scholarly journals Pathogenicity of Different Rabies Virus Variants Inversely Correlates with Apoptosis and Rabies Virus Glycoprotein Expression in Infected Primary Neuron Cultures

1999 ◽  
Vol 73 (1) ◽  
pp. 510-518 ◽  
Author(s):  
Kinjiro Morimoto ◽  
D. Craig Hooper ◽  
Sergei Spitsin ◽  
Hilary Koprowski ◽  
Bernhard Dietzschold
Keyword(s):  
Protein Expression ◽  
G Protein ◽  
Rabies Virus ◽  
Antigenic Site ◽  
Expression Levels ◽  
N Protein ◽  
Adult Mice ◽  
Glycoprotein G ◽  
Neuronal Processes ◽  
Nucleoprotein N

ABSTRACT The mouse-adapted rabies virus strain CVS-24 has stable variants, CVS-B2c and CVS-N2c, which differ greatly in their pathogenicity for normal adult mice and in their ability to infect nonneuronal cells. The glycoprotein (G protein), which has previously been implicated in rabies virus pathogenicity, shows substantial structural differences between these variants. Although prior studies have identified antigenic site III of the G protein as the major pathogenicity determinant, CVS-B2c and CVS-N2c do not vary at this site. The possibility that pathogenicity is inversely related to G protein expression levels is suggested by the finding that CVS-B2c, the less pathogenic variant, expresses at least fourfold-higher levels of G protein than CVS-N2c in infected neurons. Although there is some difference between CVS-B2c- and CVS-N2c-infected neurons in G protein mRNA expression levels, the differential expression of G protein appears to be largely determined by posttranslational mechanisms that affect G protein stability. Pulse-chase experiments indicated that the G protein of CVS-B2c is degraded more slowly than that of CVS-N2c. The accumulation of G protein correlated with the induction of programmed cell death in CVS-B2c-infected neurons. The extent of apoptosis was considerably lower in CVS-N2c-infected neurons, where G protein expression was minimal. While nucleoprotein (N protein) expression levels were similar in neurons infected with either variant, the transport of N protein into neuronal processes was strongly inhibited in CVS-B2c-infected cells. Thus, downregulation of G protein expression in neuronal cells evidently contributes to rabies virus pathogenesis by preventing apoptosis and the apparently associated failure of the axonal transport of N protein.

Mapping of epitopes and structural analysis of antigenic sites in the nucleoprotein of rabies virus

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 119-127 ◽  
Author(s):  
Hideo Goto ◽  
Nobuyuki Minamoto ◽  
Hiroshi Ito ◽  
Naoto Ito ◽  
Makoto Sugiyama ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Antigenic Site ◽  
Cytoplasmic Inclusion ◽  
The Other ◽  
N Protein ◽  
Antigenic Sites ◽  
Immature Form ◽  
Infected Cells ◽  
Cytoplasmic Inclusion Bodies ◽  
Nucleoprotein N

Linear epitopes on the rabies virus nucleoprotein (N) recognized by six MAbs raised against antigenic sites I (MAbs 6-4, 12-2 and 13-27) and IV (MAbs 6-9, 7-12 and 8-1) were investigated. Based on our previous studies on sites I and IV, 24 consecutively overlapping octapeptides and N- and C-terminal-deleted mutant N proteins were prepared. Results showed that all three site I epitopes studied and two site IV epitopes (for MAbs 8-1 and 6-9) mapped to aa 358–367, and that the other site IV epitope of MAb 7-12 mapped to aa 375–383. Tests using chimeric and truncated proteins showed that MAb 8-1 also requires the N-terminal sequence of the N protein to recognize its binding region more efficiently. Immunofluorescence studies demonstrated that all three site I-specific MAbs and one site IV-specific MAb (7-12) stained the N antigen that was diffusely distributed in the whole cytoplasm; the other two site IV-specific MAbs (6-9 and 8-1) detected only the N antigen in the cytoplasmic inclusion bodies (CIB). An antigenic site II-specific MAb (6-17) also detected CIB-associated N antigen alone. Furthermore, the level of diffuse N antigens decreased after treatment of infected cells with cycloheximide. These results suggest that epitopes at site I are expressed on the immature form of the N protein, but epitope structures of site IV MAbs 6-9 and 8-1 are created and/or exposed only after maturation of the N protein.

