The long-term goal of this renewal application is to understand the molecular basis of pathogenicity of ,viruses belonging to the negative strand RNA (nsRNA) family using vesicular stomatitis virus (VSV) as the prototype virus. The nsRNA viruses comprise a vast multitude of viruses that inflict profound damage and destruction to all living organisms including vertebrates, invertebrates, and plant kingdom. The viruses, such as rabies, measles, mumps, parainfluenza, respiratory syncitial, and many more fall in this nsRNA virus category. Recently, the emergence of novel pathogenic hanta and Ebola viruses has provided renewed impetus to delineate the molecular basis of pathogenicity of the nsRNA viruses. A thorough understanding of the mode of transcription and replication of these viruses is fundamental to develop reagents to combat these deadly pathogens. Our major emphasis toward this goal has been to establish the functions of the key viral proteins of VSV, such as L, the RNA polymerase, P, the transcription factor and N, the genome RNA-binding nucleocapsid protein. These proteins constitute the transcribing ribonucleoprotein (RNP) complex infection within the infected cells. We have been successful in expressing, in biologically active form, these polypeptides in procaryotic and eucaryotic cells using recombinant expression vectors. During the current granting period, we made several important discoveries specially with respect to the subunit composition of the L protein involving cellular translation elongation factors and the putative replicase subunit complex. In the current proposal, we intend to study in detail the structure and function of the transcriptase and replicase complexes of VSV using biochemical and reverse genetics approaches. We will investigate in detail (1) the structure and function of the L protein, specifically the role of translation elongation factor EF-1 alphabetagamma in L activity; (2) the structure and function of the putative replicase complex L-(N-P) and the roles of N-P complex and cellular functions in the replicase function; (3) the structure and function of the P protein with regard to the role of domain II phosphorylation in replication in vitro and using reverse genetics. We will undertake detail mutational studies to determine the precise functions of various domains in the P protein and characterize the unique protein kinase involved in rabies es virus P protein phosphorylation. These studies have the potential to gain insight into the biosynthetic pathways leading to transcription and replication of VSV genome RNA, which is fundamental to our understanding of the molecular basis of pathogenesis of VSV and other nsRNA viruses.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI026585-15
Application #
6631754
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Challberg, Mark D
Project Start
1988-08-01
Project End
2004-12-14
Budget Start
2003-04-01
Budget End
2004-12-14
Support Year
15
Fiscal Year
2003
Total Cost
$403,363
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Ogino, Tomoaki (2013) In vitro capping and transcription of rhabdoviruses. Methods 59:188-98
Ogino, Tomoaki; Banerjee, Amiya K (2011) An unconventional pathway of mRNA cap formation by vesiculoviruses. Virus Res 162:100-9
Ogino, Tomoaki; Yadav, Satya P; Banerjee, Amiya K (2010) Histidine-mediated RNA transfer to GDP for unique mRNA capping by vesicular stomatitis virus RNA polymerase. Proc Natl Acad Sci U S A 107:3463-8
Ogino, Tomoaki; Banerjee, Amiya K (2010) The HR motif in the RNA-dependent RNA polymerase L protein of Chandipura virus is required for unconventional mRNA-capping activity. J Gen Virol 91:1311-4
Sarkar, Anindya; Chattopadhyay, Santanu; Cox, Robert et al. (2010) Structural and functional properties of the vesicular stomatitis virus nucleoprotein-RNA complex as revealed by proteolytic digestion. Virology 401:61-9
Neznanov, Nickolay; Gorbachev, Anton V; Neznanova, Lubov et al. (2009) Anti-malaria drug blocks proteotoxic stress response: anti-cancer implications. Cell Cycle 8:3960-70
Neznanov, Nickolay; Dragunsky, Eugenia M; Chumakov, Konstantin M et al. (2008) Different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication. PLoS One 3:e1887
Ogino, Tomoaki; Banerjee, Amiya K (2008) Formation of guanosine(5')tetraphospho(5')adenosine cap structure by an unconventional mRNA capping enzyme of vesicular stomatitis virus. J Virol 82:7729-34
Chen, Mingzhou; Ogino, Tomoaki; Banerjee, Amiya K (2007) Interaction of vesicular stomatitis virus P and N proteins: identification of two overlapping domains at the N terminus of P that are involved in N0-P complex formation and encapsidation of viral genome RNA. J Virol 81:13478-85
Ogino, Tomoaki; Banerjee, Amiya K (2007) Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus. Mol Cell 25:85-97

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