Modulation of Influenza A virus NS1 expression reveals prioritization of host response antagonism at single-cell resolution.

Influenza A virus (IAV) is an important human respiratory pathogen that causes significant seasonal epidemics and potential devastating pandemics. As part of its life cycle, IAV encodes the multifunctional protein NS1, that, among many roles, prevents immune detection and limits interferon (IFN) production. As distinct host immune pathways exert different selective pressures against IAV, as replication progresses, we expect a prioritization in the host immune antagonism by NS1. In this work, we profiled bulk transcriptomic differences in a primary bronchial epithelial cell model facing IAV infections at distinct NS1 levels. We further demonstrated that, at single cell level, the intracellular amount of NS1 in-part shapes the heterogeneity of the host response. We found that modulation of NS1 levels reveal a ranking in its inhibitory roles: modest NS1 expression is sufficient to inhibit immune detection, and thus the expression of pro-inflammatory cytokines (including IFNs), but higher levels are required to inhibit IFN signaling and ISG expression. Lastly, inhibition of chaperones related to the unfolded protein response requires the highest amount of NS1, often associated with later stages of viral replication. This work demystifies some of the multiple functions ascribed to IAV NS1, highlighting the prioritization of NS1 in antagonizing the different pathways involved in the host response to IAV infection.

SARS-CoV-2 hijacks p38ß/MAPK11 to promote virus replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, drastically modifies infected cells to optimize virus replication. One such modification is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammatory cytokine production, a hallmark of severe COVID-19. We previously demonstrated that inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduced both cytokine production and viral replication. Here, we combined quantitative genetic screening, genomics, proteomics, and phosphoproteomics to better understand mechanisms underlying the dependence of SARS-CoV-2 on the p38 pathway. We found that p38ß is a critical host factor for SARS-CoV-2 replication in multiple relevant cell lines and that it functions at a step after viral mRNA expression. We identified putative host and viral p38ß substrates in the context of SARS-CoV-2 infection and found that most host substrates have intrinsic antiviral activities. Taken together, this study reveals a unique proviral function for p38ß and supports exploring p38ß inhibitor development as a strategy toward creating a new class of COVID-19 therapies. IMPORTANCE SARS-CoV-2 is the causative agent of the COVID-19 pandemic that has claimed millions of lives since its emergence in 2019. SARS-CoV-2 infection of human cells requires the activity of several cellular pathways for successful replication. One such pathway, the p38 MAPK pathway, is required for virus replication and disease pathogenesis. Here, we applied systems biology approaches to understand how MAPK pathways benefit SARS-CoV-2 replication to inform the development of novel COVID-19 drug therapies.

SARS-CoV-2 airway infection results in the development of somatosensory abnormalities in a hamster model.

Although largely confined to the airways, SARS-CoV-2 infection has been associated with sensory abnormalities that manifest in both acute and chronic phenotypes. To gain insight on the molecular basis of these sensory abnormalities, we used the golden hamster model to characterize and compare the effects of infection with SARS-CoV-2 and influenza A virus (IAV) on the sensory nervous system. We detected SARS-CoV-2 transcripts but no infectious material in the cervical and thoracic spinal cord and dorsal root ganglia (DRGs) within the first 24 hours of intranasal virus infection. SARS-CoV-2-infected hamsters exhibited mechanical hypersensitivity that was milder but prolonged compared with that observed in IAV-infected hamsters. RNA sequencing analysis of thoracic DRGs 1 to 4 days after infection suggested perturbations in predominantly neuronal signaling in SARS-CoV-2-infected animals as opposed to type I interferon signaling in IAV-infected animals. Later, 31 days after infection, a neuropathic transcriptome emerged in thoracic DRGs from SARS-CoV-2-infected animals, which coincided with SARS-CoV-2-specific mechanical hypersensitivity. These data revealed potential targets for pain management, including the RNA binding protein ILF3, which was validated in murine pain models. This work elucidates transcriptomic signatures in the DRGs triggered by SARS-CoV-2 that may underlie both short- and long-term sensory abnormalities.

Nipah Virus Bangladesh Infection Elicits Organ-Specific Innate and Inflammatory Responses in the Marmoset Model.

