publications

Preprints

1. Probing the role of membrane in neutralizing activity of antibodies against influenza virus

   Ozgulbas DG*, Tan TJC*, Wen PC, Teo QW, Lv H, Ghaemi Z, Frank M, Wu NC, Tajkhorshid E

   bioRxiv (2025)

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   Influenza poses a major health issue globally. Neutralizing antibodies targeting the highly conserved stem region of hemagglutinin (HA) of the influenza virus provide considerable protection against the infection. Using an array of advanced simulation technologies, we developed a high-resolution structural model of full-length, Fab-bound HA in a native viral membrane to characterize direct membrane interactions that govern the efficacy of the antibody. We reveal functionally important residues beyond the antibody's complementary-determining regions that contribute to its membrane binding. Mutagenesis experiments and infectivity assays confirm that deactivating the membrane-binding residues of the antibody decreases its neutralization activity. Therefore, we propose that the association with the viral membrane plays a key role in the neutralization activity of these antibodies. Given the rapid evolution of the influenza virus, the developed model provides a structural framework for the rational design and development of more effective therapeutic antibodies.

2. Systematic investigation of double emulsion dewetting dynamics for the droplet microfluidic production of giant unilamellar vesicles (GUVs) under biocompatible conditions

   Jing W*, Noh H*, Tan TJC, Wu NC, Han HS

   bioRxiv (2025)

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   Giant unilamellar vesicles (GUVs) embody biomimetic membranes with compartmentalization that serve not only as simplified models to better understand complex biochemical and biophysical processes, but also as a chassis for the bottom-up assembly of synthetic cells. Recently, double emulsion droplet microfluidics has proven to be a promising platform for their production, offering greater throughput, control, and reproducibility over traditional methods. However, the interplay of parameters—particularly under biocompatible conditions—that influence the complex multiphase fluid dynamics of the dewetting process underlying GUV production has not been thoroughly studied, limiting the democratization of the approach. In this study, we systematically investigate how lipid composition and concentration, aqueous phase conditions, droplet confinement, and fluid dynamics effects promote or impede the dewetting process. We show that the prevalent use of high concentrations of glycerol and P188 are unnecessary, and the altered dewetting dynamics with restricted surfactant usage can be tuned by adjusting chip dimensions and multi-phase compositions. Guided by these findings, we achieved robust, high throughput production of monodisperse GUVs using 0.1% P188 and no glycerol with salts. Our results improve the reliability and accessibility of droplet-microfluidics GUV platforms to catalyze advances in biophysics, synthetic biology, and drug discovery.

3. Propagation of human respiratory syncytial virus in cells derived from the black flying fox (Pteropus alecto)

   Tan TJC, Tan BH, Sugrue RJ

   bioRxiv (2023)

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   The propagation of human respiratory syncytial virus (hRSV) was evaluated in the Pteropus alecto kidney (PaKi) cell line. At 20 hrs post-infection, immunoblotting of hRSV-infected PaKi cell lysates with anti-G, anti-N, anti-P and anti-M2-1 indicated expression of the respective virus proteins of the correct size. The hRSV-infected PaKi cell were also stained using anti-F, anti-G, anti-N, anti-P and anti-M2-1 and imaged using immunofluorescence microscopy, which confirmed high levels of virus infection, and the presence of numerous virus filaments and virus-induced inclusion bodies. PaKi cell monolayers also supported multiple cycle infection when hRSV was used to infect PaKi cells using a low multiplicity of infection. These data indicate that prior adaptation of hRSV was not required for its propagation in the PaKi cell line, and suggests that PaKi cell line is a suitable cell model system with which to examine virus-host interactions involving RSV infection in fruit bats.

2025

1. Probing the functional constraints of influenza A virus NEP by deep mutational scanning

   Teo QW*, Wang Y*, Lv H*, Oade MS, Mao KJ, Tan TJC, Huan YW, Rivera-Cardona J, Shao EK, Choi D, Wang C, Dargani ZT, Brooke CB, te Velthuis AJW, Wu NC

   Cell Reports

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   The influenza A virus nuclear export protein (NEP) is a multifunctional protein that is essential for the viral life cycle and has very high sequence conservation. However, since the open reading frame of NEP largely overlaps with that of another influenza viral protein, non-structural protein 1, it is difficult to infer the functional constraints of NEP based on sequence conservation analysis. In addition, the N-terminal of NEP is structurally disordered, which further complicates the understanding of its function. Here, we systematically measure the replication fitness effects of >1,800 mutations of NEP. Our results show that the N-terminal domain has high mutational tolerance. Additional experiments show that N-terminal domain mutations affect viral transcription and replication dynamics, host cellular responses, and mammalian adaptation of avian influenza virus. Overall, our study not only advances the functional understanding of NEP but also provides insights into its evolutionary constraints.

