key: cord-0306187-alurvafb
authors: Ng, Tai L.; Olson, Erika J.; Yoo, Tae Yeon; Weiss, H. Sloane; Koide, Yukiye; Koch, Peter D.; Rollins, Nathan J.; Mach, Pia; Meisinger, Tobias; Bricken, Trenton; Chang, Timothy Z.; Molloy, Colin; Zürcher, Jérôme; Mitchison, Timothy J.; Glass, John I.; Marks, Debora S.; Way, Jeffrey C.; Silver, Pamela A.
title: High content screening and computational prediction reveal viral genes that suppress innate immune response
date: 2021-12-15
journal: bioRxiv
DOI: 10.1101/2021.12.14.472572
sha: 807a25b70d7445b27dc80fca47ce5bacaaf3e5ab
doc_id: 306187
cord_uid: alurvafb

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-κB and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins. IMPORTANCE Infectious diseases caused by viral pathogens exacerbate healthcare and economic burdens. Numerous viral biomolecules suppress the human innate immune system, enabling viruses to evade an immune response from the host. Despite our current understanding of viral replications and immune evasion, new viral proteins, including those encoded by uncultivated viruses or emerging viruses, are being unearthed at a rapid pace from large scale sequencing and surveillance projects. The use of medium- and high-throughput functional assays to characterize immunosuppressive functions of viral proteins can advance our understanding of viral replication and possibly treatment of infections. In this study we assembled a large viral gene library from diverse viral families and developed a high content assay to test for inhibition of innate immunity pathways. Our work expands the tools that can rapidly link sequence and protein function, representing a practical step towards early-stage evaluation of emerging and understudied viruses.

Overall, our library of 605 genes contains 69 viral immune inhibitors with GO annotations containing immune 124 suppressive (IMS) terms, 158 viral genes with 20% sequence similarity to the IMS genes. Additionally, this library 125 contains 42 known inhibitors determined by containing an immunosuppressive domain by Pfam classification and 126 243 predicted inhibitors using permissive hmmscan (Data Set S1). 358 genes in this library are not predicted to be 127 immunosuppressive. In total, we tested 269 proteins from DNA viruses, 335 proteins from RNA viruses (including 128 retroviruses), and 1 protein from an unclassified hudisavirus ( Figure 1C) . The COVID-19 pandemic occurred in the 129 middle of our screening effort described below, which prompted us to test 195 viral genes encoded by the 130 coronaviruses SARS-CoV-2, SARS-CoV, MERS-CoV, hCoV-229E, hCoV-NL63, hCoV-OC43, and hCoV-HKU1 nuclear/cytoplasmic localization of IRF3 and NF-B could be simultaneously visualized using non-cross-reacting 143 primary and secondary antibodies (16) . (18)). Since DNA transfection alone stimulates the cGAS-STING pathway (Figure 2e) , we 176 generated a CRISPR knockout of cGAS (BJ-5ta (cGAS − ), Methods). In this cell line, DNA transfection-mediated 177 stimulation of IRF3 nuclear transport was essentially undetectable (Figure 2b ).

To assess the effect of a particular gene on innate immune signaling pathways, BJ-5ta (cGAS − ) cells ( Figure   179 2a) were seeded in 384-well plates and co-transfected with a viral gene expression vector and a GFP expression 

High content imaging assays identified known and predicted viral inhibitors.

Using the described assays, we tested 605 viral genes from our library (Figure 1c ) and an additional 195 

The list of 79 viral proteins included numerous known inhibitors of innate immunity (Data Set S4). Specifically, transport signal fused to a red fluorescent protein with a constitutive nuclear export or import signal. For example,

we measure a viral protein's effect on nuclear import rate by expressing our viral proteins in U2OS cells that stably 280 express a photoactivatable nuclear import sequence fused to a cytoplasmic RFP protein (Figure 4b) . We find that 

Two proteins with no previously known function are a hypothetical protein with an intrinsically disordered domain 301 encoded in torque teno virus 10 (TTV10) (NCBI accession: YP_003587850) and an intrinsically disordered protein 302 from human respirovirus 3 (NCBI accession: NP_599250) (Figure 5a) . TTV is reported to be a prevalent virus 303 present in most humans, yet it is understudied along with other human anelloviruses due to difficult cultivating 304 conditions (34). We tested these proteins in A549 Dual reporter cells (Invivogen ®) that report on the expression 305 level of IRF translocation via a luciferase readout (Figure 5b) . We observed that expression of TTV hypothetical 306 protein and HRV disordered protein inhibited ISRE-driven gene expression when the cells were stimulated with 307 poly(I:C). We next immunostained the streptavidin (strep)-tagged versions of the two proteins overexpressed in BJ-308 5ta cells and observed that TTV hypothetical protein is associated with the nuclear compartment (Figure 5c) . Figure S5 : Commassie stained SDS-PAGE gels of (a) human respirovirus (HRV) 3 D protein (NCBI accession:

Other strong hits include several phenuiviral nonstructural proteins (NSs), picornarviral '3C' proteases (3C pro ), References

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This work was supported by IARPA-FunGCAT Cooperative Agreement W911NF-17-2-0092. Tai L. Ng is an Open