Characterization of novel immunomodulatory proteins encoded by parapoxvirus ORF virus

Orf or contagious ecthyma is a ubiquitous disease of sheep and goats caused by the parapoxvirus (PPV) orf virus (ORFV). The disease is characterized by focal lesions in the mucocutaneous transitions of the mouth that progress from maculae to papulae, pustules and scabs, and usually resolve in 6-8 weeks. ORFV preparations have long been used in Veterinary Medicine as a preventive and therapeutic immunomodulatory agent. However, the functions (genes, proteins, and mechanisms of action) involved in immunomodulation remain poorly understood. ORFV establishes infection of keratinocytes in a proinflammatory environment triggered by skin abrasions. Thus, it is likely ORFV evolved unique mechanisms to modulate inflammatory responses and innate immune signaling very early in infection. In this thesis, the roles in immune evasion of two genes unique to PPV, ORFV073 and ORFV113, were investigated. ORFV073 was shown to encode for a virion NF-κB inhibitor that functions very early in infected primary ovine cells (OFTu). In the present study, a role of ORFV073 in immune evasion and virulence was investigated. Infection with a ORFV in which ORFV073 has been deleted (OV-IA82Δ073) led to increased accumulation of NF-κB essential modulator (NEMO), marked phosphorylation of IκB kinase (IKK) subunits IKKα and IKKβ, IκBα and NF-κB subunit p65 (NF-κB-p65), and to early nuclear translocation of NF-κB-p65 in virus-infected cells (≤ 30 min post infection). Consistent with observed inhibition of IKK complex activation, ORFV073 interacted with the regulatory subunit of the IKK complex NEMO. Infection of sheep with OV-IA82Δ073 led to virus attenuation, indicating that ORFV073 is a virulence determinant in the natural host. ORFV073 represents the first poxviral virion-associated NF-κB inhibitor described, highlighting the significance of early inhibition of NF-κB signaling in virus infected cells. ORFV113 was shown to enhance p38 kinase phosphorylation in ORFV infected OFTu cells, cells transiently expressing ORFV113 and in cells treated with soluble ORFV113. Infection of cells with ORFV113 gene deletion virus (OV-IA82Δ113) significantly decreased p38 phosphorylation and reduced the size of ORFV plaques. Infection of cells with ORFV in the presence of p38 inhibitor markedly diminished ORFV plaque formation and ORFV replication, highlighting importance of p38 signaling during ORFV infection. Consistent with the activation of p38 signaling, ORFV113 interacted with Lysophosphatidic acid receptor 1 (LPA1), a G protein coupled receptor. ORFV113 enhancement of p38 activation was prevented by treatment of cell cultures with LPA1 inhibitor, and in LPA1 siRNA and CRISPR knock down cells. Additionally, treatment of cells with Lysophosphatidic acid (LPA) and antibody against LPA1 enhanced ORFV113 induced p38 phosphorylation. Infection of sheep with OV-IA82Δ113 led to a strikingly attenuated disease phenotype, indicating that ORFV113 is a major virulence determinant in the natural host.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

VMS - Pathobiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2020

Author dc:creator

• Khatiwada, Sushil

Contributors dc:contributor

• Rock, Daniel L
• Yoo, Dongwan
• Zuckermann, Federico
• Chen, Lin-Feng L

• Parapoxvirus
• Orf virus
• Immunomodulation
• Viral pathogenesis
• Virulence

Statement dc:rights

• Copyright 2020 Sushil Khatiwada

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/108676

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/108676