2. Use of bio-layer interferometry (BLI) to measure binding affinities of SNAREs and phosphoinositides

   Calderin JD, Zhang C Tan TJC, Wu NC, Fratti R

   Methods in Molecular Biology

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   Bio-Layer Interferometry (BLI) is a technique that uses optical biosensing to analyze interactions between molecules. The analysis of molecular interactions is measured in real-time and does not require fluorescent tags. BLI uses disposable biosensors that come in a variety of formats to bind different ligands including biotin, hexahistidine, GST, and the Fc portion of antibodies. Unlike surface plasmon resonance (SPR), BLI is an open system that does not require microfluidics, which eliminates issues that result from clogging and changes in viscosity. Importantly, BLI readings can be completed in minutes and can be formatted for high throughput screening. Here we use biotinylated short chain phosphoinositides and phosphatidic acid bound to streptavidin BLI biosensors to measure the binding of the soluble Qc SNARE Vam7 from Saccharomyces cerevisiae. Unlike most SNAREs, Vam7 lacks a transmembrane domain or lipid anchor to associate with membranes. Instead Vam7 associates to yeast vacuolar membranes using its N-terminal PX domain that binds to phosphatidylinositol 3-phosphate (PI3P) and phosphatidic acid (PA). Using full length Vam7, Vam7^Y42A, and PX domain alone, we determined and compared the dissociation constants (K_D) of each to biotinylated PI3P and PA biosensors.

3. Epistatic hotspots organize antibody fitness landscape and boost evolvability

   Schulz S*, Tan TJC*, Wang S, Wu NC

   Proceedings of the National Academy of Sciences

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   The course of evolution is strongly shaped by interaction between mutations. Such epistasis can yield rugged sequence-function maps and constrain the availability of adaptive paths. While theoretical intuition is often built on global statistics of large, homogeneous model landscapes, mutagenesis measurements necessarily probe a limited neighborhood of a reference genotype. It is unclear to what extent local topography of a real epistatic landscape represents its global shape. Here, we demonstrate that epistatic landscapes can be heterogeneously rugged and this heterogeneity may render biomolecules more evolvable. By characterizing a multi-peaked fitness landscape of a SARS-CoV-2 antibody mutant library, we show that heterogeneous ruggedness arises from sparse epistatic hotspots, whose mutation impacts the fitness effect of numerous sequence sites. Surprisingly, mutating an epistatic hotspot may enhance, rather than reduce, the accessibility of the fittest genotype, while increasing the overall ruggedness. Further, migratory constraints in real space alleviate mutational constraints in sequence space, which not only diversify direct paths taken but may also turn a road-blocking fitness peak into a stepping stone leading toward the global optimum. Our results suggest that epistatically important sequence sites organize the fitness landscape in such a way that path-orienting ruggedness confers global smoothness.

2024

1. Seasonal influenza A virus lineages exhibit divergent abilities to antagonize interferon induction and signaling

   Rivera-Cardona J, Kakuturu N, Rowland EF, Teo QW, Thayer EA, Tan TJC, Sun J, Kieffer C, Wu NC, Brooke CB

   PLoS Pathogens

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   The circulation of seasonal influenza A viruses (IAVs) in humans relies on effective evasion and subversion of the host immune response. While the evolution of seasonal H1N1 and H3N2 viruses to avoid humoral immunity is well characterized, relatively little is known about the evolution of innate immune antagonism phenotypes in these viruses. Numerous studies have established that only a small subset of infected cells is responsible for initiating the type I and type III interferon (IFN) response during IAV infection, emphasizing the importance of single cell studies to accurately characterize the IFN response during infection. We developed a flow cytometry-based method to examine transcriptional changes in IFN and interferon stimulated gene (ISG) expression at the single cell level. We observed that NS segments derived from seasonal H3N2 viruses are more efficient at antagonizing IFN signaling but less effective at suppressing IFN induction, compared to the pdm2009 H1N1 lineage. We compared a collection of NS segments spanning the natural history of the current seasonal IAV lineages and demonstrate long periods of stability in IFN antagonism potential, punctuated by occasional phenotypic shifts. Altogether, our data reveal significant differences in how seasonal and pandemic H1N1 and H3N2 viruses antagonize the human IFN response at the single cell level.

