Deep mutational scanning of influenza A virus NEP reveals pleiotropic mutations in its N-terminal domain.

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. Besides, the N-terminal of NEP is structurally disordered, which further complicates the understanding of its function. Here, we systematically measured the replication fitness effects of >1,800 mutations of NEP. Our results show that the N-terminal domain has high mutational tolerance. Additional experiments demonstrate that N-terminal domain mutations pleiotropically 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.

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

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.

Contrasting roles of MERS-CoV and SARS-CoV-2 internal proteins in pathogenesis in mice.

Betacoronaviruses encode an internal (I) gene via an alternative reading frame within the nucleocapsid gene, called ORF8b for Middle-East respiratory syndrome coronavirus (MERS-CoV) and ORF9b for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Previous reports suggested that proteins 8b and 9b are involved in evading multiple innate immune signaling pathways. However, their roles in mediating pathogenesis in infected animals have not been determined. In this study, we abrogated the expression of protein 8b in MERS-CoV and protein 9b in SARS-CoV-2. Using mouse models of MERS-CoV and SARS-CoV-2 infection, we found that MERS-CoV lacking protein 8b expression was more virulent, while SARS-CoV-2 lacking protein 9b expression was attenuated compared with the respective wild-type viruses. Upon further analysis, we detected increased levels of type I interferon and enhanced infiltration of immune cells to the lungs of mice infected with MERS-CoV lacking protein 8b expression. These data suggest that the I protein of MERS-CoV plays a role in limiting pathogenesis while that of SARS-CoV-2 enhances disease severity. IMPORTANCE The function of betacoronavirus internal protein has been relatively understudied. The earliest report on the internal protein of mouse hepatitis virus suggested that the internal protein is a structural protein without significant functions in virus replication and virulence. However, the internal proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus, and SARS-CoV-2 have been shown to evade immune responses. Despite the reported functions of the internal protein in these highly pathogenic human coronaviruses, its role in mediating pathogenesis in experimentally infected animals has not been characterized. Our data indicated that despite the similar genomic location and expression strategy of these internal proteins, their effects on virulence are vastly different and virus specific, highlighting the complexity between host-virus interaction and disease outcome.

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

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.

Usp25-Erlin1/2 activity limits cholesterol flux to restrict virus infection.

Reprogramming lipid metabolic pathways is a critical feature of activating immune responses to infection. However, how these reconfigurations occur is poorly understood. Our previous screen to identify cellular deubiquitylases (DUBs) activated during influenza virus infection revealed Usp25 as a prominent hit. Here, we show that Usp25-deleted human lung epithelial A549 cells display a >10-fold increase in pathogenic influenza virus production, which was rescued upon reconstitution with the wild type but not the catalytically deficient (C178S) variant. Proteomic analysis of Usp25 interactors revealed a strong association with Erlin1/2, which we confirmed as its substrate. Newly synthesized Erlin1/2 were degraded in Usp25-/- or Usp25C178S cells, activating Srebp2, with increased cholesterol flux and attenuated TLR3-dependent responses. Our study therefore defines the function of a deubiquitylase that serves to restrict a range of viruses by reprogramming lipid biosynthetic flux to install appropriate inflammatory responses.

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

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.

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

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 revealed 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 demonstrated that the CDR H3 sequences from many other IGHV1-69 antibodies, including those to HA stem, were 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.

Mutational fitness landscape of human influenza H3N2 neuraminidase.

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.

Age-related seroprevalence trajectories of seasonal coronaviruses in children including neonates in Guangzhou, China.

OBJECTIVES: Four seasonal coronaviruses, including human coronavirus (HCoV)-229E and HCoV-OC43, HCoV-NL63, and HCoV-HKU1 cause approximately 15-30% of common colds in adults. However, the full landscape of the immune trajectory to these viruses that covers the whole childhood period is still not well understood. METHODS: We evaluated the serological responses against the four seasonal coronaviruses in 1886 children aged under 18 years by using enzyme-linked immunosorbent assay. The optical density values against each HCoV were determined from each sample. Generalized additive models were constructed to determine the relationship between age and seroprevalence throughout the whole childhood period. The specific antibody levels against the four seasonal coronaviruses were also tested from the plasma samples of 485 pairs of postpartum women and their newborn babies. RESULTS: The immunoglobulin (Ig) G levels of the four seasonal coronaviruses in the mother and the newborn babies were highly correlated (229E: r = 0.63; OC43: r = 0.65; NL63: r = 0.69; HKU1: r = 0.63). The seroprevalences in children showed a similar trajectory in that the levels of IgG in the neonates dropped significantly and reached the lowest level after the age of around 1 year (229E: 1.18 years; OC43: 0.97 years; NL63: 1.01 years; HKU1: 1.02 years) and then resurgence in the children who aged older than 1 year. Using the lowest level from the generalized additive models as our cutoff, the seroprevalences for HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 were 98.11%, 96.23%, 96.23% and 94.34% at the age of 16-18 years. CONCLUSION: Mothers share HCoV-specific IgGs with their newborn babies and the level of maternal IgGs waned at around 1 year after birth. The resurgence of the HCoV-specific IgGs was found thereafter with the increase in age suggesting repeated infection occurred in children.