scholarly journals Moving the Glycoprotein Gene of Vesicular Stomatitis Virus to Promoter-Proximal Positions Accelerates and Enhances the Protective Immune Response

2000 ◽  
Vol 74 (17) ◽  
pp. 7895-7902 ◽  
Author(s):  
E. Brian Flanagan ◽  
L. Andrew Ball ◽  
Gail W. Wertz
Keyword(s):  
Immune Response ◽  
Protein Expression ◽  
Vesicular Stomatitis Virus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Gene Encoding ◽  
N Protein ◽  
Glycoprotein G ◽  
Vesicular Stomatitis ◽  
Negative Sense

ABSTRACT Vesicular stomatitis virus (VSV) is the prototype of the Rhabdoviridae and contains nonsegmented negative-sense RNA as its genome. The 11-kb genome encodes five genes in the order 3′-N-P-M-G-L-5′, and transcription is obligatorily sequential from the single 3′ promoter. As a result, genes at promoter-proximal positions are transcribed at higher levels than those at promoter-distal positions. Previous work demonstrated that moving the gene encoding the nucleocapsid protein N to successively more promoter-distal positions resulted in stepwise attenuation of replication and lethality for mice. In the present study we investigated whether moving the gene for the attachment glycoprotein G, which encodes the major neutralizing epitopes, from its fourth position up to first in the gene order would increase G protein expression in cells and alter the immune response in inoculated animals. In addition to moving the G gene alone, we also constructed viruses having both the G and N genes rearranged. This produced three variant viruses having the orders 3′-G-N-P-M-L-5′ (G1N2), 3′-P-M-G-N-L-5′ (G3N4), and 3′-G-P-M-N-L-5′ (G1N4), respectively. These viruses differed from one another and from wild-type virus in their levels of gene expression and replication in cell culture. The viruses also differed in their pathogenesis, immunogenicity, and level of protection of mice against challenge with wild-type VSV. Translocation of the G gene altered the kinetics and level of the antibody response in mice, and simultaneous reduction of N protein expression reduced replication and lethality for animals. These studies demonstrate that gene rearrangement can be exploited to design nonsegmented negative-sense RNA viruses that have characteristics desirable in candidates for live attenuated vaccines.

scholarly journals Change in the Single Amino Acid Site 83 in Rabies Virus Glycoprotein Enhances the BBB Permeability and Reduces Viral Pathogenicity

2021 ◽  
Vol 8 ◽  
Author(s):  
Chunfu Li ◽  
Yongzhi Wang ◽  
Huiting Liu ◽  
Xinghua Zhang ◽  
Dalai Baolige ◽  
...  
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Rabies Virus ◽  
Cell Cultures ◽  
Molecular Mechanisms ◽  
Single Amino Acid ◽  
Wild Type ◽  
Cellular Adaptation ◽  
Adult Mice ◽  
Bbb Permeability

Lab-attenuated rabies virus (RABV) is a highly cellular adaptation and less pathogenic than wild-type RABV. However, the molecular mechanisms that regulate the cellular adaptation and pathogenicity remain obscure. In this work, we isolated a wild-type RABV (CNIM1701) from a rabid bovine in northern China. The original CNIM1701 was lethal in adult mice and restricted replication in cell cultures. After 20 serial passages in the brains of suckling mice, the virus was renamed CNIM1701-P20, which was safe in adult mice and replicated well in cell cultures. In addition, sequence comparison analysis of the original CNIM1701 and CNIM1701-P20 identified 2 amino acid substitutions on G protein (Lys83 → Arg83 and Pro367 → Ser 367) related to pathogenesis and cellular adaptation. Using site-directed mutagenesis to exchange Lys83 with Arg83 and Pro367 with Ser 367 in the G protein of the RABV SAD strain, the pathogenicity of rSAD-K83R was significantly decreased. Our data indicate that the decreased pathogenicity of rSAD-K83R is due to increasing the expression of RABV-G, which also induced a higher level of apoptosis in infected cells. Furthermore, the K83 mutation induced high expression of MMP-2 and MMP-9 on DCs and promoted blood–brain barrier (BBB) permeability. These results demonstrate that the pathogenesis of RABV is partially dependent on G expression and BBB permeability, which may help in the design and development of highly safe, live-RABV vaccines.