2. An explainable language model for antibody epitope prediction using curated influenza hemagglutinin antibodies

   Wang Y*, Lv H*, Teo QW, Lei R, Gopal AB, Ouyang WO, Yeung Y-H, Tan TJC, Choi D, Shen IR, Chen X, Graham CS, Wu NC

   Immunity

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   Despite decades of antibody research, it remains challenging to predict the specificity of an antibody solely based on its sequence. Two major obstacles are the lack of appropriate models and inaccessibility of datasets for model training. In this study, we curated a dataset of >5,000 influenza hemagglutinin (HA) antibodies by mining research publications and patents, which revealed many distinct sequence features between antibodies to HA head and stem domains. We then leveraged this dataset to develop a lightweight memory B cell language model (mBLM) for sequence-based antibody specificity prediction. Model explainability analysis showed that mBLM captured key sequence motifs of HA stem antibodies. Additionally, by applying mBLM to HA antibodies with unknown epitopes, we discovered and experimentally validated many HA stem antibodies. Overall, this study not only advances our molecular understanding of antibody response to influenza virus, but also provides an invaluable resource for applying deep learning to antibody research.

3. Natural variation in neuraminidase activity influences the evolutionary potential of the seasonal H1N1 lineage hemagglutinin

   Liu T, Reiser WK, Tan TJC, Lv H, Rivera-Cardona J, Heimburger K, Wu NC, Brooke CB

   Virus Evolution

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   The antigenic evolution of the influenza A virus hemagglutinin (HA) gene poses a major challenge for the development of vaccines capable of eliciting long-term protection. Prior efforts to understand the mechanisms that govern viral antigenic evolution mainly focus on HA in isolation, ignoring the fact that HA must act in concert with the viral neuraminidase (NA) during replication and spread. Numerous studies have demonstrated that the degree to which the receptor-binding avidity of HA and receptor-cleaving activity of NA are balanced with each other influences overall viral fitness. We recently showed that changes in NA activity can significantly alter the mutational fitness landscape of HA in the context of a lab-adapted virus strain. Here, we test whether natural variation in relative NA activity can influence the evolutionary potential of HA in the context of the seasonal H1N1 lineage (pdmH1N1) that has circulated in humans since the 2009 pandemic. We observed substantial variation in the relative activities of NA proteins encoded by a panel of H1N1 vaccine strains isolated between 2009 and 2019. We comprehensively assessed the effect of NA background on the HA mutational fitness landscape in the circulating pdmH1N1 lineage using deep mutational scanning and observed pronounced epistasis between NA and residues in or near the receptor-binding site of HA. To determine whether NA variation could influence the antigenic evolution of HA, we performed neutralizing antibody selection experiments using a panel of monoclonal antibodies targeting different HA epitopes. We found that the specific antibody escape profiles of HA were highly contingent upon NA background. Overall, our results indicate that natural variation in NA activity plays a significant role in governing the evolutionary potential of HA in the currently circulating pdmH1N1 lineage.

4. Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin

   Lei R*, Liang W*, Ouyang WO*, Garcia AH*, Kikuchi C, Wang S, McBride R, Tan TJC, Sun Y, Chen C, Graham CS, Rodriguez LA, Shen IR, Choi D, Bruzzone R, Paulson JC, Nair SK, Mok CKP, Wu NC

   Nature Communications

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   The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.

