Presenting Author University of South Carolina Upstate
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 mRNAs accumulate in non-membranous vesicles, known as stress granules (SGs). This allows mRNAs to be protected until stress ceases. Recently, our lab observed that nsp1 interacts with proteins of, and accumulates in, the SGs as well. We found SGs are smaller in size in the presence of nsp1. We aimed to determine the role of nsp1 within these granules. 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 in nsp1-expressing human embryonic kidney (HEK) cells. We then further isolated the RNA following standard protocols for RNA-seq analysis. Using immunoblot, we analyzed the proteins present in SGs isolated from cells in the presence and absence of nsp1. We were able to further isolate and identify these proteins using immunoblot and found that SG core protein, Ras-GTPase activating protein (SH3 domain)-binding protein 1 or G3BP1, decreases overtime in the presence of nsp1. Since nsp1 is responsible for targeting mRNA for degradation we are currently identifying mRNAs that selectively accumulate in SGs in the presence of nsp1.