scholarly journals The Appropriate Marker for Astrocytes: Comparing the Distribution and Expression of Three Astrocytic Markers in Different Mouse Cerebral Regions

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Zengli Zhang ◽  
Zhi Ma ◽  
Wangyuan Zou ◽  
Hang Guo ◽  
Min Liu ◽  
...  
Keyword(s):  
Corpus Callosum ◽  
Protein Expression ◽  
Morphological Characteristics ◽  
Scientific Investigation ◽  
Cerebral Peduncle ◽  
Dorsal Cortex ◽  
Expression Levels ◽  
Adult Mice ◽  
Gfap Immunoreactivity

Astrocytes possess different morphological characteristics depending on the cerebral region in which they are found. However, none of the current astrocytic markers can label all subpopulations successfully. Thus, identifying the appropriate marker for a specific scientific investigation is critical. Here, we compared the distribution and protein expression of three astrocyte markers: NDRG2, GFAP, and S100β, in the cortex, hippocampus, and thalamus. NDRG2- and S100β-positive astrocytes were distributed more uniformly than GFAP-positive astrocytes throughout the whole cerebrum. NDRG2 and S100βimmunoreactivities were the strongest in the dorsal cortex and thalamus, while GFAP immunoreactivity was the strongest in the hippocampus. Moreover, protein expression levels of NDRG2, GFAP, and S100βin adult mice were the highest in the cortex, hippocampus, and thalamus, respectively. We also detected astrocyte morphology and found that, in the corpus callosum and cerebral peduncle, GFAP-positive astrocytes were found with more numerous and longer processes than NDRG2- and S100β-positive astrocytes. These results demonstrate that NDRG2 and S100βare more suitably used to visualize the overall distribution and changes in the number of astrocytes, as well as label astrocytes in the cortex and thalamus. GFAP, however, is more appropriately used to label astrocytes in the corpus callosum, cerebral peduncle, and the hippocampus. These results help to guide researchers in the choice of appropriate astrocyte marker and suggest differences in immunological qualities of astrocytes based on the tissue in which they are found.

scholarly journals Matrix Protein of Rabies Virus Is Responsible for the Assembly and Budding of Bullet-Shaped Particles and Interacts with the Transmembrane Spike Glycoprotein G

1999 ◽  
Vol 73 (1) ◽  
pp. 242-250 ◽  
Author(s):  
Teshome Mebatsion ◽  
Frank Weiland ◽  
Karl-Klaus Conzelmann
Keyword(s):  
Rabies Virus ◽  
Matrix Protein ◽  
M Protein ◽  
Virus Budding ◽  
Glycoprotein G ◽  
Severe Impairment ◽  
Rnp Complex ◽  
Long Rod ◽  
Nucleoprotein N

ABSTRACT To elucidate the functions of rhabdovirus matrix (M) protein, we determined the localization of M in rabies virus (RV) and analyzed the properties of an M-deficient RV mutant. We provide evidence that M completely covers the ribonucleoprotein (RNP) coil and keeps it in a condensed form. As determined by cosedimentation experiments, not only the M-RNP complex but also M alone was found to interact specifically with the glycoprotein G. In contrast, an interaction of G with the nucleoprotein N or M-less RNP was not observed. In the absence of M, infectious particles were mainly cell associated and the yield of cell-free infectious virus was reduced by as much as 500,000-fold, demonstrating the crucial role of M in virus budding. Supernatants from cells infected with the M-deficient RV did not contain the typical bullet-shaped rhabdovirus particles but instead contained long, rod-shaped virions, demonstrating severe impairment of the virus formation process. Complementation with M protein expressed from plasmids rescued rhabdovirus formation. These results demonstrate the pivotal role of M protein in condensing and targeting the RNP to the plasma membrane as well as in incorporation of G protein into budding virions.