5. Functional and antigenic characterization of SARS-CoV-2 spike fusion peptide by deep mutational scanning

   Lei R*, Qing E*, Odle A, Yuan M, Gunawardene CD, Tan TJC, So N, Ouywang WO, Wilson IA, Gallagher T, Perlman S, Wu NC, Wong LYR

   Nature Communications

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   The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane fusion during virus entry and is part of a broadly neutralizing epitope. However, sequence determinants at the fusion peptide and its adjacent regions for pathogenicity and antigenicity remain elusive. In this study, we perform a series of deep mutational scanning (DMS) experiments on an S2 region spanning the fusion peptide of authentic SARS-CoV-2 in different cell lines and in the presence of broadly neutralizing antibodies. We identify mutations at residue 813 of the spike protein that reduced TMPRSS2-mediated entry with decreased virulence. In addition, we show that an F823Y mutation, present in bat betacoronavirus HKU9 spike protein, confers resistance to broadly neutralizing antibodies. Our findings provide mechanistic insights into SARS-CoV-2 pathogenicity and also highlight a potential challenge in developing broadly protective S2-based coronavirus vaccines.

6. Evidence of antigenic drift in the fusion machinery core of SARS-CoV-2 spike

   Tan TJC, Verma AK, Odle A, Lei R, Meyerholz DK, Matreyek KA, Perlman S, Wong LYR, Wu NC

   Proceedings of the National Academy of Sciences

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   Antigenic drift of SARS-CoV-2 is typically defined by mutations in the N-terminal domain and receptor binding domain of spike protein. In contrast, whether antigenic drift occurs in the S2 domain remains largely elusive. Here, we perform a deep mutational scanning experiment to identify S2 mutations that affect binding of SARS-CoV-2 spike to three S2 apex public antibodies. Our results indicate that spatially diverse mutations, including D950N and Q954H, which are observed in Delta and Omicron variants, respectively, weaken the binding of spike to these antibodies. Although S2 apex antibodies are known to be nonneutralizing, we show that they confer protection in vivo through Fc-mediated effector functions. Overall, this study indicates that the S2 domain of SARS-CoV-2 spike can undergo antigenic drift, which represents a potential challenge for the development of more universal coronavirus vaccines.

2023

1. Stringent and complex sequence constraints of an IGHV1-69 broadly neutralizing antibody to influenza HA stem

   Teo QW*, Wang Y*, Lv H*, Tan TJC, Lei R, Mao KJ, Wu NC

   Cell Reports

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   IGHV1-69 is frequently utilized by broadly neutralizing influenza antibodies to the hemagglutinin (HA) stem. These IGHV1-69 HA stem antibodies have diverse complementarity-determining region (CDR) H3 sequences. Besides, their light chains have minimal to no contact with the epitope. Consequently, sequence determinants that confer IGHV1-69 antibodies with HA stem specificity remain largely elusive. Using high-throughput experiments, this study reveals the importance of light-chain sequence for the IGHV1-69 HA stem antibody CR9114, which is the broadest influenza antibody known to date. Moreover, we demonstrate that the CDR H3 sequences from many other IGHV1-69 antibodies, including those to the HA stem, are incompatible with CR9114. Along with mutagenesis and structural analysis, our results indicate that light-chain and CDR H3 sequences coordinately determine the HA stem specificity of IGHV1-69 antibodies. Overall, this work provides molecular insights into broadly neutralizing antibody responses to influenza virus, which have important implications for universal influenza vaccine development.

2. Leveraging vaccination-induced protective antibodies to define conserved epitopes on influenza N2 neuraminidase

   Lei R*, Kim W*, Lv H*, Mou Z*, Scherm MJ, Schmitz AJ, Turner JC, Tan TJC, Wang Y, Ouyang WO, Liang W, Rivera-Cardona J, Teo C, Graham CS, Brooke CB, Presti RM, Mok CKP^, Krammer F^, Dai X^, Ellebedy AH^, Wu NC^

   Immunity

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   The ability of the human immune system to generate antibodies to any given antigen can be strongly influenced by immunoglobulin V-gene allelic polymorphisms. However, previous studies have provided only limited examples. Therefore, the prevalence of this phenomenon has been unclear. By analyzing >1,000 publicly available antibody-antigen structures, we show that many V-gene allelic polymorphisms in antibody paratopes are determinants for antibody binding activity. Biolayer interferometry experiments further demonstrate that paratope allelic polymorphisms on both heavy and light chains often abolish antibody binding. We also illustrate the importance of minor V-gene allelic polymorphisms with low frequency in several broadly neutralizing antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Overall, this study not only highlights the pervasive impact of V-gene allelic polymorphisms on antibody binding but also provides mechanistic insights into the variability of antibody repertoires across individuals, which in turn have important implications for vaccine development and antibody discovery.

