The Tripartite Virions of the Brome Mosaic Virus Have Distinct Physical Properties That Affect the Timing of the Infection Process

ABSTRACT The three subsets of virions that comprise the Brome mosaic virus (BMV) were previously thought to be indistinguishable. This work tested the hypothesis that distinct capsid-RNA interactions in the BMV virions allow different rates of viral RNA release. Several results support distinct interactions between the capsid and the BMV genomic RNAs. First, the deletion of the first eight residues of the BMV coat protein (CP) resulted in the RNA1-containing particles having altered morphologies, while those containing RNA2 were unaffected. Second, subsets of the BMV particles separated by density gradients into a pool enriched for RNA1 (B1) and for RNA2 and RNA3/4 (B2.3/4) were found to have different physiochemical properties. Compared to the B2.3/4 particles, the B1 particles were more sensitive to protease digestion and had greater resistivity to nanoindentation by atomic force microscopy and increased susceptibility to nuclease digestion. Mapping studies showed that portions of the arginine-rich N-terminal tail of the CP could interact with RNA1. Mutational analysis in the putative RNA1-contacting residues severely reduced encapsidation of BMV RNA1 without affecting the encapsidation of RNA2. Finally, during infection of plants, the more easily released RNA1 accumulated to higher levels early in the infection. IMPORTANCE Viruses with genomes packaged in distinct virions could theoretically release the genomes at different times to regulate the timing of gene expression. Using an RNA virus composed of three particles, we demonstrated that the RNA in one of the virions is released more easily than the other two in vitro. The differential RNA release is due to distinct interactions between the viral capsid protein and the RNAs. The ease of RNA release is also correlated with the more rapid accumulation of that RNA in infected plants. Our study identified a novel role for capsid-RNA interactions in the regulation of a viral infection.

[1]  S. Lemon,et al.  Innate immune responses in hepatitis C virus infection , 2012, Seminars in Immunopathology.

[2]  S. Larson,et al.  Crystallographic structure of the T=1 particle of brome mosaic virus. , 2005, Journal of molecular biology.

[3]  John E. Johnson,et al.  Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics , 2006, Journal of Virology.

[4]  I. Molineux,et al.  Rate of translocation of bacteriophage T7 DNA across the membranes of Escherichia coli , 1995, Journal of bacteriology.

[5]  S. Larson,et al.  The crystallographic structure of brome mosaic virus. , 2002, Journal of molecular biology.

[6]  R. Markham,et al.  A study of the self-assembly process in a small spherical virus. Formation of organized structures from protein subunits in vitro. , 1967, Virology.

[7]  R. W. Siegel,et al.  Minimal templates directing accurate initiation of subgenomic RNA synthesis in vitro by the brome mosaic virus RNA-dependent RNA polymerase. , 1997, RNA.

[8]  Paul Ahlquist,et al.  Cytoplasmic Viral Replication Complexes , 2010, Cell Host & Microbe.

[9]  C. Kao,et al.  Norovirus RNA Synthesis Is Modulated by an Interaction between the Viral RNA-Dependent RNA Polymerase and the Major Capsid Protein, VP1 , 2012, Journal of Virology.

[10]  C. Kao,et al.  The coat protein leads the way: an update on basic and applied studies with the Brome mosaic virus coat protein. , 2011, Molecular plant pathology.

[11]  John E. Johnson,et al.  Virus capsid expansion driven by the capture of mobile surface loops. , 2008, Structure.

[12]  E. Domingo,et al.  Viral Quasispecies Evolution , 2012, Microbiology and Molecular Reviews.

[13]  A. Rao,et al.  Biological significance of the seven amino-terminal basic residues of brome mosaic virus coat protein. , 1995, Virology.

[14]  C. Kao,et al.  Interaction between Brome Mosaic Virus Proteins and RNAs: Effects on RNA Replication, Protein Expression, and RNA Stability , 2005, Journal of Virology.

[15]  C. Kao,et al.  RNA Synthesis by the Brome Mosaic Virus RNA-Dependent RNA Polymerase in Human Cells Reveals Requirements for De Novo Initiation and Protein-Protein Interaction , 2012, Journal of Virology.

[16]  C. Kao,et al.  Chemical reactivity of brome mosaic virus capsid protein. , 2012, Journal of molecular biology.

[17]  J. Taubenberger,et al.  The origin of the 1918 pandemic influenza virus: a continuing enigma. , 2003, The Journal of general virology.

[18]  Shizuo Akira,et al.  Innate immune recognition of viral infection , 2006, Nature Immunology.

[19]  K. Schulten,et al.  Squeezing protein shells: how continuum elastic models, molecular dynamics simulations, and experiments coalesce at the nanoscale. , 2010, Biophysical journal.

[20]  K. Van Reeth Avian and swine influenza viruses: our current understanding of the zoonotic risk. , 2007, Veterinary research.

[21]  Jason Cleveland,et al.  Finite optical spot size and position corrections in thermal spring constant calibration , 2004 .

[22]  Haley Hoover,et al.  The plant host can affect the encapsidation of brome mosaic virus (BMV) RNA: BMV virions are surprisingly heterogeneous. , 2014, Journal of molecular biology.

[23]  F. Niesen,et al.  The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability , 2007, Nature Protocols.

[24]  William M Gelbart,et al.  Forces and pressures in DNA packaging and release from viral capsids. , 2003, Biophysical journal.

[25]  Chandrajit L. Bajaj,et al.  VIPERdb: a relational database for structural virology , 2005, Nucleic Acids Res..

[26]  J. H. Strauss,et al.  The Alphaviruses: Gene Expression, Replication, and Evolution , 1994, Microbiological reviews.

[27]  M. Figlerowicz,et al.  Characterization of a Novel 5′ Subgenomic RNA3a Derived from RNA3 of Brome Mosaic Bromovirus , 2006, Journal of Virology.

[28]  Wah Chiu,et al.  An examination of the electrostatic interactions between the N-terminal tail of the Brome Mosaic Virus coat protein and encapsidated RNAs. , 2012, Journal of molecular biology.

[29]  P. Ahlquist,et al.  Brome mosaic virus RNA replication: revealing the role of the host in RNA virus replication. , 2003, Annual review of phytopathology.

[30]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[31]  J. Bujarski Bromovirus isolation and RNA extraction. , 1998, Methods in molecular biology.

[32]  C. Kao,et al.  Effects of amino-acid substitutions in the Brome mosaic virus capsid protein on RNA encapsidation. , 2010, Molecular plant-microbe interactions : MPMI.

[33]  W S Klug,et al.  Nanoindentation studies of full and empty viral capsids and the effects of capsid protein mutations on elasticity and strength. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. Götte,et al.  The distinct contributions of fitness and genetic barrier to the development of antiviral drug resistance. , 2012, Current opinion in virology.

[35]  P. Ni,et al.  Non-encapsidation activities of the capsid proteins of positive-strand RNA viruses , 2013, Virology.

[36]  B. Semler,et al.  Regulation of picornavirus gene expression. , 2004, Microbes and infection.

[37]  C. Kao,et al.  Mapping protein–RNA interactions , 2012 .