The common marmoset (Callithrix jacchus) is increasingly recognized as an ideal nonhuman primate (NHP) at high biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets at biosafety level 4. Infection via the intranasal and intratracheal route resulted in fatal disease in all 4 infected marmosets. Three developed pulmonary edema and hemorrhage as well as multifocal hemorrhagic lymphadenopathy, while 1 recapitulated neurologic clinical manifestations and cardiomyopathy on gross pathology. Organ-specific innate and inflammatory responses were characterized by RNA sequencing in 6 different tissues from infected and control marmosets. Notably, a unique transcriptome was revealed in the brainstem of the marmoset exhibiting neurological signs. Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.

SARS-CoV-2 Airway Infection Results in Time-dependent Sensory Abnormalities in a Hamster Model.

Despite being largely confined to the airways, SARS-CoV-2 infection has been associated with sensory abnormalities that manifest in both acute and long-lasting phenotypes. To gain insight on the molecular basis of these sensory abnormalities, we used the golden hamster infection model to characterize the effects of SARS-CoV-2 versus Influenza A virus (IAV) infection on the sensory nervous system. Efforts to detect the presence of virus in the cervical/thoracic spinal cord and dorsal root ganglia (DRGs) demonstrated detectable levels of SARS-CoV-2 by quantitative PCR and RNAscope uniquely within the first 24 hours of infection. SARS-CoV-2-infected hamsters demonstrated mechanical hypersensitivity during acute infection; intriguingly, this hypersensitivity was milder, but prolonged when compared to IAV-infected hamsters. RNA sequencing (RNA-seq) of thoracic DRGs from acute infection revealed predominantly neuron-biased signaling perturbations in SARS-CoV-2-infected animals as opposed to type I interferon signaling in tissue derived from IAV-infected animals. RNA-seq of 31dpi thoracic DRGs from SARS-CoV-2-infected animals highlighted a uniquely neuropathic transcriptomic landscape, which was consistent with substantial SARS-CoV-2-specific mechanical hypersensitivity at 28dpi. Ontology analysis of 1, 4, and 30dpi RNA-seq revealed novel targets for pain management, such as ILF3. Meta-analysis of all SARS-CoV-2 RNA-seq timepoints against preclinical pain model datasets highlighted both conserved and unique pro-nociceptive gene expression changes following infection. Overall, this work elucidates novel transcriptomic signatures triggered by SARS-CoV-2 that may underlie both short- and long-term sensory abnormalities while also highlighting several therapeutic targets for alleviation of infection-induced hypersensitivity. One Sentence Summary: SARS-CoV-2 infection results in an interferon-associated transcriptional response in sensory tissues underlying time-dependent hypersensitivity.

SARS-CoV-2 infection in hamsters and humans results in lasting and unique systemic perturbations after recovery.

The host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in prolonged pathologies collectively referred to as post-acute sequalae of COVID-19 (PASC) or long COVID. To better understand the mechanism underlying long COVID biology, we compared the short- and long-term systemic responses in the golden hamster after either SARS-CoV-2 or influenza A virus (IAV) infection. Results demonstrated that SARS-CoV-2 exceeded IAV in its capacity to cause permanent injury to the lung and kidney and uniquely affected the olfactory bulb (OB) and olfactory epithelium (OE). Despite a lack of detectable infectious virus, the OB and OE demonstrated myeloid and T cell activation, proinflammatory cytokine production, and an interferon response that correlated with behavioral changes extending a month after viral clearance. These sustained transcriptional changes could also be corroborated from tissue isolated from individuals who recovered from COVID-19. These data highlight a molecular mechanism for persistent COVID-19 symptomology and provide a small animal model to explore future therapeutics.

Immune memory from SARS-CoV-2 infection in hamsters provides variant-independent protection but still allows virus transmission.

SARS-CoV-2 has caused morbidity and mortality across the globe. As the virus spreads, new variants are arising that show enhanced capacity to bypass preexisting immunity. To understand the memory response to SARS-CoV-2, here, we monitored SARS-CoV-2­specific T and B cells in a longitudinal study of infected and recovered golden hamsters (Mesocricetus auratus). We demonstrated that engagement of the innate immune system after SARS-CoV-2 infection was delayed but was followed by a pronounced adaptive response. Moreover, T cell adoptive transfer conferred a reduction in virus levels and rapid induction of SARS-CoV-2­specific B cells, demonstrating that both lymphocyte populations contributed to the overall response. Reinfection of recovered animals with a SARS-CoV-2 variant of concern showed that SARS-CoV-2­specific T and B cells could effectively control the infection that associated with the rapid induction of neutralizing antibodies but failed to block transmission to both naïve and seroconverted animals. These data suggest that the adaptive immune response to SARS-CoV-2 is sufficient to provide protection to the host, independent of the emergence of variants.

