Roles of the Rabies Virus Phosphoprotein Isoforms in Pathogenesis

ABSTRACTRabies virus (RABV) P gene mRNA encodes five in-frame start codons, resulting in expression of full-length P protein (P1) and N-terminally truncated P proteins (tPs), designated P2, P3, P4, and P5. Despite the fact that some tPs are known as interferon (IFN) antagonists, the importance of tPs in the pathogenesis of RABV is still unclear. In this study, to examine whether tPs contribute to pathogenesis, we exploited a reverse genetics approach to generate CE(NiP)ΔP2-5, a mutant of pathogenic CE(NiP) in which the P gene was mutated by replacing all of the start codons (AUG) for tPs with AUA. We confirmed that while CE(NiP) expresses detectable levels of P2 and P3, CE(NiP)ΔP2-5 has an impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)ΔP2-5 caused significantly lower morbidity and mortality rates in mice than did CE(NiP), indicating that tPs play a critical role in RABV neuroinvasiveness. Further examinations revealed that this less neuroinvasive phenotype of CE(NiP)ΔP2-5 correlates with its impaired ability to replicate in muscle cells, indicative of the importance of tPs in viral replication in muscle cells. We also demonstrated that CE(NiP)ΔP2-5 infection induced a higher level ofIfn-βgene expression in muscle cells than did CE(NiP) infection, consistent with the results of an IFN-β promoter reporter assay suggesting that all tPs function to antagonize IFN induction in muscle cells. Taken together, our findings strongly suggest that tPs promote viral replication in muscle cells through their IFN antagonist activities and thereby support infection of peripheral nerves.IMPORTANCEDespite the fact that previous studies have demonstrated that P2 and P3 of RABV have IFN antagonist activities, the actual importance of tPs in pathogenesis has remained unclear. Here, we provide the first evidence that tPs contribute to the pathogenesis of RABV, especially its neuroinvasiveness. Our results also show the mechanism underlying the neuroinvasiveness driven by tPs, highlighting the importance of their IFN antagonist activities, which support viral replication in muscle cells.

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Structural relationship between nucleocapsid-binding activity of the rabies virus phosphoprotein (P) and exposure of epitope 402-13 located at the C terminus

Journal of General Virology ◽

10.1099/0022-1317-83-12-3035 ◽

2002 ◽

Vol 83 (12) ◽

pp. 3035-3043 ◽

Cited By ~ 14

Author(s):

Harufusa Toriumi ◽

Yoshikazu Honda ◽

Kinjiro Morimoto ◽

Tadafumi S. Tochikura ◽

Akihiko Kawai

Keyword(s):

Rabies Virus ◽

Structural Changes ◽

Binding Activity ◽

Free Form ◽

Gene Products ◽

Epitope Region ◽

N Protein ◽

P Gene ◽

P Protein ◽

P Proteins

The structural changes of the nominal phosphoprotein (P) of rabies virus using a monoclonal antibody, mAb #402-13, was investigated. This mAb recognized a linear epitope that was mapped roughly to a C-terminal region of the P protein, ranging from aa 256 to 297. The P gene products were detected by the mAb in immunoblot assays, the products of which were produced either in BHK-21 cells or in Escherichia coli cells. The mAb, however, detected very low levels of P gene products in immunoprecipitation assays. The mAb recognized the nucleocapsid (NC)-associated P proteins but recognized free P protein and free N–P complex produced in the infected cells much less efficiently. When the P proteins were released from the NC, however, they were no longer recognized by the mAb. Similar results were obtained from BHK-21 cells co-transfected with P and N cDNAs. Furthermore, studies with C-terminally truncated P protein mutants revealed that the NC-binding ability of the P protein was dependent on the presence of the C-terminal epitope region. From these results, it is thought that the 402-13 epitope region is concealed when the P protein is present in a free form or free N–P complex but is exposed when it is associated with the NC. The C-terminal epitope region seemed to be essential for the P protein to be associated with the NC but not for the formation of free N–P complexes with newly synthesized N protein.

