A SARS-CoV-2 - host proximity interactome (preprint)

Viral replication is dependent on interactions between viral polypeptides and host proteins. Identifying virus-host protein interactions can thus uncover unique opportunities for interfering with the virus life cycle via novel drug compounds or drug repurposing. Importantly, many viral-host protein interactions take place at intracellular membranes and poorly soluble organelles, which are difficult to profile using classical biochemical purification approaches. Applying proximity-dependent biotinylation (BioID) with the fast-acting miniTurbo enzyme to 27 SARS-CoV-2 proteins in a lung adenocarcinoma cell line (A549), we detected 7810 proximity interactions (7382 of which are new for SARS-CoV-2) with 2242 host proteins (results available at covid19interactome.org). These results complement and dramatically expand upon recent affinity purification-based studies identifying stable host-virus protein complexes, and offer an unparalleled view of membrane-associated processes critical for viral production. Host cell organellar markers were also subjected to BioID in parallel, allowing us to propose modes of action for several viral proteins in the context of host proteome remodelling. In summary, our dataset identifies numerous high confidence proximity partners for SARS-CoV-2 viral proteins, and describes potential mechanisms for their effects on specific host cell functions.

Global BioID-based SARS-CoV-2 proteins proximal interactome unveils novel ties between viral polypeptides and host factors involved in multiple COVID19-associated mechanisms (preprint)

The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.

Silicon Nitride Inactivates SARS-CoV-2 in vitro (preprint)

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the COVID-19 pandemic, remains viable and therefore potentially infectious on several materials. One strategy to discourage the fomite-mediated spread of COVID-19 is the development of materials whose surface chemistry can spontaneously inactivate SARS-CoV-2. Silicon nitride (Si3N4), a material used in spine fusion surgery, is one such candidate because it has been shown to inactivate several bacterial species and viral strains. This study hypothesized that contact with Si3N4 would inactivate SARS-CoV-2, while mammalian cells would remain unaffected. MaterialsSARS-CoV-2 virions (2x104 PFU/mL diluted in growth media) were exposed to 5, 10, 15, and 20% (w/v) of an aqueous suspension of sintered Si3N4 particles for durations of 1, 5, and 10 minutes, respectively. Before exposure to the virus, cytotoxicity testing of Si3N4 alone was assessed in Vero cells at 24 and 48 hour post-exposure times. Following each exposure to Si3N4, the remaining infectious virus was quantitated by plaque assay. ResultsVero cell viability increased at 5% and 10% (w/v) concentrations of Si3N4 at exposure times up to 10 minutes, and there was only minimal impact on cell health and viability up to 20% (w/v). However, the SARS-CoV-2 titers were markedly reduced when exposed to all concentrations of Si3N4; the reduction in viral titers was between 85% - 99.6%, depending on the dose and duration of exposure. ConclusionsSi3N4 was non-toxic to the Vero cells while showing strong antiviral activity against SARS-CoV-2. The viricidal effect increased with increasing concentrations of Si3N4 and longer duration of exposure. Surface treatment strategies based on Si3N4 may offer novel methods to discourage SARS-CoV-2 persistence and infectivity on surfaces and discourage the spread of COVID-19.

A SARS-CoV-2 BioID-based virus-host membrane protein interactome and virus peptide compendium: new proteomics resources for COVID-19 research (preprint)

Key steps of viral replication take place at host cell membranes, but the detection of membrane-associated protein-protein interactions using standard affinity-based approaches (e.g. immunoprecipitation coupled with mass spectrometry, IP-MS) is challenging. To learn more about SARS-CoV-2 - host protein interactions that take place at membranes, we utilized a complementary technique, proximity-dependent biotin labeling (BioID). This approach uncovered a virus-host topology network comprising 3566 proximity interactions amongst 1010 host proteins, highlighting extensive virus protein crosstalk with: (i) host protein folding and modification machinery; (ii) membrane-bound vesicles and organelles, and; (iii) lipid trafficking pathways and ER-organelle membrane contact sites. The design and implementation of sensitive mass spectrometric approaches for the analysis of complex biological samples is also important for both clinical and basic research proteomics focused on the study of COVID-19. To this end, we conducted a mass spectrometry-based characterization of the SARS-CoV-2 virion and infected cell lysates, identifying 189 unique high-confidence virus tryptic peptides derived from 17 different virus proteins, to create a high quality resource for use in targeted proteomics approaches. Together, these datasets comprise a valuable resource for MS-based SARS-CoV-2 research, and identify novel virus-host protein interactions that could be targeted in COVID-19 therapeutics.

A Flexible Genome-Scale SARS-CoV-2 Clone Resource (preprint)

The world is facing a major health crisis, the global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus, for which no approved antiviral agents or vaccines are currently available. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available via Addgene. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.