The NF-κB Transcriptional Footprint Is Essential for SARS-CoV-2 Replication.

SARS-CoV-2, the etiological agent of COVID-19, is characterized by a delay in type I interferon (IFN-I)-mediated antiviral defenses alongside robust cytokine production. Here, we investigate the underlying molecular basis for this imbalance and implicate virus-mediated activation of NF-κB in the absence of other canonical IFN-I-related transcription factors. Epigenetic and single-cell transcriptomic analyses show a selective NF-κB signature that was most prominent in infected cells. Disruption of NF-κB signaling through the silencing of the NF-κB transcription factor p65 or p50 resulted in loss of virus replication that was rescued upon reconstitution. These findings could be further corroborated with the use of NF-κB inhibitors, which reduced SARS-CoV-2 replication in vitro. These data suggest that the robust cytokine production in response to SARS-CoV-2, despite a diminished IFN-I response, is the product of a dependency on NF-κB for viral replication. IMPORTANCE The COVID-19 pandemic has caused significant mortality and morbidity around the world. Although effective vaccines have been developed, large parts of the world remain unvaccinated while new SARS-CoV-2 variants keep emerging. Furthermore, despite extensive efforts and large-scale drug screenings, no fully effective antiviral treatment options have been discovered yet. Therefore, it is of the utmost importance to gain a better understanding of essential factors driving SARS-CoV-2 replication to be able to develop novel approaches to target SARS-CoV-2 biology.

Leveraging the antiviral type I interferon system as a first line of defense against SARS-CoV-2 pathogenicity.

The emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global morbidity, mortality, and societal disruption. A better understanding of virus-host interactions may potentiate therapeutic insights toward limiting this infection. Here we investigated the dynamics of the systemic response to SARS-CoV-2 in hamsters by histological analysis and transcriptional profiling. Infection resulted in consistently high levels of virus in the upper and lower respiratory tracts and sporadic occurrence in other distal tissues. A longitudinal cohort revealed a wave of inflammation, including a type I interferon (IFN-I) response, that was evident in all tissues regardless of viral presence but was insufficient to prevent disease progression. Bolstering the antiviral response with intranasal administration of recombinant IFN-I reduced viral disease, prevented transmission, and lowered inflammation in vivo. This study defines the systemic host response to SARS-CoV-2 infection and supports use of intranasal IFN-I as an effective means of early treatment.

Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition.

Negative-sense RNA viruses (NSVs) rely on prepackaged viral RNA-dependent RNA polymerases (RdRp) to replicate and transcribe their viral genomes. Their replication machinery consists of an RdRp bound to viral RNA which is wound around a nucleoprotein (NP) scaffold, forming a viral ribonucleoprotein complex. NSV NP is known to regulate transcription and replication of genomic RNA; however, its role in maintaining and protecting the viral genetic material is unknown. Here, we exploited host microRNA expression to target NP of influenza A virus and Sendai virus to ascertain how this would impact genomic levels and the host response to infection. We find that in addition to inducing a drastic decrease in genome replication, the antiviral host response in the absence of NP is dramatically enhanced. Additionally, our data show that insufficient levels of NP prevent the replication machinery of these NSVs to process full-length genomes, resulting in aberrant replication products which form pathogen-associated molecular patterns in the process. These dynamics facilitate immune recognition by cellular pattern recognition receptors leading to a strong host antiviral response. Moreover, we observe that the consequences of limiting NP levels are universal among NSVs, including Ebola virus, Lassa virus, and measles virus. Overall, these results provide new insights into viral genome replication of negative-sense RNA viruses and highlight novel avenues for developing effective antiviral strategies, adjuvants, and/or live-attenuated vaccines.IMPORTANCE Negative-sense RNA viruses comprise some of the most important known human pathogens, including influenza A virus, measles virus, and Ebola virus. These viruses possess RNA genomes that are unreadable to the host, as they require specific viral RNA-dependent RNA polymerases in conjunction with other viral proteins, such as nucleoprotein, to be replicated and transcribed. As this process generates a significant amount of pathogen-associated molecular patterns, this phylum of viruses can result in a robust induction of the intrinsic host cellular response. To circumvent these defenses, these viruses form tightly regulated ribonucleoprotein replication complexes in order to protect their genomes from detection and to prevent excessive aberrant replication. Here, we demonstrate the balance that negative-sense RNA viruses must achieve both to replicate efficiently and to avoid induction of the host defenses.