3. Widespread impact of immunoglobulin V gene allelic polymorphisms on antibody reactivity

   Yuan M, Feng Z, Lv H, So N, Shen IR, Tan TJC, Teo QW, Ouyang WO, Talmage L, Wilson IA, Wu NC

   Cell Reports

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   The ability of the human immune system to generate antibodies to any given antigen can be strongly influenced by immunoglobulin V-gene allelic polymorphisms. However, previous studies have provided only limited examples. Therefore, the prevalence of this phenomenon has been unclear. By analyzing >1,000 publicly available antibody-antigen structures, we show that many V-gene allelic polymorphisms in antibody paratopes are determinants for antibody binding activity. Biolayer interferometry experiments further demonstrate that paratope allelic polymorphisms on both heavy and light chains often abolish antibody binding. We also illustrate the importance of minor V-gene allelic polymorphisms with low frequency in several broadly neutralizing antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Overall, this study not only highlights the pervasive impact of V-gene allelic polymorphisms on antibody binding but also provides mechanistic insights into the variability of antibody repertoires across individuals, which in turn have important implications for vaccine development and antibody discovery.

4. Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH

   Schulte T*, Panas MD*, Han X, Williams L, Kedersha N, Fleck JS,  Tan TJC, Dopico XC, Olsson A, Morro AM, Hanke L, Nilvebrant J, Giang KA, Nygren P-Å, Anderson P, Achour A^, McInerney GM^

   Open Biology

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   G3BP is the central node within stress-induced protein–RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.

5. High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike

   Tan TJC, Mou Z, Lei R, Ouyang WO, Yuan M, Song G, Andrabi R, Wilson IA, Kieffer C, Dai X, Matreyek KA, Wu NC

   Nature Communications

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   Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All COVID-19 vaccines in the US encode spike with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering prefusion-stabilized spike immunogens involve tedious experimental work and heavily rely on structural information. Here, we establish a systematic and unbiased method of identifying mutations that concomitantly improve expression and stabilize the prefusion conformation of the SARS-CoV-2 spike. Our method integrates a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, we apply this method to a region in the S2 domain that includes the first heptad repeat and central helix. Our results reveal that besides K986P and V987P, several mutations simultaneously improve expression and significantly lower the fusogenicity of the spike. As prefusion stabilization is a common challenge for viral immunogen design, this work will help accelerate vaccine development against different viruses.

6. Mutational fitness landscape of human influenza H3N2 neuraminidase

   Lei R, Hernandez Garcia A, Tan TJC, Teo QW, Wang Y, Zhang X, Luo S, Nair SK, Peng J, Wu NC

   Cell Reports

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   Influenza neuraminidase (NA) has received increasing attention as an effective vaccine target. However, its mutational tolerance is not well characterized. Here, the fitness effects of >6,000 mutations in human H3N2 NA are probed using deep mutational scanning. Our result shows that while its antigenic regions have high mutational tolerance, there are solvent-exposed regions with low mutational tolerance. We also find that protein stability is a major determinant of NA mutational fitness. The deep mutational scanning result correlates well with mutational fitness inferred from natural sequences using a protein language model, substantiating the relevance of our findings to the natural evolution of circulating strains. Additional analysis further suggests that human H3N2 NA is far from running out of mutations despite already evolving for >50 years. Overall, this study advances our understanding of the evolutionary potential of NA and the underlying biophysical constraints, which in turn provide insights into NA-based vaccine design.

2022

1. Probing the biophysical constraints of SARS-CoV-2 spike N-terminal domain using deep mutational scanning

   Ouyang WO*, Tan TJC*, Lei R, Song G, Kieffer C, Andrabi R, Matreyek KA, Wu NC

   Science Advances

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   Increasing the expression level of the SARS-CoV-2 spike (S) protein has been critical for COVID-19 vaccine development. While previous efforts largely focused on engineering the receptor-binding domain (RBD) and the S2 subunit, the N-terminal domain (NTD) has been long overlooked due to the limited understanding of its biophysical constraints. In this study, the effects of thousands of NTD single mutations on S protein expression were quantified by deep mutational scanning. Our results revealed that in terms of S protein expression, the mutational tolerability of NTD residues was inversely correlated with their proximity to the RBD and S2. We also identified NTD mutations at the interdomain interface that increased S protein expression without altering its antigenicity. Overall, this study not only advances the understanding of the biophysical constraints of the NTD, but also provides invaluable insights into S-based immunogen design.