scholarly journals Indigenous Wildlife Rabies in Taiwan: Ferret Badgers, a Long Term Terrestrial Reservoir

2017 ◽  
Vol 2017 ◽  
pp. 1-6 ◽  
Author(s):  
Yu-Ching Lan ◽  
Tzai-Hung Wen ◽  
Chao-chin Chang ◽  
Hsin-Fu Liu ◽  
Pei-Fen Lee ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Temporal Dynamics ◽  
Animal Health ◽  
Glycoprotein G ◽  
Coalescent Approach ◽  
Coalescent Tree ◽  
Southern Taiwan ◽  
World Organization ◽  
Nucleoprotein N

The emerging disease of rabies was confirmed in Taiwan ferret badgers (FBs) and reported to the World Organization for Animal Health (OIE) on July 17, 2013. The spread of wildlife rabies can be related to neighborhood countries in Asia. The phylogenetic analysis was conducted by maximum likelihood (ML) methods and the Bayesian coalescent approach based on the glycoprotein (G) and nucleoprotein (N) genes. The phylogeographic and spatial temporal dynamics of viral transmission were determined by using SPREAD, QGIS. Therefore, the origin and the change with time of the viruses can be identified. Results showed the rabies virus of FB strains in Taiwan is a unique clade among other strains in Asia. According to the phylogeographic coalescent tree, three major genotypes of the FB rabies virus have circulated in three different geographical areas in Taiwan. Two genotypes have distributed into central and southern Taiwan between two ecological river barriers. The third genotype has been limited in southeastern Taiwan by the natural mountain barrier. The diversity of FB rabies viruses indicates that the biological profile of FBs could vary in different geographical areas in Taiwan. An enhanced surveillance system needs to be established near the currently identified natural barriers for early warnings of the rabies virus outbreak in Taiwan.

scholarly journals Definition of the immune evasion-replication interface of rabies virus P protein

2021 ◽  
Vol 17 (7) ◽  
pp. e1009729
Author(s):  
Jingyu Zhan ◽  
Angela R. Harrison ◽  
Stephanie Portelli ◽  
Thanh Binh Nguyen ◽  
Isshu Kojima ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Immune Evasion ◽  
Structural Data ◽  
Direct Analysis ◽  
Multifunctional Protein ◽  
N Protein ◽  
Binding Interface ◽  
P Protein ◽  
Definition Of ◽  
Nucleoprotein N

Rabies virus phosphoprotein (P protein) is a multifunctional protein that plays key roles in replication as the polymerase cofactor that binds to the complex of viral genomic RNA and the nucleoprotein (N protein), and in evading the innate immune response by binding to STAT transcription factors. These interactions are mediated by the C-terminal domain of P (PCTD). The colocation of these binding sites in the small globular PCTD raises the question of how these interactions underlying replication and immune evasion, central to viral infection, are coordinated and, potentially, coregulated. While direct data on the binding interface of the PCTD for STAT1 is available, the lack of direct structural data on the sites that bind N protein limits our understanding of this interaction hub. The PCTD was proposed to bind via two sites to a flexible loop of N protein (Npep) that is not visible in crystal structures, but no direct analysis of this interaction has been reported. Here we use Nuclear Magnetic Resonance, and molecular modelling to show N protein residues, Leu381, Asp383, Asp384 and phosphor-Ser389, are likely to bind to a ‘positive patch’ of the PCTD formed by Lys211, Lys214 and Arg260. Furthermore, in contrast to previous predictions we identify a single site of interaction on the PCTD by this Npep. Intriguingly, this site is proximal to the defined STAT1 binding site that includes Ile201 to Phe209. However, cell-based assays indicate that STAT1 and N protein do not compete for P protein. Thus, it appears that interactions critical to replication and immune evasion can occur simultaneously with the same molecules of P protein so that the binding of P protein to activated STAT1 can potentially occur without interrupting interactions involved in replication. These data suggest that replication complexes might be directly involved in STAT1 antagonism.

