A Novel Nuclear Trafficking Module Regulates the Nucleocytoplasmic Localization of the Rabies Virus Interferon Antagonist, P Protein

Bats are reservoirs of many pathogenic viruses including the lyssaviruses rabies virus (RABV) and Australian bat lyssavirus (ABLV). Lyssavirus strains are closely associated with particular host reservoir species, with evidence of specific adaptation. Associated phenotypic changes remain poorly understood but are likely to involve P protein, a key mediator of the intracellular virus-host interface. Here, we examine the phenotype of P protein of ABLV, which circulates as two defined lineages associated with frugivorous and insectivorous bats, providing the opportunity compare proteins of viruses adapted to divergent bat species. We report that key functions of P protein in interferon/STAT1 antagonism and the capacity of P protein to undergo nuclear trafficking differ between lineages. Molecular mapping indicates that these differences are functionally distinct, and appear to involve modulatory effects on regulatory regions or structural impact, rather than changes to defined interaction sequences. This results in partial but significant phenotypic divergence, consistent with ‘fine-tuning’ to host biology, and with potentially distinct properties in the virus-host interface between bat families that represent key zoonotic reservoirs.

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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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1H, 15N and 13C resonance assignments of the C-terminal domain of the P protein of the Nishigahara strain of rabies virus

Biomolecular NMR Assignments ◽

10.1007/s12104-018-9841-4 ◽

2018 ◽

Vol 13 (1) ◽

pp. 5-8 ◽

Cited By ~ 1

Author(s):

Jingyu Zhan ◽

Md. Alamgir Hossain ◽

Ashish Sethi ◽

Toyoyuki Ose ◽

Gregory W. Moseley ◽

...

Keyword(s):

Rabies Virus ◽

Resonance Assignments ◽

Terminal Domain ◽

P Protein

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Interaction of the Rabies Virus P Protein with the LC8 Dynein Light Chain

Journal of Virology ◽

10.1128/jvi.74.21.10212-10216.2000 ◽

2000 ◽

Vol 74 (21) ◽

pp. 10212-10216 ◽

Cited By ~ 214

Author(s):

Hélène Raux ◽

Anne Flamand ◽

Danielle Blondel

Keyword(s):

Rabies Virus ◽

Light Chain ◽

Cytoplasmic Dynein ◽

Dynein Light Chain ◽

Directed Movement ◽

Virus Particles ◽

P Protein ◽

Infected Cells ◽

Transcription And Replication

ABSTRACT The rabies virus P protein is involved in viral transcription and replication but its precise function is not clear. We investigated the role of P (CVS strain) by searching for cellular partners by using a two-hybrid screening of a PC12 cDNA library. We isolated a cDNA encoding a 10-kDa dynein light chain (LC8). LC8 is a component of cytoplasmic dynein involved in the minus end-directed movement of organelles along microtubules. We confirmed that this molecule interacts with P by coimmunoprecipitation in infected cells and in cells transfected with a plasmid encoding P protein. LC8 was also detected in virus particles. Series of deletions from the N- and C-terminal ends of P protein were used to map the LC8-binding domain to the central part of P (residues 138 to 172). These results are relevant to speculate that dynein may be involved in the axonal transport of rabies virus along microtubules through neuron cells.

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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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Interaction of Rabies Virus P-Protein With STAT Proteins is Critical to Lethal Rabies Disease

The Journal of Infectious Diseases ◽

10.1093/infdis/jit829 ◽

2013 ◽

Vol 209 (11) ◽

pp. 1744-1753 ◽

Cited By ~ 40

Author(s):

Linda Wiltzer ◽

Kazuma Okada ◽

Satoko Yamaoka ◽

Florence Larrous ◽

Henna Veera Kuusisto ◽

...

Keyword(s):

Rabies Virus ◽

Stat Proteins ◽

P Protein

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Identification of a Role for Nucleolin in Rabies Virus Infection

Journal of Virology ◽

10.1128/jvi.03320-14 ◽

2014 ◽

Vol 89 (3) ◽

pp. 1939-1943 ◽

Cited By ~ 20

Author(s):

S. Oksayan ◽

J. Nikolic ◽

C. T. David ◽

D. Blondel ◽

D. A. Jans ◽

...

Keyword(s):

Rabies Virus ◽

Infectious Virus ◽

Virus Production ◽

Host Cells ◽

Nucleocytoplasmic Trafficking ◽

P Protein ◽

Rabies Virus Infection ◽

Infected Cells ◽

Protein Isoform ◽

Nucleolin Expression

Rabies virus replicates in the cytoplasm of host cells, but rabies virus phosphoprotein (P-protein) undergoes active nucleocytoplasmic trafficking. Here we show that the largely nuclear P-protein isoform P3 can localize to nucleoli and forms specific interactions with nucleolin. Importantly, depletion of nucleolin expression inhibits viral protein expression and infectious virus production by infected cells. This provides the first evidence that lyssaviruses interact with nucleolin and that nucleolin is important to lyssavirus infection.

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