2. Molecular analysis of a public cross-neutralizing antibody response to SARS-CoV-2

   Yuan M, Wang Y, Lv H, Tan TJC, Wilson IA, Wu NC

   Cell Reports

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   As SARS-CoV-2 variants of concerns (VOCs) continue to emerge, cross-neutralizing antibody responses become key towards next-generation design of a more universal COVID-19 vaccine. By analyzing published data from the literature, we report here that the combination of germline genes IGHV2-5/IGLV2-14 represents a public antibody response to the receptor-binding domain (RBD) that potently cross-neutralizes all VOCs to date, including Omicron and its sub-lineages. Detailed molecular analysis shows that the complementarity-determining region H3 sequences of IGHV2-5/IGLV2-14-encoded RBD antibodies have a preferred length of 11 amino acids and a conserved HxIxxI motif. In addition, these antibodies have a strong allelic preference due to an allelic polymorphism at amino-acid residue 54 of IGHV2-5, which locates at the paratope. These findings have important implications for understanding cross-neutralizing antibody responses to SARS-CoV-2 and its heterogenicity at the population level as well as the development of a universal COVID-19 vaccine.

3. Prevalence and mechanisms of evolutionary contingency in human influenza H3N2 neuraminidase

   Lei R, Tan TJC, Hernandez Garcia A, Wang Y, Diefenbacher M, Teo C, Gopan G, Dargani ZT, Teo QW, Graham CS, Brooke CB, Nair SK, Wu NC

   Nature Communications

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   Neuraminidase (NA) of human influenza H3N2 virus has evolved rapidly and been accumulating mutations for more than half-century. However, biophysical constraints that govern the evolutionary trajectories of NA remain largely elusive. Here, we show that among 70 natural mutations that are present in the NA of a recent human H3N2 strain, >10% are deleterious for an ancestral strain. By mapping the permissive mutations using combinatorial mutagenesis and next-generation sequencing, an extensive epistatic network is revealed. Biophysical and structural analyses further demonstrate that certain epistatic interactions can be explained by non-additive stability effect, which in turn modulates membrane trafficking and enzymatic activity of NA. Additionally, our results suggest that other biophysical mechanisms also contribute to epistasis in NA evolution. Overall, these findings not only provide mechanistic insights into the evolution of human influenza NA and elucidate its sequence-structure-function relationship, but also have important implications for the development of next-generation influenza vaccines.

4. The evolutionary potential of the influenza A virus hemagglutinin is highly constrained by intersegment epistasis

   Liu T, Wang Y, Tan TJC, Wu NC, Brooke CB

   Cell Host & Microbe

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   The ongoing antigenic evolution of the influenza A virus (IAV) hemagglutinin (HA) gene limits efforts to effectively control the spread of the virus in the human population through vaccination. The factors that influence and constrain the evolutionary potential of the HA gene remain poorly understood. Efforts to understand the mechanisms that govern HA antigenic evolution typically examine the HA gene in isolation and ignore the importance of balancing HA receptor-binding activities with the receptor-destroying activities of the viral neuraminidase (NA) for maintaining viral fitness. We hypothesized that the need to maintain functional balance with NA significantly constrains the evolutionary potential of the HA gene. We used deep mutational scanning to show that variation in NA activity significantly reshapes the HA fitness landscape by modulating the overall mutational robustness of the HA protein. Consistent with this, we observe that different NA backgrounds support the emergence of distinct repertoires of HA escape variants under neutralizing antibody pressure. Our results reveal a critical role for intersegment epistatic interactions in shaping the evolutionary potential of the HA gene.

5. Egg-adaptation pathway of human influenza H3N2 virus is contingent on natural evolution

   Liang W, Tan TJC, Wang Y, Lv H, Sun Y, Bruzzone R, Mok CKP, Wu NC

   PLoS Pathogens

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   Egg-adaptive mutations in influenza hemagglutinin (HA) often emerge during the production of egg-based seasonal influenza vaccines, which contribute to the largest share in the global influenza vaccine market. While some egg-adaptive mutations have minimal impact on the HA antigenicity (e.g. G186V), others can alter it (e.g. L194P). Here, we show that the preference of egg-adaptation pathway in human H3N2 HA is strain-dependent. In particular, Thr160 and Asn190, which are found in many recent H3N2 strains, restrict the emergence of L194P but not G186V. Our results further suggest that natural amino acid variants at other HA residues also play a role in determining the egg-adaptation pathway. Consistently, recent human H3N2 strains from different clades acquire different mutations during egg passaging. Overall, these results demonstrate that natural mutations in human H3N2 HA can influence the egg-adaption pathway, which has important implications in seed strain selection for egg-based influenza vaccine.

