Narnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods, and nematodes. Though initially described as simple single-gene nonsegmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as "ambigrammatic" harbor a unique genomic configuration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquito-infecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2-kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8-kb "Robin" RNA. We developed a system to launch CxNV1 in a naive mosquito cell line and then showed that functional RdRp is required for persistence of both segments, and an intact reverse ORF is required on the RdRp segment for persistence. Mass spectrometry of persistently CxNV1-infected cells provided evidence for translation of this reverse ORF. Finally, ribosome profiling yielded a striking pattern of footprints for all four CxNV1 RNA strands that was distinct from actively translating ribosomes on host mRNA or coinfecting RNA viruses. Taken together, these data raise the possibility that the process of translation itself is important for persistence of ambigrammatic narnaviruses, potentially by protecting viral RNA with ribosomes, thus suggesting a heretofore undescribed viral tactic for replication and transmission. IMPORTANCE Fundamental to our understanding of RNA viruses is a description of which strand(s) of RNA are transmitted as the viral genome relative to which encode the viral proteins. Ambigrammatic narnaviruses break the mold. These viruses, found broadly in fungi, plants, and insects, have the unique feature of two overlapping genes encoded on opposite strands, comprising nearly the full length of the viral genome. Such extensive overlap is not seen in other RNA viruses and comes at the cost of reduced evolutionary flexibility in the sequence. The present study is motivated by investigating the benefits which balance that cost. We show for the first time a functional requirement for the ambigrammatic genome configuration in Culex narnavirus 1, which suggests a model for how translation of both strands might benefit this virus. Our work highlights a new blueprint for viral persistence, distinct from strategies defined by canonical definitions of the coding strand.

Lymphocytic choriomeningitis virus (LCMV) is a well-studied mammarenavirus that can be fatal in congenital infections. However, our understanding of LCMV and its interactions with human host factors remains incomplete. Here, host determinants affecting LCMV infection were investigated through a genome-wide CRISPR knockout screen in A549 cells, a human lung adenocarcinoma line. We identified and validated a variety of novel host factors that play a functional role in LCMV infection. Among these, knockout of the sialomucin CD164, a heavily glycosylated transmembrane protein, was found to ablate infection with multiple LCMV strains but not other hemorrhagic mammarenaviruses in several cell types. Further characterization revealed a dependency of LCMV entry on the cysteine-rich domain of CD164, including an N-linked glycosylation site at residue 104 in that region. Given the documented role of LCMV with respect to transplacental human infections, CD164 expression was investigated in human placental tissue and placental cell lines. CD164 was found to be highly expressed in the cytotrophoblast cells, an initial contact site for pathogens within the placenta, and LCMV infection in placental cells was effectively blocked using a monoclonal antibody specific to the cysteine-rich domain of CD164. Together, this study identifies novel factors associated with LCMV infection of human tissues and highlights the importance of CD164, a sialomucin that previously had not been associated with viral infection. IMPORTANCE Lymphocytic choriomeningitis virus (LCMV) is a human-pathogenic mammarenavirus that can be fatal in congenital infections. Although frequently used in the study of persistent infections in the field of immunology, aspects of this virus's life cycle remain incomplete. For example, while viral entry has been shown to depend on a cell adhesion molecule, DAG1, genetic knockout of this gene allows for residual viral infection, implying that additional receptors can mediate cell entry. The significance of our study is the identification of host factors important for successful infection, including the sialomucin CD164, which had not been previously associated with viral infection. We demonstrated that CD164 is essential for LCMV entry into human cells and can serve as a possible therapeutic target for treatment of congenital infection.

The identification of autoantigens remains a critical challenge for understanding and treating autoimmune diseases. Autoimmune polyendocrine syndrome type 1 (APS1), a rare monogenic form of autoimmunity, presents as widespread autoimmunity with T and B cell responses to multiple organs. Importantly, autoantibody discovery in APS1 can illuminate fundamental disease pathogenesis, and many of the antigens found in APS1 extend to more common autoimmune diseases. Here, we performed proteome-wide programmable phage-display (PhIP-Seq) on sera from a cohort of people with APS1 and discovered multiple common antibody targets. These novel APS1 autoantigens exhibit tissue-restricted expression, including expression in enteroendocrine cells, pineal gland, and dental enamel. Using detailed clinical phenotyping, we find novel associations between autoantibodies and organ-restricted autoimmunity, including a link between anti-KHDC3L autoantibodies and premature ovarian insufficiency, and between anti-RFX6 autoantibodies and diarrheal-type intestinal dysfunction. Our study highlights the utility of PhIP-Seq for extensively interrogating antigenic repertoires in human autoimmunity and the importance of antigen discovery for improved understanding of disease mechanisms.