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Role of Interferon Antagonist Activity of Rabies Virus Phosphoprotein in Viral Pathogenicity

Journal of Virology ◽

10.1128/jvi.00011-10 ◽

2010 ◽

Vol 84 (13) ◽

pp. 6699-6710 ◽

Cited By ~ 61

Author(s):

Naoto Ito ◽

Gregory W. Moseley ◽

Danielle Blondel ◽

Kenta Shimizu ◽

Caitlin L. Rowe ◽

...

Keyword(s):

Rabies Virus ◽

Nuclear Export ◽

Nuclear Translocation ◽

Chicken Embryo Fibroblast ◽

Fibroblast Cell ◽

Chicken Embryo Fibroblast Cell ◽

Adult Mice ◽

P Gene ◽

P Protein ◽

P Proteins

ABSTRACT The fixed rabies virus (RV) strain Nishigahara kills adult mice after intracerebral inoculation, whereas the chicken embryo fibroblast cell-adapted strain Ni-CE causes nonlethal infection in adult mice. We previously reported that the chimeric CE(NiP) strain, which has the phosphoprotein (P protein) gene from the Nishigahara strain in the genetic background of the Ni-CE strain, causes lethal infection in adult mice, indicating that the P gene is responsible for the different pathogenicities of the Nishigahara and Ni-CE strains. Previous studies demonstrated that RV P protein binds to the interferon (IFN)-activated transcription factor STAT1 and blocks IFN signaling by preventing its translocation to the nucleus. In this study, we examine the molecular mechanism by which RV P protein determines viral pathogenicity by comparing the IFN antagonist activities of the Nishigahara and Ni-CE P proteins. The results, obtained from both RV-infected cells and cells transfected to express P protein only, show that Ni-CE P protein is significantly impaired for its capacity to block IFN-activated STAT1 nuclear translocation and, consequently, inhibits IFN signaling less efficiently than Nishigahara P protein. Further, it was demonstrated that a defect in the nuclear export of Ni-CE P protein correlates with a defect in its ability to cause the mislocalization of STAT1. These data provide the first evidence that the capacity of the RV P protein to inhibit STAT1 nuclear translocation and IFN signaling correlates with the viral pathogenicity.

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Interferon-Inducible GTPase 1 Impedes the Dimerization of Rabies Virus Phosphoprotein and Restricts Viral Replication

Journal of Virology ◽

10.1128/jvi.01203-20 ◽

2020 ◽

Vol 94 (21) ◽

Author(s):

Bin Tian ◽

Yueming Yuan ◽

Yu Yang ◽

Zhaochen Luo ◽

Baokun Sui ◽

...

Keyword(s):

Viral Replication ◽

Viral Infection ◽

Rabies Virus ◽

Parasite Infection ◽

Major Public Health Problem ◽

Intracellular Parasite ◽

Neurotropic Virus ◽

Pathogen Invasion ◽

P Protein

ABSTRACT Rabies, caused by rabies virus (RABV), is an ancient zoonosis and still a major public health problem for humans, especially in developing countries. RABV can be recognized by specific innate recognition receptors, resulting in the production of hundreds of interferon-stimulated genes (ISGs), which can inhibit viral replication at different stages. Interferon-inducible GTPase 1 (IIGP1) is a mouse-specific ISG and belongs to the immunity-related GTPases (IRGs) family. IIGP is reported to constrain intracellular parasite infection by disrupting the parasitophorous vacuole membrane. However, the role of IIGP1 in restricting viral replication has not been reported. In this present study, we found that IIGP1 was upregulated in cells and mouse brains upon RABV infection. Overexpression of IIGP1 limited RABV replication in cell lines and reduced viral pathogenicity in a mouse model. Consistently, deficiency of IIGP1 enhanced RABV replication in different parts of mouse brains. Furthermore, we found that IIGP1 could interact with RABV phosphoprotein (P protein). Mutation and immunoprecipitation analyses revealed that the Y128 site of P protein is critical for its interaction with IIGP1. Further study demonstrated that this interaction impeded the dimerization of P protein and thus suppressed RABV replication. Collectively, our findings for the first reveal a novel role of IIGP1 in restricting a typical neurotropic virus, RABV, which will provide fresh insight into the function of this mouse-specific ISG. IMPORTANCE Interferon and its downstream products, ISGs, are essential in defending against pathogen invasion. One of the ISGs, IIGP1, has been found to constrain intracellular parasite infection by disrupting their vacuole membranes. However, the role of IIGP1 in limiting viral infection is unclear. In this study, we show that infection with a typical neurotropic virus, RABV, can induce upregulation of IIGP1, which, in turn, suppresses RABV by interacting with its phosphoprotein (P protein) and thus blocking the dimerization of P protein. Our study provides the first evidence that IIGP1 functions in limiting viral infection and provides a basis for comprehensive understanding of this important ISG.