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19.

Viral pandemics, such as the one caused by SARS-CoV-2, pose an imminent threat to humanity. Because of its recent emergence, there is a paucity of information regarding viral behavior and host response following SARS-CoV-2 infection. Here we offer an in-depth analysis of the transcriptional response to SARS-CoV-2 compared with other respiratory viruses. Cell and animal models of SARS-CoV-2 infection, in addition to transcriptional and serum profiling of COVID-19 patients, consistently revealed a unique and inappropriate inflammatory response. This response is defined by low levels of type I and III interferons juxtaposed to elevated chemokines and high expression of IL-6. We propose that reduced innate antiviral defenses coupled with exuberant inflammatory cytokine production are the defining and driving features of COVID-19.

Viral Fitness Landscapes in Diverse Host Species Reveal Multiple Evolutionary Lines for the NS1 Gene of Influenza A Viruses.

Influenza A viruses (IAVs) have a remarkable tropism in their ability to circulate in both mammalian and avian species. The IAV NS1 protein is a multifunctional virulence factor that inhibits the type I interferon host response through a myriad of mechanisms. How NS1 has evolved to enable this remarkable property across species and its specific impact in the overall replication, pathogenicity, and host preference remain unknown. Here we analyze the NS1 evolutionary landscape and host tropism using a barcoded library of recombinant IAVs. Results show a surprisingly great variety of NS1 phenotypes according to their ability to replicate in different hosts. The IAV NS1 genes appear to have taken diverse and random evolutionary pathways within their multiple phylogenetic lineages. In summary, the high evolutionary plasticity of this viral protein underscores the ability of IAVs to adapt to multiple hosts and aids in our understanding of its global prevalence.

Type I interferon response impairs differentiation potential of pluripotent stem cells.

Upon virus infection, pluripotent stem cells neither induce nor respond to canonical type I interferons (IFN-I). To better understand this biology, we characterized induced pluripotent stem cells (iPSCs) as well as their differentiated parental or rederived counterparts. We confirmed that only iPSCs failed to respond to viral RNA, IFN-I, or viral infection. This lack of response could be phenocopied in fibroblasts with the expression of a reprogramming factor which repressed the capacity to induce canonical antiviral pathways. To ascertain the consequences of restoring the antiviral response in the context of pluripotency, we engineered a system to engage these defenses in iPSCs. Inducible expression of a recombinant virus-activated transcription factor resulted in the successful reconstitution of antiviral defenses through the direct up-regulation of IFN-I-stimulated genes. Induction of the antiviral signature in iPSCs, even for a short duration, resulted in the dysregulation of genes associated with all three germ layers despite maintaining pluripotency markers. Trilineage differentiation of these same cells showed that engagement of the antiviral defenses compromised ectoderm and endoderm formation and dysregulated the development of mesodermal sublineages. In all, these data suggest that the temporal induction of the antiviral response primes iPSCs away from pluripotency and induces numerous aberrant gene products upon differentiation. Together these results suggest that the IFN-I system and pluripotency may be incompatible with each other and thus explain why stem cells do not utilize the canonical antiviral system.

Homologous recombination is an intrinsic defense against antiviral RNA interference.

RNA interference (RNAi) is the major antiviral defense mechanism of plants and invertebrates, rendering the capacity to evade it a defining factor in shaping the viral landscape. Here we sought to determine whether different virus replication strategies provided any inherent capacity to evade RNAi in the absence of an antagonist. Through the exploitation of host microRNAs, we recreated an RNAi-like environment in vertebrates and directly compared the capacity of positive- and negative-stranded RNA viruses to cope with this selective pressure. Applying this defense against four distinct viral families revealed that the capacity to undergo homologous recombination was the defining attribute that enabled evasion of this defense. Independent of gene expression strategy, positive-stranded RNA viruses that could undergo strand switching rapidly excised genomic material, while negative-stranded viruses were effectively targeted and cleared upon RNAi-based selection. These data suggest a dynamic relationship between host antiviral defenses and the biology of virus replication in shaping pathogen prevalence.

miRNA-mediated targeting of human cytomegalovirus reveals biological host and viral targets of IE2.

