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| Status |
Public on Oct 03, 2023 |
| Title |
SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9 |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 specifically binds to the 5'-end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RNA-binding protein and direct SND1 interaction partner, is covalently linked to the 5'-ends of positive and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.
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| Overall design |
We mapped direct protein-RNA interaction sites of the host protein SND1 in SARS-CoV-2 infected cells using enhanced crosslinking and immunoprecipitation (eCLIP) in combination with RNA-sequencing. Covalent linkages between the non-structural viral protein NSP9 and SARS-CoV-2 RNA were mapped by covalent RNA immunoprecipitation (cRIP), followed by RNA-sequencing.
We used thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) in human cells infected with SARS-CoV-2 at different time points to quantitatively assess the effect of SND1 depletion on the levels of newly synthesized and pre-existing viral RNA.
We identified RNA sequences crosslinked to proteins purified by RNA antisense purification (RAP-MS) for all viral (ALL), genomic (gRNAs), and subgenomic mRNAs (sgmRNA) by following the procedure described in the PROTOCOLS section.
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| Contributor(s) |
Munschauer M, Gabel A |
| Citation(s) |
37794589 |
| |
| Submission date |
Nov 07, 2022 |
| Last update date |
Nov 16, 2023 |
| Contact name |
Alexander Gabel |
| E-mail(s) |
alexander.gabel@helmholtz-hiri.de
|
| Organization name |
Helmholtz Institute for RNA-based Infection Research
|
| Lab |
LncRNA and Infection Biology (LRIB)
|
| Street address |
Josef-Schneider-Str. 2 / D15
|
| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
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| Platforms (1) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (60)
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| Relations |
| BioProject |
PRJNA899002 |
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