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

Journal of Virology ◽

10.1128/jvi.00826-17 ◽

2017 ◽

Vol 91 (20) ◽

Cited By ~ 4

Author(s):

Kento Nakagawa ◽

Yuki Kobayashi ◽

Naoto Ito ◽

Yoshiyuki Suzuki ◽

Kazuma Okada ◽

...

Keyword(s):

Rna Polymerase ◽

Rabies Virus ◽

Reverse Genetics ◽

Function Analysis ◽

Therapeutic Drug ◽

Molecular Function ◽

Content Type ◽

Complementation Assay ◽

L Protein ◽

P Protein

ABSTRACT 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.

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Defect of rabies virus phosphoprotein in its interferon-antagonist activity negatively affects viral replication in muscle cells

Journal of Veterinary Medical Science ◽

10.1292/jvms.17-0054 ◽

2017 ◽

Vol 79 (8) ◽

pp. 1394-1397 ◽

Cited By ~ 4

Author(s):

Satoko YAMAOKA ◽

Kazuma OKADA ◽

Naoto ITO ◽

Kota OKADERA ◽

Hiromichi MITAKE ◽

...

Keyword(s):

Viral Replication ◽

Rabies Virus ◽

Muscle Cells ◽

Antagonist Activity

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‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein

Australian Journal of Chemistry ◽

10.1071/ch19571 ◽

2020 ◽

Vol 73 (8) ◽

pp. 686

Author(s):

Ashley M. Rozario ◽

Fabian Zwettler ◽

Sam Duwé ◽

Riley B. Hargreaves ◽

Aaron Brice ◽

...

Keyword(s):

Rabies Virus ◽

Super Resolution ◽

Time Lapse ◽

Live Cells ◽

Conventional Imaging ◽

P Protein ◽

Conventional Microscopy ◽

P Proteins ◽

Super Resolution Microscopy

The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.

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The Phosphoprotein of Rabies Virus Is Phosphorylated by a Unique Cellular Protein Kinase and Specific Isomers of Protein Kinase C

Journal of Virology ◽

10.1128/jvi.74.1.91-98.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 91-98 ◽

Cited By ~ 63

Author(s):

Ashim K. Gupta ◽

Danielle Blondel ◽

Suresh Choudhary ◽

Amiya K. Banerjee

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinases ◽

Rabies Virus ◽

Cell Extract ◽

Site Directed Mutagenesis ◽

Virus Protein ◽

P Gene ◽

Kinase C ◽

P Protein

ABSTRACT The phosphoprotein (P) gene of rabies virus (CVS strain) was cloned and expressed in bacteria. The purified protein was used as the substrate for phosphorylation by the protein kinase(s) present in cell extract prepared from rat brain. Two distinct types of protein kinases, staurosporin sensitive and heparin sensitive, were found to phosphorylate the P protein in vitro by the cell extract. Interestingly, the heparin-sensitive kinase was not the ubiquitous casein kinase II present in a variety of cell types. Further purification of the cell fractions revealed that the protein kinase C (PKC) isomers constitute the staurosporin-sensitive kinases α, β, γ, and ζ, with the PKCγ isomer being the most effective in phosphorylating the P protein. A unique heparin-sensitive kinase was characterized as a 71-kDa protein with biochemical properties not demonstrated by any known protein kinases stored in the protein data bank. This protein kinase, designated RVPK (rabies virus protein kinase), phosphorylates P protein (36 kDa) and alters its mobility in gel to migrate at 40 kDa. In contrast, the PKC isoforms do not change the mobility of unphosphorylated P protein. RVPK appears to be packaged in the purified virions, to display biochemical characteristics similar to those of the cell-purified RVPK, and to similarly alter the mobility of endogenous P protein upon phosphorylation. By site-directed mutagenesis, the sites of phosphorylation of RVPK were mapped at S63 and S64, whereas PKC isomers phosphorylated at S162, S210, and S271. Involvement of a unique protein kinase in phosphorylating rabies virus P protein indicates its important role in the structure and function of the protein and consequently in the life cycle of the virus.