Impaired baseline lung function is associated with mortality after pediatric allogeneic hematopoietic cell transplantation (HCT), yet limited knowledge of the molecular pathways that characterize pretransplant lung function has hindered the development of lung-targeted interventions. In this study, we quantified the association between bronchoalveolar lavage (BAL) metatranscriptomes and paired pulmonary function tests performed a median of 1 to 2 weeks before allogeneic HCT in 104 children in The Netherlands. Abnormal pulmonary function was recorded in more than half the cohort, consisted most commonly of restriction and impaired diffusion, and was associated with both all-cause and lung injury-related mortality after HCT. Depletion of commensal supraglottic taxa, such as Haemophilus, and enrichment of nasal and skin taxa, such as Staphylococcus, in the BAL microbiome were associated with worse measures of lung capacity and gas diffusion. In addition, BAL gene expression signatures of alveolar epithelial activation, epithelial-mesenchymal transition, and down-regulated immunity were associated with impaired lung capacity and diffusion, suggesting a postinjury profibrotic response. Detection of microbial depletion and abnormal epithelial gene expression in BAL enhanced the prognostic utility of pre-HCT pulmonary function tests for the outcome of post-HCT mortality. These findings suggest a potentially actionable connection between microbiome depletion, alveolar injury, and pulmonary fibrosis in the pathogenesis of pre-HCT lung dysfunction.

Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-κB inhibitor IκBα (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells.

The wide spectrum of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with phenotypes impacting transmission and antibody sensitivity necessitates investigation of immune responses to different spike protein versions. Here, we compare neutralization of variants of concern, including B.1.617.2 (delta) and B.1.1.529 (omicron), in sera from individuals exposed to variant infection, vaccination, or both. We demonstrate that neutralizing antibody responses are strongest against variants sharing certain spike mutations with the immunizing exposure, and exposure to multiple spike variants increases breadth of variant cross-neutralization. These findings contribute to understanding relationships between exposures and antibody responses and may inform booster vaccination strategies.

We evaluated the performance of the Abbott BinaxNOW rapid antigen test for coronavirus disease 2019 (Binax-CoV2) to detect virus among persons, regardless of symptoms, at a public plaza site of ongoing community transmission. Titration with cultured severe acute respiratory syndrome coronavirus 2 yielded a human observable threshold between 1.6 × 104-4.3 × 104 viral RNA copies (cycle threshold [Ct], 30.3-28.8). Among 878 subjects tested, 3% (26 of 878) were positive by reverse-transcription polymerase chain reaction, of whom 15 of 26 had a Ct <30, indicating high viral load; of these, 40% (6 of 15) were asymptomatic. Using this Ct threshold (<30) for Binax-CoV2 evaluation, the sensitivity of Binax-CoV2 was 93.3% (95% confidence interval, 68.1%-99.8%) (14 of 15) and the specificity was 99.9% (99.4%-99.9%) (855 of 856).

SARS-CoV-2 infection has led to a global health crisis, and yet our understanding of the disease and potential treatment options remains limited. The infection occurs through binding of the virus with angiotensin converting enzyme 2 (ACE2) on the cell membrane. Here, we established a screening strategy to identify drugs that reduce ACE2 levels in human embryonic stem cell (hESC)-derived cardiac cells and lung organoids. Target analysis of hit compounds revealed androgen signaling as a key modulator of ACE2 levels. Treatment with antiandrogenic drugs reduced ACE2 expression and protected hESC-derived lung organoids against SARS-CoV-2 infection. Finally, clinical data on COVID-19 patients demonstrated that prostate diseases, which are linked to elevated androgen, are significant risk factors and that genetic variants that increase androgen levels are associated with higher disease severity. These findings offer insights on the mechanism of disproportionate disease susceptibility in men and identify antiandrogenic drugs as candidate therapeutics for COVID-19.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and μMT) were used to model nonspecific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate 3 distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from nonobese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, was enriched in peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.