6. Interactions between influenza A virus nucleoprotein and gene segment UTRs facilitate selective modulation of viral gene expression

   Diefenbacher M, Tan TJC, Bauer DLV, Stadtmueller B, Wu NC, Brooke CB

   Journal of Virology

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   The influenza A virus (IAV) genome is divided into eight negative-sense, single-stranded RNA segments. Each segment exhibits a unique level and temporal pattern of expression, however the exact mechanisms underlying the patterns of individual gene segment expression are poorly understood. We previously demonstrated that a single substitution in the viral nucleoprotein (NP:F346S) selectively modulates neuraminidase (NA) gene segment expression while leaving other segments largely unaffected. Given what is currently known about NP function, there is no obvious explanation for how changes in NP can selectively modulate the replication of individual gene segments. We found that the specificity of this effect for the NA segment is virus strain specific and depends on the UTR sequences of the NA segment. While the NP:F346S substitution did not significantly alter the RNA binding or oligomerization activities of NP in vitro, it specifically decreased the ability of NP to promote NA segment vRNA synthesis. In addition to NP residue F346, we identified two other adjacent aromatic residues in NP (Y385 & F479) capable of similarly regulating NA gene segment expression, suggesting a larger role for this domain in gene-segment specific regulation. Our findings reveal a new role for NP in selective regulation of viral gene segment replication and demonstrate how the expression patterns of individual viral gene segments can be modulated during adaptation to new host environments.

2021

1. Arabidopsis thaliana G3BP ortholog rescues mammalian stress granule phenotype across kingdoms

   Reuper H, Götte B, Williams L, Tan TJC, McInerney GM, Panas MD^, Krenz B^

   International Journal of Molecular Sciences

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   Stress granules (SGs) are dynamic RNA–protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.

2. Sequence signatures of two IGHV3-53/3-66 public clonotypes to SARS-CoV-2 receptor binding domain

   Tan TJC*, Yuan M*, Kuzelka K, Padron GC, Beal JR, Chen X, Wang Y, Rivera-Cardona J, Zhu X, Stadtmueller BM, Brooke CB, Wilson IA^, Wu NC^

   Nature Communications

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   Since the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short complementarity-determining region (CDR) H3. Germline-encoded sequence motifs in heavy chain CDRs H1 and H2 have a major function, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, is not clear. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that seem to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. These results advance understanding of the antibody response to SARS-CoV-2.

3. Virus-induced activation of the rac1 protein at the site of respiratory syncytial virus assembly is a requirement for virus particle assembly on infected cells

   Ravi LI, Tan TJC, Tan BH, Sugrue RJ

   Virology

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   The distributions of the rac1, rhoA and cdc42 proteins in respiratory syncytial virus (RSV) infected cells was examined. All three rhoGTPases were detected within inclusion bodies, and while the rhoA and rac1 proteins were associated with virus filaments, only the rac1 protein was localised throughout the virus filaments. RSV infection led to increased rac1 protein activation, and using the rac1 protein inhibitor NS23766 we provided evidence that the increased rac1 activation occurred at the site of RSV assembly and facilitated F-actin remodeling during virus morphogenesis. A non-infectious virus-like particle (VLP) assay showed that the RSV VLPs formed in lipid-raft microdomains containing the rac1 protein, together with F-actin and filamin-1 (cell proteins associated with virus filaments). This provided evidence that the virus envelope proteins are trafficked to membrane microdomains containing the rac1 protein. Collectively, these data provide evidence that the rac1 protein plays a direct role in the RSV assembly process.

4. An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants

   Chan KK, Tan TJC, Narayanan KK, Procko E

   Science Advances

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   The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, because of close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find that an engineered decoy receptor, sACE2_2.v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild-type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild-type receptor. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.