolved. We report that nucleocytoplasmic localization of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derived strain, Ni-CE, differs significantly, with nuclear accumulation defective for Ni-CE-P3. Molecular mapping indicates that altered localization derives from a coordinated effect, including two residue substitutions that independently disable nuclear localization and augment nuclear export signals, collectively promoting nuclear exclusion. Intriguingly, this appears to relate to effects on protein conformation or regulatory mechanisms, rather than direct modification of defined trafficking signal sequences. These data provide new insights into the role of regulated nuclear trafficking of a viral protein in the pathogenicity of a virus that replicates in the cytoplasm.

Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus.

Rabies virus (RABV) causes fatal neurological encephalitis and results in approximately 6000 human death cases worldwide every year. The large (L) protein of RABV, possessing conserved domains, is considered as the target for detection. In this study, three monoclonal antibodies (mAbs), designated as 3F3, 3A6 and L-C, against L protein were generated by using the recombinant truncated L protein (aa 1431–1754) and the epitopes were also identified using a series of overlapping truncated polypeptides for testing the reactivity of mAbs with different RABV strains. The 1479EIFSIP1484, 1659RALSK1663 and 1724VFNSL1728 were identified as the minimal linear epitopes recognized by mAbs 3F3, 3A6 and L-C, respectively. Amino acid alignment showed epitope 1724VFNSL1728 recognized by mAb L-C is completely conserved among RABV strains, indicating that mAb L-C could be used to detect all of the RABV strains. Epitope 1479EIFSIP1484 is highly conserved among RABV strains except for a P1484S substitution in a China I sub-lineage strain of Asian lineage, which eliminated the reactivity of the epitope with mAb 3F3. However, the epitope 1659RALSK1663 was only completely conserved in the Africa-2 and Indian lineages, and a single A1660T substitution, mainly appeared in strains of the China I belonging to Asian lineage and a Cosmopolitan lineage strain, still retained the reactivity of the epitope with mAb 3A6. While both A1660T and K1663R substitutions in a China I lineage strain, single K1663R/Q substitution in some China II strains of Asian lineage and some Arctic-like lineage strains and R1659Q mutation in a strain of Africa-3 lineage eliminated the reactivity of the epitope with mAb 3A6, suggesting mAb 3A6 could be used for differentiation of variable epitopes of some strains in different lineages. Thus, variability and conservation of the three epitopes of L protein showed the reactive difference of mAbs among RABV strains of different lineages. These results may facilitate future studies in development of detection methods for RABV infection, the structure and function of RABV L protein.

Targeted Brain Delivery of RVG29-Modified Rifampicin-Loaded Nanoparticles for Treatment and Diagnosis of Alzheimer Disease

Alzheimer's disease (AD) is a neurodegenerative disease related to age. The main pathological features of AD are β-amyloid protein (Aβ) deposition and tau protein hyperphosphorylation. Currently, there are not an effective drug for the etiological treatment of AD. Rifampicin (RIF) is a semi-synthetic broad-spectrum antibiotic with anti-β-amyloid deposition, anti-inflammatory, anti-apoptosis and neuroprotective effects, but its application in AD treatment has been limited by its strong hydrophobicity, high toxicity, short half-life, low bioavailability and blood-brain barrier (BBB) hindrance. We designed a novel brain-targeted and MRI-characteristic nanomedicine through loading rabies virus protein 29 (RVG29), rifampicin and Gd on poly(L-lactide) nanoparticles (RIF@PLA-PEG-Gd/Mal-RVG29). The cytotoxicity assay demonstrated that RIF@PLA-PEG-Gd/Mal-RVG29 had good biocompatibility and security. The results of cellular uptake and fluorescence in vivo imaging showed that PLA-PEG-Gd/Mal-RVG29 could deliver rifampicin into brain by enhancing cellular uptake and brain targeting performance, so that improving the bioavailability of rifampicin. In vivo study, compared with rifampicin, RIF@PLA-PEG-Gd/Mal-RVG29 improved the spatial learning and memory capability of APP/PS1 mice in the Morris water maze. Immunofluorescence, TEM, immunoblotting and HE test showed that RIF@PLA-PEG-Gd/Mal-RVG29 could reduce Aβ deposition in hippocampal and cortex of APP/PS1 mice, improve the damage of synaptic ultrastructure, increase the expression level of PSD95 and SYP, as well as reduce the necrosis of neurons. These findings suggest that RIF@PLA-PEG-Gd/Mal-RVG29 may be an effective strategy for the treatment and diagnosis of AD.

