Studies of an Influenza A Virus Temperature-Sensitive Mutant Identify a Late Role for NP in the Formation of Infectious Virions (cid:1)

The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperature-sensitive ( ts ) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited

[1]  Pang-Chui Shaw,et al.  Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design , 2008, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[2]  J. Gog,et al.  Mutational Analysis of cis-Acting RNA Signals in Segment 7 of Influenza A Virus , 2008, Journal of Virology.

[3]  R. Lamb,et al.  The Influenza Virus M2 Protein Cytoplasmic Tail Interacts with the M1 Protein and Influences Virus Assembly at the Site of Virus Budding , 2008, Journal of Virology.

[4]  Radostin Danev,et al.  Zernike phase contrast electron microscopy of ice-embedded influenza A virus. , 2008, Journal of structural biology.

[5]  R. Lamb,et al.  Mechanisms for enveloped virus budding: can some viruses do without an ESCRT? , 2008, Virology.

[6]  Yuko Morikawa,et al.  Visualization of microtubule-mediated transport of influenza viral progeny ribonucleoprotein. , 2007, Microbes and infection.

[7]  E. Medcalf,et al.  Identification of the domains of the influenza A virus M1 matrix protein required for NP binding, oligomerization and incorporation into virions , 2007, The Journal of general virology.

[8]  R. Lamb,et al.  Influenza Virus Hemagglutinin and Neuraminidase, but Not the Matrix Protein, Are Required for Assembly and Budding of Plasmid-Derived Virus-Like Particles , 2007, Journal of Virology.

[9]  S. Boulo,et al.  Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes. , 2007, Virus research.

[10]  J. Gog,et al.  Codon conservation in the influenza A virus genome defines RNA packaging signals , 2007, Nucleic acids research.

[11]  R. Pettersson,et al.  The Glycoprotein Cytoplasmic Tail of Uukuniemi Virus (Bunyaviridae) Interacts with Ribonucleoproteins and Is Critical for Genome Packaging , 2007, Journal of Virology.

[12]  M. Amorim,et al.  Nuclear Export of Influenza A Virus mRNAs Requires Ongoing RNA Polymerase II Activity , 2007, Traffic.

[13]  R. Krug,et al.  The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA , 2006, Nature.

[14]  Giovanni Cardone,et al.  Influenza virus pleiomorphy characterized by cryoelectron tomography , 2006, Proceedings of the National Academy of Sciences.

[15]  E. Medcalf,et al.  Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexes , 2006, Virology Journal.

[16]  A. Pekosz,et al.  Distinct Domains of the Influenza A Virus M2 Protein Cytoplasmic Tail Mediate Binding to the M1 Protein and Facilitate Infectious Virus Production , 2006, Journal of Virology.

[17]  Yukiko Muramoto,et al.  The Cytoplasmic Tail of the Influenza A Virus M2 Protein Plays a Role in Viral Assembly , 2006, Journal of Virology.

[18]  M. Amorim,et al.  ‘Genome gating’; polarized intranuclear trafficking of influenza virus RNPs , 2005, Biology Letters.

[19]  Andrew Pekosz,et al.  The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging , 2005, Journal of Virology.

[20]  J. Skehel,et al.  Influenza A Viruses with Mutations in the M1 Helix Six Domain Display a Wide Variety of Morphological Phenotypes , 2005, Journal of Virology.

[21]  M. Amorim,et al.  Increased amounts of the influenza virus nucleoprotein do not promote higher levels of viral genome replication. , 2004, The Journal of general virology.

[22]  M. Amorim,et al.  Lipid Raft‐Dependent Targeting of the Influenza A Virus Nucleoprotein to the Apical Plasma Membrane , 2004, Traffic.

[23]  Shinji Watanabe,et al.  Influenza B Virus BM2 Protein Is a Crucial Component for Incorporation of Viral Ribonucleoprotein Complex into Virions during Virus Assembly , 2004, Journal of Virology.

[24]  Z. Ye,et al.  Introduction of a Temperature-Sensitive Phenotype into Influenza A/WSN/33 Virus by Altering the Basic Amino Acid Domain of Influenza Virus Matrix Protein , 2004, Journal of Virology.

[25]  G. Brownlee,et al.  Model Suggesting that Replication of Influenza Virus Is Regulated by Stabilization of Replicative Intermediates , 2004, Journal of Virology.

[26]  A. Osterhaus,et al.  Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments. , 2004, Virus research.

[27]  P. Digard,et al.  Functional domains of the influenza A virus PB2 protein: identification of NP- and PB1-binding sites. , 2004, Virology.

[28]  W. Barclay,et al.  The M1 matrix protein controls the filamentous phenotype of influenza A virus. , 2004, Virology.

[29]  C. Müller,et al.  Crystal structure of the M1 protein‐binding domain of the influenza A virus nuclear export protein (NEP/NS2) , 2003, The EMBO journal.

[30]  A. García-Sastre,et al.  Reverse genetics studies on the filamentous morphology of influenza A virus. , 2003, The Journal of general virology.

[31]  Z. Ye,et al.  Association of influenza virus matrix protein with ribonucleoproteins may control viral growth and morphology. , 2002, Virology.

[32]  D. Ellis,et al.  A functional link between the actin cytoskeleton and lipid rafts during budding of filamentous influenza virions. , 2002, Virology.

[33]  P. Digard,et al.  The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. , 2002, The Journal of general virology.

[34]  P. Digard,et al.  Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase. , 2002, Nucleic acids research.

