Studies on the interaction between porcine reproductive and respiratory syndrome virus and its natural host cell

Chen, Wei Yu

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https://hdl.handle.net/2142/89219 Copy

Description

Title

Studies on the interaction between porcine reproductive and respiratory syndrome virus and its natural host cell

Author(s)

Chen, Wei Yu

Issue Date

2015-12-01

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

Zuckermann, Federico

Doctoral Committee Chair(s)

Zuckermann, Federico

Committee Member(s)

• Rock, Daniel
• Segre, Mariangela
• Shisler, Joanna

Department of Study

Pathobiology

Discipline

VMS - Veterinary Pathobiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Alveolar Macrophages
• Innate immunity
• endoplasmic reticulum
• Unfolded Protein Response
• IRE1
• eIF2
• type I interferon
• tumor necrosis factor alpha (TNFa)
• double stranded ribonucleic acid (dsRNA)

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, causes a costly global disease of swine. PRRSV infects their alveolar macrophages (AMΦ) resulting in an interstitial pneumonia. The ability of PRRSV to modulate the production of interferon (IFN)a and tumor necrosis factor (TNF)a by affected AMΦ is implicated in the virus’s pathogenesis. In this regard, infection of porcine AMΦ with PRRSV reduced by >50% the amount of IFN-α otherwise produced following the cells’ exposure to synthetic dsRNA. Interestingly, there was no corresponding impairment of the activation of either interferon regulatory factor 3 (IRF3) or signal transducer and activator of transcription 1 (STAT1), or of the transcription of the IFN-α, IFN-b, or IRF7 genes. Rather, the reduction correlated with the phosphorylation of eukaryotic translation initiation factor 2 (eIF2)a via the protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) and the appearance of stress granules, indicating translational attenuation. Likewise, a TNF-α response to lipopolysaccharide (LPS) was inhibited when this toll-like receptor (TLR)4 agonist was introduced at 6 h post-infection, when peak TNF-α synthesis and eIF2α phosphorylation would coincide. In contrast, a synergistic TNFα response, due to NFkB activation via the inositol-requiring protein-1α (IRE-1α), was observed if LPS exposure occurred 4 h earlier enabling subsequent TNFα production to be unaffected by later eIF2α phosphorylation. Thus, representatives of two branches of the unfolded protein response (UPR) of AMΦ to PRRSV replication, IRE-1α and PERK, can enhance or suppress cytokine production by triggering the activation of NF-κB or eIF2a, respectively. It worth noting that the modulatory activities became critical when the PRRSV-infected cells were super-exposed to secondary stimulation of poly(I:C) or LPS. In which case exacerbated inflammation response in the lung will develop and produce significant morbidity and death. In the case of PRRSV infection alone, these cytokine responses such IFN-α are barely detected in spite of massive dsRNA produced in PRRSV-infected porcine AMΦ cells. As dsRNA is currently considered to be the most potent type I interferon (IFN) agonist, it has been suggested that sequestration of viral dsRNA may also help viruses evade host innate immune detection by reducing exposure of viral dsRNA to viral nucleic acids cytoplasmic sensors. This predicted type of immune evasion by virus-induced intracellular membrane structures had been confirmed for flaviviruses. By coupling a selective permeablization technique with immunostaining analysis, a positive correlation between the cytosolic exposure of virus dsRNA and a host type I IFN response was demonstrated. In this case, the extent of flavivirus dsRNA exposure was dependent on both the virus species and host cell type. Despite lacking definitive experimental evidence regarding members of the nidovirales family including arteriviruses and coronaviruses, it has been suggested that these viruses also utilize a similar immune evasion strategy. In this regard, the development of double membrane vesicle (DMV) structures has been associated with the activity of arterivirus nsp2 and its coronavirus equivalent, nsp3. Moreover, in our previous study, the arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV) was shown to be incapable of inducing type I IFN synthesis upon infecting porcine alveolar macrophages. Thus, PRRSV appears to utilize a passive innate immunity evading mechanism. In the present study, in conjunction with the application of selective permeabilization, our immuno-staining results demonstrated for the first time that during PRRSV infection the viral dsRNA is sequestered inside intracellular membranes, which could prevent detection of virus dsRNA by host’s viral nucleic acid sensor. Evidence that it is indeed the case was provided by a laboratory PRRSV strain with a unique and deleterious mutation in the N-terminal region of NSP2, for which the virus losses the ability to sequester its dsRNA inside intracellular membrane structures and thus away from detection by viral nucleic acid sensors, resulting in a substantial type I IFN response upon infecting alveolar macrophages.

Graduation Semester

2015-12

Type of Resource

text

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http://hdl.handle.net/2142/89219

Copyright and License Information

Copyright 2015 Wei Yu Chen

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