The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption 1,2 . There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

Highlights d Phosphoproteomics analysis of SARS-CoV-2-infected cells uncovers signaling rewiring d Infection promotes host p38 MAPK cascade activity and shutdown of mitotic kinases d Infection stimulates CK2-containing filopodial protrusions with budding virus d Kinase activity analysis identifies potent antiviral drugs and compounds

The COVID-19 (Coronavirus disease-2019) pandemic, caused by the SARS-CoV-2 coronavirus, is a significant threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and MERS-CoV. Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analysis for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 Orf9b, an interaction we structurally characterized using cryo-EM. Combining genetically-validated host factors with both COVID-19 patient genetic data and medical billing records identified important molecular mechanisms and potential drug treatments that merit further molecular and clinical study.

ABSTRACTAn outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

In eukaryotes, protein phosphorylation is specifically catalyzed by numerous protein kinases (PKs), faithfully orchestrates various biological processes, and reversibly determines cellular dynamics and plasticity. Here we report an updated algorithm of Group-based Prediction System (GPS) 5.0 to improve the performance for predicting kinase-specific phosphorylation sites (p-sites). Two novel methods, position weight determination (PWD) and scoring matrix optimization (SMO), were developed. Compared with other existing tools, GPS 5.0 exhibits a highly competitive accuracy. Besides serine/threonine or tyrosine kinases, GPS 5.0 also supports the prediction of dual-specificity kinase -specific p-sites. In the classical module of GPS 5.0, 617 individual predictors were constructed for predicting p-sites of 479 human PKs. To extend the application of GPS 5.0, a species-specific module was implemented to predict kinase-specific p-sites for 44,795 PKs in 161 eukaryotes. The online service and local packages of GPS 5.0 are freely available for academic research at http://gps.biocuckoo.cn .

The negative regulators in the interferon (IFN) signaling pathway inhibit intrahepatic immune response, resulting in suboptimal therapeutic response to IFNα treatment in chronic hepatitis B (CHB) patients. Identifying the key negative factors and elucidating the regulating mechanism are essential for improving anti‐HBV (hepatitis B virus) efficacy of IFNα. From the Gene Expression Omnibus (GEO) database, we downloaded and analyzed gene expression profiles of CHB patients with different responses to IFNα (GSE54747), and found that innate immune status was associated with the IFNα‐based therapeutic response in CHB patients. Through PCR array, we found higher baseline level of IFN‐induced transmembrane protein 2 (IFITM2) mRNA and lower baseline level of IFNα mRNA in peripheral blood mononuclear cells (PBMCs) of CHB patients with suboptimal response to IFNα treatment. Increased IFITM2 protein was also found in the serum of IFNα nonresponsive patients. With further experiments, we found that overexpressing IFITM2 in Huh7 cells suppressed endogenous IFNα synthesis by inhibiting phosphorylation of extracellular signal–regulated kinase (ERK), TANK‐binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3); knocking out IFITM2 enhanced activation of the endogenous IFNα synthesis pathway, exhibiting better inhibition on HBV replication. We also found that IFITM2 protein was shuttled by exosomes to dendritic cells (DCs), the main source of endogenous IFNα. Exosome‐mediated transport of IFITM2 inhibited synthesis of endogenous IFNα in DCs whereas the inhibitory effect was abolished when IFITM2 was knocked out. Furthermore, we demonstrated that both palmitoylation inhibitor and mutation on 70/71 sites of IFITM2 protein influenced its incorporation into exosomes. Mutated IFITM2 protein increased the effect of IFNα against HBV. Conclusion: Exosome‐mediated transport of IFITM2 to DCs inhibits IFNα pathway activation and blocks anti‐HBV efficacy of exogenous IFNα. The findings provide an explanation to the suboptimal response of CHB patients to IFNα treatment.

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that is causing massive economic loss in the Chinese duck industry. To obtain a live vaccine candidate against the disease, the DTMUV isolate FX2010 was passaged serially in chicken embryo fibroblasts (CEFs). Characterization of FX2010-180P revealed that it was unable to replicate efficiently in chicken embryonated eggs, nor intranasally infect mice or shelducks at high doses of 5.5log10 tissue culture infectious doses (TCID50). FX2010-180P did not induce clinical symptoms, or pathological lesions in ducks at a dose of 5.5log10TCID50. The attenuation of FX2010-180P was due to 19 amino acid changes and 15 synonymous mutations. Importantly, FX2010-180P elicited good immune responses in ducks inoculated at low doses (3.5log10TCID50) and provided complete protection against challenge with a virulent strain. These results indicate that FX2010-180P is a promising candidate live vaccine for prevention of duck Tembusu viral disease.

The routes of transmission of a newly emerged Tembusu virus (TMUV, Flavivirus) in ducks in China remain unclear. Our epidemiological data show that TMUV is spread in winter, when mosquitos are inactive, which suggests that nonvector transmission routes are involved in the spread of TMUV. Furthermore, in vivo studies indicate that TMUV can be transmitted efficiently among ducks by both direct contact and aerosol transmission. This finding has important implications for the control of infection with this novel TMUV in the field. In April 2010, a novel Tembusu virus (TMUV)-associated disease, characterized by retarded growth, high fever, loss of appetite, decline in egg production, and death, emerged in ducks in China (1-5). Since then, TMUV has continuously infected ducks and caused significant economic loss in China. TMUV is a member of the Ntaya virus group in the genus Flavivirus of the family Flaviviridae (6, 7). TMUV was first isolated from mosquitoes in Kuala Lumpur, Malaysia, in 1955; since then, TMUV has been isolated from Culex species mosquitos in Malaysia and Thailand (6, 7). However, the natural reservoir, transmission route, and epidemic situation of TMUV in China remain unclear. To better understand the prevalence and potential transmission route of TMUV in China, we conducted active surveillance of TMUV in ducks at different time points in China.A total of 1,200 serum samples were collected randomly from 53 duck farms in Shandong and Hebei Provinces. Samples were collected from 27-to 380-day-old ducks from 25 farms in the fall (October to December 2011) and 53 farms in the late winter (February and March 2012), respectively. The farms investigated in winter included all of the fall sites (except one farm in Laiwu) (see Fig. S1 in the supplemental material). The presence of TMUVspecific antibody was determined by a blocking enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody (MAb) 1F5, which is a neutralizing MAb that binds specifically to the E protein of TMUV. A blocking ELISA based on this MAb was developed for the detection of neutralization-related antibodies against TMUV (8). TMUV antibody titers are expressed as percent inhibition (PI) of blocking of MAb 1F5 at a test serum dilution of 1:10. The selected cutoff PI value for positive serum was 18.4%. All serum samples were prepared and tested as described previously (8). In October and December 2011, only 11.8% of the farms investigated (2/17) were TMUV seropositive in Shandong Province, and none of the 8 farms investigated in Hebei Province were seropositive (Fig. 1A). Surprisingly, 3 to 4 months later (February and March 2012), 53.7% (22/41) and 50.0% (6/12) of the farms investigated in Shandong and Hebei Provinces became TMUV seropositive. During October and December 2011, the mean rates of seropositivity of the serum samples collected were 11.7 and 0% in Shandong and Hebei Provinces, respectively, which increased to 41.0 and 54.5% during February and March 2012. At most (78.1%) of the TMUV-positive farms, the seropositivi...