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Role of Integrated Stress Response pathway in fish cells during VHSV Ia infection

Shetty, Adarsh G
http://orcid.org/0000-0001-8184-8086
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1661512096975219

Abstract Details

2022, Master of Science, University of Toledo, Biology (Cell-Molecular Biology).
Eukaryotic cells have evolved the ability to generate immune responses when they detect pathogens such as viruses and bacteria. The innate immune response is initiated by detection of pathogen-associated molecular patterns (PAMPs) by specialized pattern recognition receptors (PRRs) in host cells. PRRs such as RIG-I and MDA5 detect cytoplasmic viral dsRNA and distinguish between self- and non-self (viral) RNA. Virus infection activates the integrated stress response (ISR) pathway that allows the cell to clear infections. Both dsRNA-dependent protein kinase R (PKR) and PKR-like ER kinase (PERK) are stress kinases that are activated during viral infection. Phosphorylation and activation of PKR by cytosolic dsRNA or PERK due to ER stress phosphorylates the α-subunit of a eukaryotic translational initiator protein, eIF2 (eIF2α). Phosphorylation of eIF2α stalls cap dependent translation, which in turn prevents viral replication. Translational arrest results in accumulation of stalled ribosomes and mRNA into unique membraneless aggregates known as stress granules (SG). SG can be induced by a variety of stress stimuli including oxidative stress, heat shock stress, misfolded proteins, and viral infection. A specific subset of stress granules known as antiviral stress granules form in response to viral infection and are enriched with host antiviral proteins like RIG-I, PKR, OAS and RNase L. Viral Hemorrhagic Septicemia Virus (VHSV) is an aquatic pathogen that has caused widespread loss to both wild and aquaculture fishes. VHSV Ia has caused massive deaths of rainbow trout fishes in Europe. Different viral proteins are known to play an important role to evade host immune responses and carryout successful viral replication. Previous studies from our lab has shown that the matrix (M) protein from VHSV IVb suppresses the IFN production and the non-virion (NV) protein augments the IFN production. Several fish cell lines, including fathead minnow epithelial (EPC) cells and rainbow trout (RTG2, RTGill) cells contain a homolog to the human G3BP1 protein, which is essential for stress granule formation. We further demonstrated that rainbow trout cells form stress granules in response to oxidative stress, heat shock stress and viral infection. The ISR pathway is well characterized in mammalian cells, but in response to viral infection in fish cells it is relatively unknown. VHSV Ia infection in RTG2 and RTGill induces the IFN expression by 24h before suppressing the expression. Overexpression through fish MAVS is also inhibited by the Ia virus. Previous studies had shown that VHSV IVb activates the ISR by phosphorylating eIF2α through the PERK pathway. Similarly, VHSV Ia also activates the ISR via PERK and SG formation and inhibiting PERK through pharmacological inhibitors reduced the eIF2α phosphorylation levels, viral protein expression, and stress granule formation in infected cells. Expressing individual VHSV Ia protein ectopically in RTG2 and RTGill cells failed to form SG which suggests that combination of more than one viral protein and/or viral replication is required to induce the SGs. We confirmed that infecting RTG2 and RTGill cells with UV inactivated VHSV Ia prevented SG formation when compared to active VHSV Ia which induced SG by 36hpi (hours post infection). Reduced viral protein expression along with lack of SG formation using PERK inhibitor suggested that VHSV uses SG to support replication. To determine this, we successfully knocked down G3BP1 in RTGill cells through CRISPR/Cas9 method. When RTGill G3BP1 KD cells were infected with VHSV Ia, eIF2α phosphorylation levels was significantly reduced along with viral protein expression. VHSV Ia in RTGill WT cells inhibits proteins like RIG-I, MAVS, IRF3 and PERK which are involved in IFN response by the end of 48hpi. Interestingly, in RTGill G3BP1 KD cells MAVS, MDA5, RIG-I, IRF3, PKR and PERK are constitutively expressed. Through RT-PCR, we determined that levels of IFN mRNA in RTGill WT cells goes down by 36hpi, whereas the levels are almost 80-fold increase in RTGill G3BP1 KD cells. Similarly, IFN-stimulated gene MX1 mRNA levels also increased 80-fold in RTGill G3BP1 KD cells when compared to RTGill WT cells. This confirms that VHSV Ia inhibits the IFN response and G3BP1 regulates IFN levels during Ia infection. Using the IFN bioassay, we showed that RTGill G3BP1 KD cells expressed higher levels of IFN compared to RTGill WT cells. In RTGill G3BP1 KD cells, the VHSV mRNA levels for viral proteins were significantly higher than RTGill WT cells suggesting that VHSV requires G3BP1 for its viral protein translation. This correlated with our viral yield data, which showed lower viral titers in RTGill G3BP1 KD cells compared to the RTGill WT cells. These results suggest that VHSV Ia may use SG to support replication and rely on G3BP1 for its viral protein translation. Further studies will identify proteins and RNA that localize to SG during VHSV Ia infection and the role of G3BP1 in VHSV Ia pathogenesis.
Malathi Krishnamurthy (Advisor)
Scott Crawley (Committee Member)
Scott Leisner (Committee Member)
101 p.
Biology
Integrated Stress Response pathway; Stress Granules; VHSV Ia replication; G3BP1; IFN response; Fish cells; eIF2α

Recommended Citations

Citations

  • Shetty, A. G. (2022). Role of Integrated Stress Response pathway in fish cells during VHSV Ia infection [Master's thesis, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1661512096975219

    APA Style (7th edition)

  • Shetty, Adarsh. Role of Integrated Stress Response pathway in fish cells during VHSV Ia infection. 2022. University of Toledo, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1661512096975219.

    MLA Style (8th edition)

  • Shetty, Adarsh. "Role of Integrated Stress Response pathway in fish cells during VHSV Ia infection." Master's thesis, University of Toledo, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1661512096975219

    Chicago Manual of Style (17th edition)

toledo1661512096975219
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© 2022, some rights reserved.Creative Commons License Role of Integrated Stress Response pathway in fish cells during VHSV Ia infection by Adarsh G Shetty is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Toledo and OhioLINK.