[35]  J. Galarza,et al.  Formation of Wild-Type and Chimeric Influenza Virus-Like Particles following Simultaneous Expression of Only Four Structural Proteins , 2001, Journal of Virology.

[36]  J. McCauley,et al.  Edinburgh Research Explorer Interaction of the influenza virus nucleoprotein with the cellular CRM1-mediated nuclear export pathway , 2022 .

[37]  P. Gómez-Puertas,et al.  Influenza Virus Matrix Protein Is the Major Driving Force in Virus Budding , 2000, Journal of Virology.

[38]  Ayub Ali,et al.  Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein , 2000, Journal of Virology.

[39]  R. Lamb,et al.  The cytoplasmic tails of the influenza virus spike glycoproteins are required for normal genome packaging. , 2000, Virology.

[40]  P. Digard,et al.  Temperature-Sensitive Lesions in Two Influenza A Viruses Defective for Replicative Transcription Disrupt RNA Binding by the Nucleoprotein , 1999, Journal of Virology.

[41]  P. Digard,et al.  Edinburgh Research Explorer Identification of amino acid residues of influenza virus nucleoprotein essential for RNA binding , 2022 .

[42]  Z. Ye,et al.  Association of Influenza Virus Matrix Protein with Ribonucleoproteins , 1999, Journal of Virology.

[43]  E. Medcalf,et al.  Modulation of Nuclear Localization of the Influenza Virus Nucleoprotein through Interaction with Actin Filaments , 1999, Journal of Virology.

[44]  P. L. Boutz,et al.  Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins , 1998, Journal of Virology.

[45]  D. Nayak,et al.  Association of influenza virus NP and M1 proteins with cellular cytoskeletal elements in influenza virus-infected cells , 1997, Journal of virology.

[46]  R. Lamb,et al.  Influenza virus hemagglutinin and neuraminidase cytoplasmic tails control particle shape , 1997, The EMBO journal.

[47]  R. Lamb,et al.  Characterization of the membrane association of the influenza virus matrix protein in living cells. , 1996, Virology.

[48]  G. Blobel,et al.  Nuclear Import of Influenza Virus RNA Can Be Mediated by Viral Nucleoprotein and Transport Factors Required for Protein Import (*) , 1995, The Journal of Biological Chemistry.

[49]  A. Kendal,et al.  Role of the M2 protein in influenza virus membrane fusion: effects of amantadine and monensin on fusion kinetics. , 1993, Virology.

[50]  K. Martin,et al.  Nuclear transport of influenza virus ribonucleoproteins: The viral matrix protein (M1) promotes export and inhibits import , 1991, Cell.

[51]  C. Scholtissek,et al.  Mutants and revertants of an avian influenza A virus with temperature-sensitive defects in the nucleoprotein and PB2. , 1991, Virology.

[52]  A Helenius,et al.  Transport of incoming influenza virus nucleocapsids into the nucleus , 1991, Journal of virology.

[53]  P. Palese,et al.  Determination of influenza virus proteins required for genome replication , 1990, Journal of virology.

[54]  A. Ishihama,et al.  Reconstitution of influenza virus RNA-nucleoprotein complexes structurally resembling native viral ribonucleoprotein cores. , 1990, The Journal of biological chemistry.

[55]  C. Scholtissek,et al.  Localisation of the temperature-sensitive defect in the nucleoprotein of an influenza A/FPV/Rostock/34 virus. , 1989, Virus research.

[56]  A. Ishihama,et al.  RNA polymerase of influenza virus: role of NP in RNA chain elongation. , 1988, Journal of Biochemistry (Tokyo).

[57]  R. Krug,et al.  Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an added primer , 1988, Journal of virology.

[58]  R. Krug,et al.  Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5' capped end. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[59]  P. Krijgsman,et al.  Natural heterogeneity of shape, infectivity and protein composition in an influenza A (H3N2) virus preparation. , 1985, Virus research.

[60]  I. Roditi,et al.  Nucleotide sequence of RNA segment 5, encoding the nucleoprotein, of influenza A/FPV/Rostock/34 , 1984 .

[61]  O. Danos,et al.  Use of specific single stranded DNA probes cloned in M13 to study the RNA synthesis of four temperature-sensitive mutants of HK/68 influenza virus. , 1982, Nucleic acids research.

[62]  Y. Ghendon,et al.  Influence of membrane (M) protein on influenza A virus virion transcriptase activity in vitro and its susceptibility to rimantadine , 1980, Journal of virology.

[63]  D. McGeoch,et al.  Temperature-sensitive mutants of fowl plague virus: isolation and genetic characterization. , 1979, Virology.

[64]  D. McGeoch,et al.  Method for assigning temperature-sensitive mutations of influenza viruses to individual segments of the genome. , 1977, Virology.

[65]  C. Scholtissek,et al.  Isolation and characterization of temperature-sensitive mutants of fowl plague virus. , 1975, Virology.

[66]  R. Krug,et al.  Temperature-sensitive mutants of influenza WSN virus defective in virus-specific RNA synthesis , 1975, Journal of virology.

[67]  I. Schulze,et al.  The structure of influenza virus. II. A model based on the morphology and composition of subviral particles. , 1972, Virology.

[68]  C. Scholtissek,et al.  Binding of ribonucleic acids to the RNP-antigen protein of influenza viruses. , 1971, The Journal of general virology.

[69]  R. Chanock,et al.  Mutants of influenza virus. , 1969, British medical journal.

[70]  A. Isaacs,et al.  Counts of influenza virus particles. , 1954, Journal of general microbiology.