Unique amongst the DNA viruses, poxviruses complete their replicative cycles entirely within the cytoplasmic compartment of infected cells. This property requires that these viruses be able to express, activate, localize and concentrate their encoded gene products at the correct intracellular sites to enable the assembly of infectious progeny. The process becomes more complicated due to the fact that poxiviruses produce a number of different virion forms (IMV, intracellular mature virus; IEV, intracellular enveloped virus: CEV, cell-associated enveloped virus; and EEV, extacellular enveloped virus), all of which may play different roles in vivo. Furthermore, some poxviruses (e.g. Cowpox) also produce yet another form of virion which is occluded in an inclusion body, presumably to facilitate the stability and dissemination of infectious particles in nature. To help direct viral protein traffic, poxviruses such as vaccinia virus have adopted many of the same protein modification, activation and targeting pathways used by their cellular hosts including glycosylation, phosphorylation, proteolytic processing, and in particular, acylation. During the previous grant period, our laboratory identified more than a dozen vaccinia acylproteins, both myristylproteins and palmitylproteins and demonstrated that they have key roles in the assembly of vaccinia virions. In the experiments detailed for the upcoming grant period, we now propose to focus and extend our studies in this area by concentrating on three vaccinia virus acyl protein which play central roles in VV replication processes: L1R(a 25 kDa N-terminally myristylated protein involved in IMV maturation and/or penetration), F13L(a 37 kDa palmitylated protein required for EEV envelopment), and the ATI gene product (a 92kDa protein homologue of the CPV 160 kDa ATI protein involved in the occlusion process, which contains a novel internal myristylation modification). For each protein we will use a combination of genetic, biochemical and molecular biological approaches: i) to study the nature, site and enzymology of the modification reaction; (ii) to examine what the function of the acyl modification is relative to the location and activity of the protein; and iii) to determine what biological role these proteins play during the VV replicative process itself.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI021335-13
Application #
2442415
Study Section
Experimental Virology Study Section (EVR)
Project Start
1983-12-01
Project End
2001-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
13
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Oregon State University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
053599908
City
Corvallis
State
OR
Country
United States
Zip Code
97339
Alzhanova, Dina; Hruby, Dennis E (2007) A host cell membrane protein, golgin-97, is essential for poxvirus morphogenesis. Virology 362:421-7
Alzhanova, Dina; Hruby, Dennis E (2006) A trans-Golgi network resident protein, golgin-97, accumulates in viral factories and incorporates into virions during poxvirus infection. J Virol 80:11520-7
Blouch, Robert E; Byrd, Chelsea M; Hruby, Dennis E (2005) Importance of disulphide bonds for vaccinia virus L1R protein function. Virol J 2:91
Yoder, Jennifer D; Chen, Tsefang; Hruby, Dennis E (2004) Sequence-independent acylation of the vaccinia virus A-type inclusion protein. Biochemistry 43:8297-302
Chen, Tsefang F; Yoder, Jennifer D; Hruby, Dennis E (2004) Mass spectrometry analysis of synthetically myristoylated peptides. Eur J Mass Spectrom (Chichester, Eng) 10:501-8
Chen, Tsefang S; Yoder, Jennifer D; Hruby, Dennis E (2003) Preparation of a large hydrophobic protein for mass spectrometry analysis: vaccina virus ATI protein. Anal Biochem 315:277-80
Grosenbach, D W; Hansen, S G; Hruby, D E (2000) Identification and analysis of vaccinia virus palmitylproteins. Virology 275:193-206
Hansen, S G; Grosenbach, D W; Hruby, D E (1999) Analysis of the site occupancy constraints of primary amino acid sequences in the motif directing palmitylation of the vaccinia virus 37-kDa envelope protein. Virology 254:124-37
Martin, K H; Franke, C A; Hruby, D E (1999) Novel acylation of poxvirus A-type inclusion proteins. Virus Res 60:147-57
Grosenbach, D W; Hruby, D E (1998) Analysis of a vaccinia virus mutant expressing a nonpalmitylated form of p37, a mediator of virion envelopment. J Virol 72:5108-20

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