Periodic Reporting for period 1 - COVIDecode (Interrogating RNA-protein interactions underlying SARS-CoV-2 infection and antiviral defense)
Période du rapport: 2022-05-01 au 2024-10-31
The COVID-19 pandemic underscores the need to better understand its causative agent, SARS-CoV-2, and the various other emerging viruses threatening human health. Like many human pathogenic viruses, SARS-CoV-2 utilizes RNA as its replicated genetic material and its template for translating the virus’s proteins. Ongoing research into SARS-CoV-2 and other RNA viruses has largely focused on understanding the function of their encoded proteins, revealing key roles in host cell entry, viral replication, and immune suppression. In contrast, little is known about the set of viral RNAs and how they interact with host machinery as part of a virus’s replication cycle in infected cells.
We recently discovered a large collection of virus and host proteins that bind and regulate the RNA of SARS-CoV-2 during infection. This collection of proteins provides an excellent starting point to dissect how RNA-binding proteins shape the viral RNA life cycle and contribute to the host's antiviral defense. The central hypothesis is that SARS-CoV-2 dynamically modulates RNA-protein interactions in the host to facilitate the functions of different viral RNAs generated at distinct stages of the replication cycle. To test this hypothesis, we devised the following objectives: (i) decode mechanisms of host-mediated control over the life cycle of SARS-CoV-2 RNAs, (ii) map with temporal resolution which host cell proteins engage each SARS-CoV-2 RNA type, and (iii) elucidate the role of unconventional proteins that moonlight as RNA binders in SARS-CoV-2 infections.
This project aims to identify novel pro- and antiviral host factors in SARS-CoV-2 infection and reveal underlying RNA regulatory mechanisms. Together, these insights will provide an RNA-centric view of viral infections and identify candidate factors and pathways for the rational design of novel antiviral strategies in the future.
We recently discovered a large collection of virus and host proteins that bind and regulate the RNA of SARS-CoV-2 during infection. This collection of proteins provides an excellent starting point to dissect how RNA-binding proteins shape the viral RNA life cycle and contribute to the host's antiviral defense. The central hypothesis is that SARS-CoV-2 dynamically modulates RNA-protein interactions in the host to facilitate the functions of different viral RNAs generated at distinct stages of the replication cycle. To test this hypothesis, we devised the following objectives: (i) decode mechanisms of host-mediated control over the life cycle of SARS-CoV-2 RNAs, (ii) map with temporal resolution which host cell proteins engage each SARS-CoV-2 RNA type, and (iii) elucidate the role of unconventional proteins that moonlight as RNA binders in SARS-CoV-2 infections.
This project aims to identify novel pro- and antiviral host factors in SARS-CoV-2 infection and reveal underlying RNA regulatory mechanisms. Together, these insights will provide an RNA-centric view of viral infections and identify candidate factors and pathways for the rational design of novel antiviral strategies in the future.
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.
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.
This project produced several results beyond the state-of-the-art. First, we discovered that SARS-CoV-2 uses NSP9 as a protein primer to initiate viral RNA synthesis in human cells. This protein priming mechanism was not previously recognized and likely also applies to other coronaviruses. Unexpectedly, we also discovered a host factor controlling the protein priming activity of NSP9 by directly interacting with NSP9 and negative-sense RNA. These findings collectively expand our understanding of the RNA synthesis mechanism utilized by SARS-CoV-2 and also provide a starting point for the rational design of novel antivirals.
Beyond the mechanistic insights described above, this project also led to the development of novel technology. Specifically, we developed SHIFTR, the first method to enable identifying proteins directly bound to a specific region within any endogenous or viral RNA. In addition to delivering region-resolution, SHIFTR requires orders of magnitude lower input material compared to the state-of-the-art, thus removing a critical bottleneck. SHIFTR will be a revolutionary tool enabling the systematic mapping of proteins bound to any regulatory RNA region in any cell type.
Beyond the mechanistic insights described above, this project also led to the development of novel technology. Specifically, we developed SHIFTR, the first method to enable identifying proteins directly bound to a specific region within any endogenous or viral RNA. In addition to delivering region-resolution, SHIFTR requires orders of magnitude lower input material compared to the state-of-the-art, thus removing a critical bottleneck. SHIFTR will be a revolutionary tool enabling the systematic mapping of proteins bound to any regulatory RNA region in any cell type.