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

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.

Neutralizing Antibody Response to Sarbecovirus Is Delayed in Sequential Heterologous Immunization.

Antigenic imprinting, which describes the bias of the antibody response due to previous immune history, can influence vaccine effectiveness. While this phenomenon has been reported for viruses such as influenza, there is little understanding of how prior immune history affects the antibody response to SARS-CoV-2. This study provides evidence for antigenic imprinting through immunization with two Sarbecoviruses, the subgenus that includes SARS-CoV-2. Mice were immunized subsequently with two antigenically distinct Sarbecovirus strains, namely SARS-CoV-1 and SARS-CoV-2. We found that sequential heterologous immunization induced cross-reactive binding antibodies for both viruses and delayed the emergence of neutralizing antibody responses against the booster strain. Our results provide fundamental knowledge about the immune response to Sarbecovirus and important insights into the development of pan-sarbecovirus vaccines and guiding therapeutic interventions.

Ring finger protein 213 assembles into a sensor for ISGylated proteins with antimicrobial activity.

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.

Homologous and heterologous serological response to the N-terminal domain of SARS-CoV-2 in humans and mice.

The increasing numbers of infected cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses serious threats to public health and the global economy. Most SARS-CoV-2 neutralizing antibodies target the receptor binding domain (RBD) and some the N-terminal domain (NTD) of the spike protein, which is the major antigen of SARS-CoV-2. While the antibody response to RBD has been extensively characterized, the antigenicity and immunogenicity of the NTD protein are less well studied. Using 227 plasma samples from COVID-19 patients, we showed that SARS-CoV-2 NTD-specific antibodies could be induced during infection. As compared to the results of SARS-CoV-2 RBD, the serological response of SARS-CoV-2 NTD is less cross-reactive with SARS-CoV, a pandemic strain that was identified in 2003. Furthermore, neutralizing antibodies are rarely elicited in a mice model when NTD is used as an immunogen. We subsequently demonstrate that NTD has an altered antigenicity when expressed alone. Overall, our results suggest that while NTD offers a supplementary strategy for serology testing, it may not be suitable as an immunogen for vaccine development.

Escaping the Lion's Den: redirecting autophagy for unconventional release and spread of viruses.

Autophagy is an evolutionarily conserved process, designed to maintain cellular homeostasis during a range of internal and external stimuli. Conventionally, autophagy is known for coordinated degradation and recycling of intracellular components and removal of cytosolic pathogens. More recently, several lines of evidence have indicated an unconventional, nondegradative role of autophagy for secretion of cargo that lacks a signal peptide. This process referred to as secretory autophagy has also been implicated in the infection cycle of several virus species. This review focuses on the current evidence available on the nondegradative features of autophagy, emphasizing its potential role and unresolved questions in the release and spread of (-) and (+) RNA viruses.

OTUB1 Is a Key Regulator of RIG-I-Dependent Immune Signaling and Is Targeted for Proteasomal Degradation by Influenza A NS1.

Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses.

Dmrt5 controls corticotrope and gonadotrope differentiation in the zebrafish pituitary.

Dmrt transcription factors control sex determination or sex-specific differentiation across all invertebrate and vertebrate species, in which they have been studied so far. In addition to important functions in the reproductive system, also nongonadal roles have been assigned to several dmrt family members. One example is dmrt5, which was shown to guide neurogenesis in the forebrain of some vertebrates including fish. Here we show that in zebrafish, dmrt5 is also expressed adjacent to the pituitary anlage and later in the anterior pars distalis in which it organizes differentiation of endocrine cells. We find that pituitary induction, cell survival, proliferation, and early lineage specification in the pituitary is independent of dmrt5. Instead, dmrt5 is required for terminal differentiation of corticotropes and gonadotropes. Gene knockdown and mutant analysis revealed that dmrt5 promotes corticotrope differentiation via tbx19 expression, whereas it prevents gonadotrope differentiation in the anterior pars distalis. In dmrt5 morphants and mutants, reduced corticotrope numbers may result in irregular positioning and reduced maintenance of lactotropes. In conclusion, our study establishes a novel function for dmrt5 for cell differentiation in the anterior pituitary. Intriguingly, its effect on gonadotrope numbers defines a first nongonadal role for a dmrt family member that appears crucial for the activity of the reproductive system.