The project progressed extremely well and important advances have been made, particularly in Objectives 1 and 2. Work on Objective 3 has begun as well, but is expected to take a more central role during the next reporting period.
The goal of Objective 1 was to mechanistically characterize the susceptibility of SARS-CoV-2 to host control mechanisms, focusing on two types of host factors: (1) regulators of mRNA translation/stability and (2) regulators of RNA synthesis. We previously found that the RNA-binding protein LARP1 directly binds SARS-CoV-2 RNAs and restricts viral replication. Using ribosome profiling experiments to quantify translation states for virus and host mRNAs, we now show that LARP1 represses SARS-CoV-2 at the level of mRNA translation. Interestingly, viral genes located at the 5’ end of the SARS-CoV-2 genome are most susceptible to repression by LARP1.
Next, we focused on potential regulators of viral RNA synthesis. As part of Objective 2, we developed DiVi-RAP-MS to analyze proteins bound to different viral RNA species. Using DiVi-RAP-MS we recorded the most comprehensive collection proteins bound to SARS-CoV-2 RNAs available to date. We identified the host protein SND1 among proteins with a clear preference for binding subgenomic viral RNAs. Mapping SND1 binding sites in infected cells revealed an unusual pattern: SND1 selectively binds the lowly abundant negative-sense RNA of SARS-CoV-2. Since negative-sense RNA serves as an amplification template for producing new viral RNA, we measured nascent viral RNA synthesis and found a dramatic loss of new RNA production in SND1 depleted cells. Consistently, double-membrane vesicles that make up the viral replication factories, were significantly smaller in SND1 depleted cells. We found that SND1 directly interacts with the viral protein NSP9. Interestingly, the viral RNA polymerase attaches a single nucleoside monophosphate (NMP) to the N-terminus of NSP9. Our data indicates that NMPylated NSP9 serves as a starting point to initiate viral RNA synthesis, leading to the covalent attachment of NSP9 to SARS-CoV-2 positive- and negative-sense RNA. Hence, we identified a novel protein priming mechanism utilized by SARS-CoV-2. We could further demonstrate that the priming activity of NSP9 is regulated by the host factor SND1. These insights represent a fundamental advance in our understanding of the SARS-CoV-2 RNA synthesis mechanism.
To facilitate the temporally-resolved mapping of RNA-protein interactions at different stages of the viral replication cycle in Objective 2, we developed an entirely novel RNA interactome capture technology: SHIFTR. SHIFTR relies on the highly efficient enrichment of crosslinked protein-RNA complexes in interphases during organic phase extraction and uses a sequence-specific RNA depletion procedure to selectively remove a single RNA species or RNA region. Digestion of the RNA component releases the protein, which then shifts to the organic phase (hence SHIFTR), from where the it can be identified by mass spectrometry. SHIFTR is a revolutionary tool because it is the only methodology that can interrogate specific RNA regions and their interacting protein in any endogenously expressed RNA in its native configuration without the need for genetic manipulation. We applied SHIFTR to SARS-CoV-2 and, for the first time, map interactions of known regulatory regions in authentic viral RNA, such as the 5’ leader sequence or the 3’ UTR.
