Event Title

CB1 -- The Role of SARS-CoV Non-structural Protein 1 in Cytoplasmic Stress Granule Formation

Location

URC Greatroom

Start Date

8-4-2022 10:30 AM

Description

Severe acute respiratory syndrome coronavirus (SARS-CoV and SARS-CoV-2) encoded nonstructural protein 1 (nsp1) is known to suppress host gene expression, known as host shutoff. Nsp1 interacts with host proteins that facilitate nsp1 to block host protein synthesis by cellular protein translation factors. Nsp1 also triggers the degradation of host messenger RNA (mRNA) while keeping the viral RNA intact. During viral infection, stalled host mRNA accumulates in non-membranous vesicles, known as stress granules (SGs). This allows mRNA to be protected until stress ceases. Previously, we used BioID2-mediated proximity labeling followed by LC-MS/MS to identify proteins that have transient interaction with nsp1. This method identified about 114 strong interactors including multiple members of the stress granule complex. Given the role of nsp1 in degrading mRNAs, we suspected that this viral protein may modify the composition of SGs to prevent it from protecting host mRNAs. To identify any compositional changes by nsp1, we successfully adapted and optimized SG isolation methods using human embryonic kidney (HEK) cells, with and without nsp1 expression. We found both nsp1 and G3BP1 accumulate in isolated SGs while GAPDH protein was excluded, as expected. Using immunoblot, we analyzed the proteins present in SGs from cells with and without nsp1 expression after different time intervals of stress induction. We found that SG-associated protein G3BP1 dissociates from SGs overtime in the presence of nsp1. We further isolated the RNA from these SGs following standard protocols. Using mRNAs that specifically accumulate in SGs in normal cells, we are now studying the effect of nsp1 and mRNA trafficking to SGs in the presence of nsp1. We expect our results to uncover the mechanism that enables host mRNAs to be degraded during viral infection.

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Apr 8th, 10:30 AM

CB1 -- The Role of SARS-CoV Non-structural Protein 1 in Cytoplasmic Stress Granule Formation

URC Greatroom

Severe acute respiratory syndrome coronavirus (SARS-CoV and SARS-CoV-2) encoded nonstructural protein 1 (nsp1) is known to suppress host gene expression, known as host shutoff. Nsp1 interacts with host proteins that facilitate nsp1 to block host protein synthesis by cellular protein translation factors. Nsp1 also triggers the degradation of host messenger RNA (mRNA) while keeping the viral RNA intact. During viral infection, stalled host mRNA accumulates in non-membranous vesicles, known as stress granules (SGs). This allows mRNA to be protected until stress ceases. Previously, we used BioID2-mediated proximity labeling followed by LC-MS/MS to identify proteins that have transient interaction with nsp1. This method identified about 114 strong interactors including multiple members of the stress granule complex. Given the role of nsp1 in degrading mRNAs, we suspected that this viral protein may modify the composition of SGs to prevent it from protecting host mRNAs. To identify any compositional changes by nsp1, we successfully adapted and optimized SG isolation methods using human embryonic kidney (HEK) cells, with and without nsp1 expression. We found both nsp1 and G3BP1 accumulate in isolated SGs while GAPDH protein was excluded, as expected. Using immunoblot, we analyzed the proteins present in SGs from cells with and without nsp1 expression after different time intervals of stress induction. We found that SG-associated protein G3BP1 dissociates from SGs overtime in the presence of nsp1. We further isolated the RNA from these SGs following standard protocols. Using mRNAs that specifically accumulate in SGs in normal cells, we are now studying the effect of nsp1 and mRNA trafficking to SGs in the presence of nsp1. We expect our results to uncover the mechanism that enables host mRNAs to be degraded during viral infection.