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Effective pandemic preparedness relies on anticipating viral mutations that are able to evade host immune responses to facilitate vaccine and therapeutic design. However, current strategies for viral evolution prediction are not available early in a pandemic-experimental approaches require host polyclonal antibodies to test against 1-16 , and existing computational methods draw heavily from current strain prevalence to make reliable predictions of variants of concern 17-19 . To address this, we developed EVEscape, a generalizable modular framework that combines fitness predictions from a deep learning model of historical sequences with biophysical and structural information. EVEscape quantifies the viral escape potential of mutations at scale and has the advantage of being applicable before surveillance sequencing, experimental scans or three-dimensional structures of antibody complexes are available. We demonstrate that EVEscape, trained on sequences available before 2020, is as accurate as high-throughput experimental scans at anticipating pandemic variation for SARS-CoV-2 and is generalizable to other viruses including influenza, HIV and understudied viruses with pandemic potential such as Lassa and Nipah. We provide continually revised escape scores for all current strains of SARS-CoV-2 and predict probable further mutations to forecast emerging strains as a tool for continuing vaccine development ( evescape.org )., (© 2023. The Author(s).)

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 or -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 virus genes. We found 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 interferon regulatory factor 3 (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 health care 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 toward early-stage evaluation of emerging and understudied viruses.

Many organisms can survive extreme conditions and successfully recover to normal life. This extremotolerant behavior has been attributed in part to repetitive, amphipathic, and intrinsically disordered proteins that are upregulated in the protected state. Here, we assemble a library of approximately 300 naturally occurring and designed extremotolerance-associated proteins to assess their ability to protect human cells from chemically induced apoptosis. We show that several proteins from tardigrades, nematodes, and the Chinese giant salamander are apoptosis-protective. Notably, we identify a region of the human ApoE protein with similarity to extremotolerance-associated proteins that also protects against apoptosis. This region mirrors the phase separation behavior seen with such proteins, like the tardigrade protein CAHS2. Moreover, we identify a synthetic protein, DHR81, that shares this combination of elevated phase separation propensity and apoptosis protection. Finally, we demonstrate that driving protective proteins into the condensate state increases apoptosis protection, and highlights the ability of DHR81 condensates to sequester caspase-7. Taken together, this work draws a link between extremotolerance-associated proteins, condensate formation, and designing human cellular protection.

Objectives: To evaluate potential consequences of expanded carrier screening (ECS) for reproductive risk on health care utilization among women who are not at increased reproductive risk., Study Design: Women planning pregnancy were randomized to usual care carrier screening or ECS to assess reproductive risks. Electronic health record (EHR) data were used to evaluate the effects of ECS on pregnancy-related utilization and general health care utilization among all study participants who did not receive positive ECS results of at least a 25% risk (ie, received negative [normal] ECS results)., Methods: EHR data were extracted through research-ready databases and extensive chart review for 304 participants. We analyzed the effect of ECS for women who were not found to be at increased reproductive risk on (1) utilization of mental health services in the period between randomization and initial results disclosure; (2) utilization of general outpatient and inpatient services, specialty services, and mental health-related services in the year following randomization; and (3) utilization and refusal of pregnancy-related services among pregnant women (n = 129) prior to and following randomization., Results: No significant differences in health care utilization were found between women randomized to receive ECS and those receiving usual care. Women who received negative ECS results did not refuse recommended screening for conditions that are not identified via ECS at a higher rate than women in the usual care arm., Conclusions: These results suggest that ECS does not have unintended negative impacts on the health care system for the majority of patients who are not at increased reproductive risk.

Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR's pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease., Competing Interests: MC, NR, JS, KS, KM, MV, SS, FR, DD, DM, AR, DF No competing interests declared, (© 2020, Chiasson et al.)

We describe an experimental method of three-dimensional (3D) structure determination that exploits the increasing ease of high-throughput mutational scans. Inspired by the success of using natural, evolutionary sequence covariation to compute protein and RNA folds, we explored whether 'laboratory', synthetic sequence variation might also yield 3D structures. We analyzed five large-scale mutational scans and discovered that the pairs of residues with the largest positive epistasis in the experiments are sufficient to determine the 3D fold. We show that the strongest epistatic pairings from genetic screens of three proteins, a ribozyme and a protein interaction reveal 3D contacts within and between macromolecules. Using these experimental epistatic pairs, we compute ab initio folds for a GB1 domain (within 1.8 Å of the crystal structure) and a WW domain (2.1 Å). We propose strategies that reduce the number of mutants needed for contact prediction, suggesting that genomics-based techniques can efficiently predict 3D structure.

Steroids can be difficult to modify through traditional organic synthesis methods, but many enzymes regio- and stereoselectively process a wide variety of steroid substrates. We tested whether steroid-modifying enzymes could make novel steroids from non-native substrates. Numerous genes encoding steroid-modifying enzymes, including some bacterial enzymes, were expressed in mammalian cells by transient transfection and found to be active. We made three unusual steroids by stable expression, in HEK293 cells, of the 7α-hydroxylase CYP7B1, which was selected because of its high native product yield. These cells made 7α,17α-dihydroxypregnenolone and 7β,17α-dihydroxypregnenolone from 17α-hydroxypregnenolone and produced 11α,16α-dihydroxyprogesterone from 16α-hydroxyprogesterone. The last two products were the result of CYP7B1-catalyzed hydroxylation at previously unobserved sites. A Rosetta docking model of CYP7B1 suggested that these substrates' D-ring hydroxy groups might prevent them from binding in the same way as the native substrates, bringing different carbon atoms close to the active ferryl oxygen atom. This new approach could potentially use other enzymes and substrates to produce many novel steroids for drug candidate testing., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)