EXCEED THE SPACE PROVIDED. The three critical events in the pathogenesis of varicella-zoster virus (VZV) infection and its transmission are viremia, cutaneous replication and neural latency. Our overall objectives are to define regulatory/tegument gene requirements for each of these pathogenic phases using in vitro methods and our SCIDhu model of VZV skin and T cell tropism, to develop a SCIDhu neural cell model to study VZV neurotropism and to further characterize the attenuated vaccine Oka (V-Oka) virus. We will use human neural implants established within the anterior chamber of the SCID mouse eye as the primary system for VZV neurotropism studies. As an alternative, we will explore VZV inoculation of SCID animals engrafted with neuronal stem cells. Development of one or both models will make it possible to assess whether mutations altering VZV replication in skin or T cells also affect VZV neurotropism in vivo. Investigations of regulatory/tegument proteins will focus on the immediate early (IE) proteins, IE62, IE63 and IE4. ORF62 encodes the major viral transactivator of VZV and IE63 appears to have accessory transactivating activity. We have shown that one copy of IE63 is essential and have made a single copy IE63 recombinant; work is in progress to generate a single copy ORF62 recombinant. These single copy constructs will be used to introduce ORF62 and ORF63 mutations into the viral genome using parent Oka (P-Oka) cosmids. Mutagenesis targets will be selected by mapping sites of IE62tlE63 interaction and identifying putative functional regions of ORFs 62 and 63 from sequence motifs or by conservation in alphaherpesvirus genes. Domains will be defined as essential or dispensable for replication in cell culture. We propose to characterize domains in IE4 protein related to IE62 binding, dimerization, transactivation, and nuclear/cytoplasmic localization. These analyses will define IE4 regions that must be intact for infectivity. Viable recombinants that have targeted mutations in IE62, IE63 or IE4 will be evaluated for effects on VZV replication in differentiated human cells in vivo in the SCIDhu model. In order to further investigate V-Oka attenuation, chimeric recombinants made from V-Oka and P-Oka cosmids will be evaluated in the SCIDhu skin and T cell xenografts. Finally, we will exploit the model to examine structural characteristics of P-Oka virions and to compare P-Oka and V-Oka virions. Using these experimental approaches, it should be possible to create VZV recombinants that lack the capacity to disseminate by infecting T cells, or to establish persistent infection in neural cells, while retaining the capacity to replicate in skin. A better understanding of the genetic mechanisms that are required for VZV virulence in skin, T cells and neural cells will guide the design of 'second generation' live attenuated varicella vaccines. PERFORMANCESITE( ========================================Section End===========================================

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI053846-03
Application #
6840396
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Beisel, Christopher E
Project Start
2003-01-01
Project End
2007-12-31
Budget Start
2005-01-01
Budget End
2005-12-31
Support Year
3
Fiscal Year
2005
Total Cost
$417,796
Indirect Cost
Name
Stanford University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Zerboni, Leigh; Sung, Phillip; Sommer, Marvin et al. (2018) The C-terminus of varicella-zoster virus glycoprotein M contains trafficking motifs that mediate skin virulence in the SCID-human model of VZV pathogenesis. Virology 523:110-120
Khalil, Mohamed I; Che, Xibing; Sung, Phillip et al. (2016) Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication. Virology 492:82-91
Sen, Nandini; Mukherjee, Gourab; Arvin, Ann M (2015) Single cell mass cytometry reveals remodeling of human T cell phenotypes by varicella zoster virus. Methods 90:85-94
Xing, Yi; Oliver, Stefan L; Nguyen, TuongVi et al. (2015) A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL. Proc Natl Acad Sci U S A 112:6056-61
Khalil, Mohamed I; Sommer, Marvin H; Hay, John et al. (2015) Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription. Virology 481:179-86
Khalil, Mohamed I; Ruyechan, William T; Hay, John et al. (2015) Differential effects of Sp cellular transcription factors on viral promoter activation by varicella-zoster virus (VZV) IE62 protein. Virology 485:47-57
Zerboni, Leigh; Sen, Nandini; Oliver, Stefan L et al. (2014) Molecular mechanisms of varicella zoster virus pathogenesis. Nat Rev Microbiol 12:197-210
Sen, Nandini; Mukherjee, Gourab; Sen, Adrish et al. (2014) Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus. Cell Rep 8:633-45
Khalil, Mohamed I; Sommer, Marvin; Arvin, Ann et al. (2014) Cellular transcription factor YY1 mediates the varicella-zoster virus (VZV) IE62 transcriptional activation. Virology 449:244-53
Wang, Li; Rajamani, Jaya; Sommer, Marvin et al. (2013) Identification of a hydrophobic domain in varicella-zoster virus ORF61 necessary for ORF61 self-interaction, viral replication, and skin pathogenesis. J Virol 87:4075-9

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