scholarly journals Structural and Evolutionary Insights Into the Binding of Host Receptors by the Rabies Virus Glycoprotein

2021 ◽  
Vol 11 ◽  
Author(s):  
Manar E. Khalifa ◽  
Leonie Unterholzner ◽  
Muhammad Munir
Keyword(s):  
G Protein ◽  
Rabies Virus ◽  
Viral Evolution ◽  
Bioinformatic Analysis ◽  
Susceptible Host ◽  
Acetyl Choline ◽  
Host Interactions ◽  
Protein Protein Interaction ◽  
Glycoprotein G ◽  
Typical Model

Rabies represents a typical model for spillover of zoonotic viral diseases among multiple hosts. Understanding the success of rabies virus (RV) in switching hosts requires the analysis of viral evolution and host interactions. In this study, we have investigated the structural and sequence analysis of host receptors among different RV susceptible host species. Our extensive bioinformatic analysis revealed the absence of the integrin plexin domain in the integrin β1 (ITGB1) receptor of the black fruit bats in the current annotation of the genome. Interestingly, the nicotinic acetyl choline receptor (nAChR) interaction site with the glycoprotein (G) of RV was conserved among different species. To study the interaction dynamics between RV-G protein and the RV receptors, we constructed and analyzed structures of RV receptors and G proteins using homology modeling. The molecular docking of protein-protein interaction between RV-G protein and different host receptors highlighted the variability of interacting residues between RV receptors of different species. These in silico structural analysis and interaction mapping of viral protein and host receptors establish the foundation to understand complex entry mechanisms of RV entry, which may facilitate the understanding of receptor mediated spillover events in RV infections and guide the development of novel vaccines to contain the infection.

scholarly journals An Avirulent Mutant of Rabies Virus Is Unable To Infect Motoneurons In Vivo and In Vitro

1998 ◽  
Vol 72 (1) ◽  
pp. 273-278 ◽  
Author(s):  
Patrice Coulon ◽  
Jean-Pierre Ternaux ◽  
Anne Flamand ◽  
Christine Tuffereau
Keyword(s):  
Rabies Virus ◽  
Double Mutant ◽  
Antigenic Site ◽  
Lower Efficiency ◽  
Challenge Virus ◽  
Virus Challenge ◽  
Continuous Cell Lines ◽  
Adult Mice

ABSTRACT An antigenic double mutant of rabies virus (challenge virus standard [CVS] strain) was selected by successive use of two neutralizing antiglycoprotein monoclonal antibodies, both specific for antigenic site III. This mutant differed from the original virus strain by two amino acid substitutions in the ectodomain of the glycoprotein. The lysine in position 330 and the arginine in position 333 were replaced by asparagine and methionine, respectively. This double mutant was not pathogenic for adult mice. When injected intramuscularly into the forelimbs of adult mice, this virus could not penetrate the nervous system, either by the motor or by the sensory route, while respective single mutants infected motoneurons in the spinal cord and sensory neurons in the dorsal root ganglia. In vitro experiments showed that the double mutant was able to infect BHK cells, neuroblastoma cells, and freshly prepared embryonic motoneurons, albeit with a lower efficiency than the CVS strain. Upon further incubation at 37°C, the motoneurons became resistant to infection by the mutant while remaining permissive to CVS infection. These results suggest that rabies virus uses different types of receptors: a molecule which is ubiquitously expressed at the surface of continuous cell lines and which is recognized by both CVS and the double mutant and a neuron-specific molecule which is not recognized by the double mutant.

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