Immunobioinformatics of rabies virus in various countries of Asia: Glycoprotein gene

he purpose of this study was to analyze the genomic information of the glycoprotein gene of rabies virus (RABV) in several countries of Asia. The genomics information of vaccine virus strain and some local isolates were retrieved from GeneBank® (National Center for Biotechnology Information, USA). Ten genomics informations are consist of ERA vaccine virus, two viruses from Indonesia, a virus from Myanmar, India, Nepal, South Korea, Japan, Sri Lanka, and Iran. This bioinformatics process the ten genomics data in order to find pattern in 1,575 bp of glycoprotein gene of RABV. In conclusion, this study have revealed the data of homology, B-cell epitope prediction, genetic distance, and molecular phylogenic analysis of RABV in various countries of Asia. We indicated that the glycoprotein, the immunogenic surface protein of RABV, has important part for vaccine design Keywords: Bioinformatics Lyssavirus glycoprotein rabies vaccine

Rabies virus matrix protein targets host actin cytoskeleton: a protein-protein interaction analysis.

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis-cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.

Expression of the rabies virus nucleoprotein and matrix protein in a prokaryotic system at high-levels: An efficacious production method

Rabies virus (RABV) causes a zoonotic and fatal infectious disease among all warm-blooded animals including humans that continues to give a great burden on public health and the global economy especially in developing countries such as Iran. In this study, we describe a convenient and low-cost system to express the nucleoprotein (NP) and matrix protein (MP) of rabies virus. NP gene (Pasteur virus (PV) strain) and MP gene (challenge virus standard (CVS) strain) were amplified and cloned into pET28a expression vector separately. Then both genes were expressed in Escherichia coli, Rosetta strain, after induction with IPTG. Expression of the proteins was analyzed by SDS-PAGE and western blot. NP and MP were purified by Anti His Tag Column chromatography. Our results indicated the N and M genes had been successfully cloned into the expression vector and verified purification of recombinant NP and MP as 53 and 27 kDa proteins. This study showed that the NP and MP of the rabies virus can be expressed in a reproducible prokaryotic expression system using the pET28a expression vector. NP and MP can be used in vaccine production and research fields.

Comprehensive analysis of the ubiquitome in rabies virus-infected brain tissue of Mus musculus

Ubiquitination is an important post-translational modification (PTM) that plays a key role in almost every aspect of cellular processes and many signaling pathways in eukaryotes. In this study, we performed a quantitative ubiquitome study to identify the global change of ubiquitination induced by rabies virus (RABV) infection in the mouse brain tissue. 4,243 ubiquitinated sites, mapping to 1,626 proteins were identified; using a cutoff of fold change >2, 644 and 70 ubiquitinated proteins were up- and down-regulated, respectively. GO analysis indicated that the differentially ubiquitinated proteins (DUPs) were significantly enriched in the myelin sheath of cells and binding activity. KEGG pathway analysis indicated that the identified proteins were related to biosynthesis of amino acids. Of note, ubiquitination was observed on all five RABV proteins by both proteomics and biochemical approaches. Our study revealed the global ubiquitome of RABV-infected mice and provides a valuable resource for investigating the pathogenic mechanisms of RABV.

Transcriptional Control and mRNA Capping by the GDP Polyribonucleotidyltransferase Domain of the Rabies Virus Large Protein.

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.