BC-74 Investigating the role of nonstructural protein 1 inside the host cell nucleus

Nonstructural protein 1 (nsp1) of recent zoonotic coronaviruses (SARS-CoV-1, SARS-CoV-2, MERS-CoV) acts as a shutoff protein that selectively inhibits host mRNA translation by binding to the mRNA binding site on the 40s ribosomal subunit simultaneously inducing its degradation. In contrast the viral RNA is translated to aid viral propagation. Recent research focuses on nsp1’s predominant role in the cytoplasm of the host cell. Interestingly, proximity-labeling experiments conducted in our lab showed that nsp1 interacts with nuclear proteins and may also impact pre-mRNA maturation inside the nucleus. To study the role of nsp1 in the nucleus, V5-tagged nsp1 was expressed in human embryonic kidney (HEK) cells and lung epithelial cancer (A549) for 18, 24, and 48 hours followed by cellular fractionation to separate cytoplasmic extracts and nuclear extracts. The expression of nsp1 in each fraction was analyzed using Western blotting, with GAPDH as a control to confirm fraction purity. Our results indicated that nsp1 was present in both nuclear and cytoplasmic fractions of HEK and A549 cells to test which amino acid residues of nsp1 aid in nuclear localization, we used multiple known nsp1 mutants (D33R, K58E, R99E, K164A H165A, R124A K125A) with functional defects followed by western blot analysis to see if any of these structural variations prevented nsp1 from localizing inside the nucleus. Our results showed that none of the aforementioned mutations showed any difference, and all of them were found to localize in the nucleus. Currently we are investigating other large-scale mutation of nsp1 and investigating its role in RNA 3′-end processing.