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Differential Transcription Attenuation of Rabies Virus Genes by Intergenic Regions: Generation of Recombinant Viruses Overexpressing the Polymerase Gene

Journal of Virology ◽

10.1128/jvi.74.16.7261-7269.2000 ◽

2000 ◽

Vol 74 (16) ◽

pp. 7261-7269 ◽

Cited By ~ 72

Author(s):

Stefan Finke ◽

James H. Cox ◽

Karl-Klaus Conzelmann

Keyword(s):

Gene Expression ◽

Rabies Virus ◽

Reverse Genetics ◽

Virus Protein ◽

Polymerase Gene ◽

P Gene ◽

Gene Delivery Vectors ◽

Intergenic Regions ◽

Transcription Attenuation

ABSTRACT 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.

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Corrigendum to: ‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein

Australian Journal of Chemistry ◽

10.1071/ch19571_co ◽

2020 ◽

Vol 73 (8) ◽

pp. 822

Author(s):

Ashley M. Rozario ◽

Fabian Zwettler ◽

Sam Duwé ◽

Riley B. Hargreaves ◽

Aaron Brice ◽

...

Keyword(s):

Rabies Virus ◽

Super Resolution ◽

Time Lapse ◽

Live Cells ◽

Conventional Imaging ◽

P Protein ◽

Conventional Microscopy ◽

P Proteins ◽

Super Resolution Microscopy

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The Nucleocytoplasmic Rabies Virus P Protein Counteracts Interferon Signaling by Inhibiting both Nuclear Accumulation and DNA Binding of STAT1

Journal of Virology ◽

10.1128/jvi.01930-06 ◽

2007 ◽

Vol 81 (8) ◽

pp. 4255-4263 ◽

Cited By ~ 98

Author(s):

Aurore Vidy ◽

Jamila El Bougrini ◽

Mounira K. Chelbi-Alix ◽

Danielle Blondel

Keyword(s):

Dna Binding ◽

Rabies Virus ◽

Binding Activity ◽

Nuclear Accumulation ◽

Mobility Shift ◽

P Gene ◽

Cell Extracts ◽

P Protein ◽

Ifn Γ ◽

In Cells

ABSTRACT Rabies virus P protein inhibits alpha interferon (IFN-α)- and IFN-γ-stimulated Jak-STAT signaling by retaining phosphorylated STAT1 in the cytoplasm. Here, we show that P also blocks an intranuclear step that is the STAT1 binding to the DNA promoter of IFN-responsive genes. As P is a nucleocytoplasmic shuttling protein, we first investigated the effect of the cellular distribution of P on the localization of STAT1 and consequently on IFN signaling. We show that the localization of STAT1 is correlated with the localization of P: in cells expressing a nuclear form of P (the short P3 isoform or the complete P in the presence of the export inhibitor leptomycin B), STAT1 is nuclear, whereas in cells expressing a cytoplasmic form of P, STAT1 is cytoplasmic. However, the expression of nuclear forms of P inhibits the signaling of both IFN-γ and IFN-α, demonstrating that the retention of STAT1 in the cytoplasm is not the only mechanism involved in the inhibition of IFN signaling. Electrophoretic mobility shift analysis indicates that P expression in the cell extracts of infected cells or in stable cell lines prevents IFN-induced DNA binding of STAT1. The loss of the DNA binding of STAT1 and ISGF3 was also observed when purified recombinant P or P3 was added to the extracts of IFN-γ- or IFN-α-treated cells, indicating that P directly affects the DNA binding activity of STAT1. Then products of the rabies virus P gene are able to counteract IFN signaling by creating both cytoplasmic and nuclear blocks for STAT1.

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