Evolution of STAT2 resistance to flavivirus NS5 occurred multiple times despite genetic constraints.

Zika and dengue virus nonstructural protein 5 antagonism of STAT2, a critical interferon signaling transcription factor, to suppress the host interferon response is required for viremia and pathogenesis in a vertebrate host. This affects viral species tropism, as mouse STAT2 resistance renders only immunocompromised or humanized STAT2 mice infectable. Here, we explore how STAT2 evolution impacts antagonism. By measuring the susceptibility of 38 diverse STAT2 proteins, we demonstrate that resistance arose numerous times in mammalian evolution. In four species, resistance requires distinct sets of multiple amino acid changes that often individually disrupt STAT2 signaling. This reflects an evolutionary ridge where progressive resistance is balanced by the need to maintain STAT2 function. Furthermore, resistance may come with a fitness cost, as resistance that arose early in lemur evolution was subsequently lost in some lemur lineages. These findings underscore that while it is possible to evolve resistance to antagonism, complex evolutionary trajectories are required to avoid detrimental host fitness consequences.

Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). scRNA-Seq library preparation methods and data processing workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq library preparation methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We show that compared to 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') libraries or 10X Genomics Chromium Next GEM Single Cell V(D)J (10X 5') libraries sequenced with standard read configurations, 10X 5' libraries sequenced with an extended length read 1 (R1) that covers both cell barcode and transcript sequence (termed "10X 5' with extended R1") increase the number of unambiguous reads spanning leader-sgmRNA junction sites. We further present a data processing workflow, single-cell coronavirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to viral sgmRNAs or viral genomic RNA (gRNA). We find that combining 10X 5' with extended R1 library preparation/sequencing and scCoVseq data processing maximizes the number of viral UMIs per cell quantified by scRNA-Seq. Corresponding sgmRNA expression levels are highly correlated with expression in matched bulk RNA-Seq data sets quantified with established tools for SARS-CoV-2 analysis. Using this scRNA-Seq approach, we find that SARS-CoV-2 gene expression is highly correlated across individual infected cells, which suggests that the proportion of viral sgmRNAs remains generally consistent throughout infection. Taken together, these results and corresponding data processing workflow enable robust quantification of coronavirus sgmRNA expression at single-cell resolution, thereby supporting high-resolution studies of viral RNA processes in individual cells. IMPORTANCE Single-cell RNA sequencing (scRNA-Seq) has emerged as a valuable tool to study host-virus interactions, especially for coronavirus disease 2019 (COVID-19). Here we compare the performance of different scRNA-Seq library preparation methods and sequencing strategies to detect SARS-CoV-2 RNAs and develop a data processing workflow to quantify unambiguous sequence reads derived from SARS-CoV-2 genomic RNA and subgenomic mRNAs. After establishing a workflow that maximizes the detection of SARS-CoV-2 subgenomic mRNAs, we explore patterns of SARS-CoV-2 gene expression across cells with variable levels of total viral RNA, assess host gene expression differences between infected and bystander cells, and identify non-canonical and lowly abundant SARS-CoV-2 RNAs. The sequencing and data processing strategies developed here can enhance studies of coronavirus RNA biology at single-cell resolution and thereby contribute to our understanding of viral pathogenesis.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2.

Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single cell RNA sequencing.

Single cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNA-Seq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') and Chromium Next GEM Single Cell V(D)J (10X 5') sequencing, we find that 10X 5' with an extended read 1 (R1) sequencing strategy maximizes the detection of sgmRNAs by increasing the number of unambiguous reads spanning leader-sgmRNA junction sites. Using this method, we show that viral gene expression is highly correlated across cells suggesting a relatively consistent proportion of viral sgmRNA production throughout infection. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro , and in the Syrian golden hamster model in vivo . In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARY: SARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

Unbalanced Regulation of α7 nAChRs by Ly6h and NACHO Contributes to Neurotoxicity in Alzheimer's Disease.

α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain where they promote fast cholinergic synaptic transmission and serve important neuromodulatory functions. However, their high permeability to Ca2+ also predisposes them to contribute to disease states. Here, using transfected HEK-tsa cells and primary cultured hippocampal neurons from male and female rats, we demonstrate that two proteins called Ly6h and NACHO compete for access to α7 subunits, operating together but in opposition to maintain α7 assembly and activity within a narrow range that is optimal for neuronal function and viability. Using mixed gender human temporal cortex and cultured hippocampal neurons from rats we further show that this balance is perturbed during Alzheimer's disease (AD) because of amyloid ß (Aß)-driven reduction in Ly6h, with severe reduction leading to increased phosphorylated tau and α7-mediated neurotoxicity. Ly6h release into human CSF is also correlated with AD severity. Thus, Ly6h links cholinergic signaling, Aß and phosphorylated tau and may serve as a novel marker for AD progression.SIGNIFICANCE STATEMENT One of the earliest and most persistent hypotheses regarding Alzheimer's disease (AD) attributes cognitive impairment to loss of cholinergic signaling. More recently, interest has focused on crucial roles for amyloid ß (Aß) and phosphorylated tau in Alzheimer's pathogenesis. Here, we demonstrate that these elements are linked by Ly6h and its counterpart, NACHO, functioning in opposition to maintain assembly of nicotinic acetylcholine receptors (nAChRs) within the physiological range. Our data suggests that Aß shifts the balance away from Ly6h and toward NACHO, resulting in increased assembly of Ca2+-permeable nAChRs and thus a conversion of basal cholinergic to neurotoxic signaling.