Innate immune modulation and pathogenic basis for porcine reproductive and respiratory syndrome virus

Ke, Hanzhong

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https://hdl.handle.net/2142/104997 Copy

Description

Title

Innate immune modulation and pathogenic basis for porcine reproductive and respiratory syndrome virus

Author(s)

Ke, Hanzhong

Issue Date

2019-04-09

Director of Research (if dissertation) or Advisor (if thesis)

Yoo, Dongwan

Doctoral Committee Chair(s)

Yoo, Dongwan

Committee Member(s)

• Rock, Daniel L
• Zuckermann, Federico A.
• Brooke, Christopher Byron

Department of Study

Pathobiology

Discipline

VMS - Pathobiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Porcine reproductive and respiratory syndrome virus
• PRRSV
• Innate immune
• NF-kB
• pathogenesis
• SAP motif
• Nucleoporin 62
• Nup62
• PIAS1
• type I IFN
• pro-inflammatory cytokines

Abstract

Porcine reproductive and respiratory syndrome virus causes PRRS which is one of the most economically significant diseases in the swine industry worldwide. PRRSV has the ability to suppress the type I interferon induction to facilitate its survival during infection, and the nsp1 protein of PRRSV has been identified as the potent IFN antagonist. The nsp1β subunit of nsp1 blocks the host mRNA nuclear export and this is one of the viral mechanisms to inhibit host antiviral protein translation. The functional motif for both IFN suppression and host mRNA nuclear retention were identified in nsp1β, and mutations in the motif results in the IFN-suppression-negative and host-mRNA nuclear retention-negative. To examine the pathogenic consequence of these functions in the natural host animals, two mutant PRRS viruses (vL126A and vL135A) were generated using infectious clones. These mutant viruses retained the infectivity, and their phenotype was IFN suppression-negative and host mRNA nuclear retention-negative. Pigs infected with vL126A or vL135A exhibited milder severe clinical signs with lower viral titers and shorter duration of viremia when compared to those of WT-infected pigs. The levels of PRRSV-specific antibody remained comparable in all infected groups but the neutralizing antibody titers were high in vL126A-infected or vL135A-infected animals. The IFN-α concentration was also high in pigs infected with the mutant PRRSV. Subsequently, the nsp1β mediated inhibitory mechanism was investigated. Nsp1β was found to specifically interact with nucleoporin 62 (Nup62). Nup62 is one of the major components of the nuclear pore complex (NPC) that form channels spanning the double lipid bilayer of the nuclear envelope for nucleocytoplasmic transport of cellular molecules including host mRNAs. A region representing the C-terminal 328-522 residues of Nup62 was identified as the binding domain to nsp1β. Mutational studies revealed that leucine 126 of nsp1β was the critical residue for Nup62 interaction. Nsp1β L126A did not bind to Nup62, and host mRNA nuclear export occurred normally. In nsp1β-overexpressing cells or siRNA-mediated Nup62 knock-down cells, viral growth was improved. This is likely attributed to nsp1β-mediated viral protein production enhancement and reduction of antiviral proteins in the cytoplasm. One of the mechanisms that PRRSV causes respiratory disease is the onset of inflammation at the site of infection. PRRSV activates NF-kB during infection, and the nucleocapsid (N) protein has been identified as the major NF-kB activator. The protein inhibitor of activated STAT1 (PIAS1) was identified as a molecular partner of N. PIAS1 binds to RelA (p65) and prevents NF-kB activation by interfering p65-DNA binding in the nucleus and thus functions as a negative regulator of NF-kB. The binding domain of PIAS1 was mapped to the N-terminal domain, and this domain was sufficient to bind to p65 and prevent its binding to the kB site, demonstrating the competitive binding between N-PIAS1 and NF-kB-PIAS1. For N, the region between 37 and 72 amino acids was found to interact with PIAS1, and this region overlapped the nuclear localization signal (NLS) of N. A nuclear localization signal (NLS) knock-out mutant N did not activate NF-κB, and this is likely due to the lack of its interaction with PIAS1 in the nucleus, demonstrating the positive correlation between the binding of N to PIAS1 and the NF-κB activation. In conclusion, the current study dissects the molecular basis of innate immune suppression and NF-kB activation of PRRSV and identifies the leucine 126 of nsp1β and NLS of N are essential residues. The innate immune modulation and NF-kB activation together delineate the pathogenic basis of PRRSV.

Graduation Semester

2019-05

Type of Resource

text

Permalink

http://hdl.handle.net/2142/104997

Copyright and License Information

Copyright 2019 Hanzhong Ke

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