Identification of the Nuclear Export Signal and STAT-Binding Domains of the Nipah Virus V Protein Reveals Mechanisms Underlying Interferon Evasion

ABSTRACT The V proteins of Nipah virus and Hendra virus have been demonstrated to bind to cellular STAT1 and STAT2 proteins to form high-molecular-weight complexes that inhibit interferon (IFN)-induced antiviral transcription by preventing STAT nuclear accumulation. Analysis of the Nipah virus V protein has revealed a region between amino acids 174 and 192 that functions as a CRM1-dependent nuclear export signal (NES). This peptide is sufficient to complement an export-defective human immunodeficiency virus Rev protein, and deletion and substitution mutagenesis revealed that this peptide is necessary for both V protein shuttling and cytoplasmic retention of STAT1 and STAT2 proteins. However, the NES is not required for V-dependent IFN signaling inhibition. IFN signaling is blocked primarily by interaction between Nipah virus V residues 100 to 160 and STAT1 residues 509 to 712. Interaction with STAT2 requires a larger Nipah virus V segment between amino acids 100 and 300, but deletion of residues 230 to 237 greatly reduced STAT2 coprecipitation. Further, V protein interactions with cellular STAT1 is a prerequisite for STAT2 binding, and sequential immunoprecipitations demonstrate that V, STAT1, and STAT2 can form a tripartite complex. These findings characterize essential regions for Henipavirus V proteins that represent potential targets for therapeutic intervention.

[1]  Edward Yang,et al.  Dissociation Time from DNA Determines Transcriptional Function in a STAT1 Linker Mutant* , 2002, The Journal of Biological Chemistry.

[2]  C. Horvath,et al.  STAT2 Acts as a Host Range Determinant for Species-Specific Paramyxovirus Interferon Antagonism and Simian Virus 5 Replication , 2002, Journal of Virology.

[3]  C. Horvath,et al.  Paramyxoviruses SV5 and HPIV2 assemble STAT protein ubiquitin ligase complexes from cellular components. , 2002, Virology.

[4]  S. Goodbourn,et al.  Sendai Virus and Simian Virus 5 Block Activation of Interferon-Responsive Genes: Importance for Virus Pathogenesis , 1999, Journal of Virology.

[5]  C. Horvath,et al.  Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation , 2002, Journal of Virology.

[6]  K. Nagata,et al.  Measles virus V protein blocks interferon (IFN)‐α/β but not IFN‐γ signaling by inhibiting STAT1 and STAT2 phosphorylation , 2003 .

[7]  C. Horvath,et al.  STAT Protein Interference and Suppression of Cytokine Signal Transduction by Measles Virus V Protein , 2003, Journal of Virology.

[8]  Søren Brunak,et al.  NESbase version 1.0: a database of nuclear export signals , 2003, Nucleic Acids Res..

[9]  R. Lamb,et al.  The paramyxovirus SV5 V protein binds two atoms of zinc and is a structural component of virions. , 1995, Virology.

[10]  R. Lamb,et al.  The V proteins of simian virus 5 and other paramyxoviruses inhibit induction of interferon-beta. , 2002, Virology.

[11]  C. Samuel,et al.  Antiviral Actions of Interferons , 2001, Clinical Microbiology Reviews.

[12]  K. Nagata,et al.  Measles virus V protein blocks interferon (IFN)-alpha/beta but not IFN-gamma signaling by inhibiting STAT1 and STAT2 phosphorylation. , 2003, FEBS letters.

[13]  R. Lamb,et al.  The Paramyxovirus Simian Virus 5 V Protein Slows Progression of the Cell Cycle , 2000, Journal of Virology.

[14]  C. Horvath,et al.  Selective STAT Protein Degradation Induced by Paramyxoviruses Requires both STAT1 and STAT2 but Is Independent of Alpha/Beta Interferon Signal Transduction , 2002, Journal of Virology.

[15]  R. Lamb,et al.  The V protein of the paramyxovirus SV5 interacts with damage-specific DNA binding protein. , 1998, Virology.

[16]  J. Darnell,et al.  Crystal Structure of a Tyrosine Phosphorylated STAT-1 Dimer Bound to DNA , 1998, Cell.

[17]  J. Darnell,et al.  Signalling: STATs: transcriptional control and biological impact , 2002, Nature Reviews Molecular Cell Biology.

[18]  R. Lamb,et al.  The V protein of human parainfluenza virus 2 antagonizes type I interferon responses by destabilizing signal transducer and activator of transcription 2. , 2001, Virology.

[19]  H. Field,et al.  Nipah virus: a recently emergent deadly paramyxovirus. , 2000, Science.

[20]  S. Goodbourn,et al.  The V Protein of Simian Virus 5 Inhibits Interferon Signalling by Targeting STAT1 for Proteasome-Mediated Degradation , 1999, Journal of Virology.

[21]  T. Kubota,et al.  C terminal CYS-RICH region of mumps virus structural V protein correlates with block of interferon alpha and gamma signal transduction pathway through decrease of STAT 1-alpha. , 2001, Biochemical and biophysical research communications.

[22]  Nicole M. Bouvier,et al.  Newcastle Disease Virus (NDV)-Based Assay Demonstrates Interferon-Antagonist Activity for the NDV V Protein and the Nipah Virus V, W, and C Proteins , 2003, Journal of Virology.

[23]  C. Horvath,et al.  Hendra Virus V Protein Inhibits Interferon Signaling by Preventing STAT1 and STAT2 Nuclear Accumulation , 2003, Journal of Virology.

[24]  R. Lamb,et al.  Single Amino Acid Substitution in the V Protein of Simian Virus 5 Differentiates Its Ability To Block Interferon Signaling in Human and Murine Cells , 2001, Journal of Virology.

[25]  D. Aaronson,et al.  A Road Map for Those Who Don't Know JAK-STAT , 2002, Science.

[26]  R. Lamb,et al.  Recovery of paramyxovirus simian virus 5 with a V protein lacking the conserved cysteine-rich domain: the multifunctional V protein blocks both interferon-beta induction and interferon signaling. , 2002, Virology.

[27]  D. Levy,et al.  The virus battles: IFN induction of the antiviral state and mechanisms of viral evasion. , 2001, Cytokine & growth factor reviews.

[28]  J. Darnell,et al.  A STAT protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain. , 1995, Genes & development.

[29]  M. Tsurudome,et al.  High Resistance of Human Parainfluenza Type 2 Virus Protein-Expressing Cells to the Antiviral and Anti-Cell Proliferative Activities of Alpha/Beta Interferons: Cysteine-Rich V-Specific Domain Is Required for High Resistance to the Interferons , 2001, Journal of Virology.

[30]  C. Horvath,et al.  STAT3 Ubiquitylation and Degradation by Mumps Virus Suppress Cytokine and Oncogene Signaling , 2003, Journal of Virology.

[31]  Y. Ito,et al.  Recovery of infectious human parainfluenza type 2 virus from cDNA clones and properties of the defective virus without V-specific cysteine-rich domain. , 2001, Virology.

[32]  B. Henderson,et al.  A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals. , 2000, Experimental cell research.