Human cytomegalovirus (HCMV) impacts more than one-half of the human population owing to its capacity to manipulate the cell and create latent reservoirs in the host. Despite an extensive understanding of HCMV biology during acute infection in fibroblasts, the molecular basis for latency in myeloid cells remains incomplete. This knowledge gap is due largely to the fact that the existing genetic systems require virus rescue in fibroblasts, precluding the study of genes that are essential during acute infection, yet likely play unique roles in myeloid cells or the establishment of latency. Here we present a solution to address this restriction. Through the exploitation of a hematopoietic-specific microRNA, we demonstrate a one-step recombineering approach that enables gene silencing only in cells associated with latency. As a proof of concept, here we describe a TB40/E variant that undergoes hematopoietic targeting of the Immediate Early-2 (IE2) gene to explore its function during infection of myeloid cells. While virus replication of the hematopoietic-targeted IE2 variant was unimpaired in fibroblasts, we observed a >100-fold increase in virus titers in myeloid cells. Virus replication in myeloid cells demonstrated that IE2 has a significant transcriptional footprint on both viral and host genes. These data implicate IE2 as an essential mediator of virus biology in myeloid cells and illustrate the utility of cell-specific microRNA-based targeting.

RNase III nucleases from diverse kingdoms serve as antiviral effectors.

In contrast to the DNA-based viruses in prokaryotes, the emergence of eukaryotes provided the necessary compartmentalization and membranous environment for RNA viruses to flourish, creating the need for an RNA-targeting antiviral system. Present day eukaryotes employ at least two main defence strategies that emerged as a result of this viral shift, namely antiviral RNA interference and the interferon system. Here we demonstrate that Drosha and related RNase III ribonucleases from all three domains of life also elicit a unique RNA-targeting antiviral activity. Systemic evolution of ligands by exponential enrichment of this class of proteins illustrates the recognition of unbranched RNA stem loops. Biochemical analyses reveal that, in this context, Drosha functions as an antiviral clamp, conferring steric hindrance on the RNA-dependent RNA polymerases of diverse positive-stranded RNA viruses. We present evidence for cytoplasmic translocation of RNase III nucleases in response to virus in diverse eukaryotes including plants, arthropods, fish, and mammals. These data implicate RNase III recognition of viral RNA as an antiviral defence that is independent of, and possibly predates, other known eukaryotic antiviral systems.

In Vivo RNAi Screening Identifies MDA5 as a Significant Contributor to the Cellular Defense against Influenza A Virus.

Responding to an influenza A virus (IAV) infection demands an effective intrinsic cellular defense strategy to slow replication. To identify contributing host factors to this defense, we exploited the host microRNA pathway to perform an in vivo RNAi screen. To this end, IAV, lacking a functional NS1 antagonist, was engineered to encode individual siRNAs against antiviral host genes in an effort to rescue attenuation. This screening platform resulted in the enrichment of strains targeting virus-activated transcription factors, specific antiviral effectors, and intracellular pattern recognition receptors (PRRs). Interestingly, in addition to RIG-I, the PRR for IAV, a virus with the capacity to silence MDA5 also emerged as a dominant strain in wild-type, but not in MDA5-deficient mice. Transcriptional profiling of infected knockout cells confirmed RIG-I to be the primary PRR for IAV but implicated MDA5 as a significant contributor to the cellular defense against influenza A virus.

An in vivo RNAi screening approach to identify host determinants of virus replication.

RNA interference (RNAi) has been extensively used to identify host factors affecting virus infection but requires exogenous delivery of short interfering RNAs (siRNAs), thus limiting the technique to nonphysiological infection models and a single defined cell type. We report an alternative screening approach using siRNA delivery via infection with a replication-competent RNA virus. In this system, natural selection, defined by siRNA production, permits the identification of host restriction factors through virus enrichment during a physiological infection. We validate this approach with a large-scale siRNA screen in the context of an in vivo alphavirus infection. Monitoring virus evolution across four independent screens identified two categories of enriched siRNAs: specific effectors of the direct antiviral arsenal and host factors that indirectly dampened the overall antiviral response. These results suggest that pathogenicity may be defined by the ability of the virus to antagonize broad cellular responses and specific antiviral factors.

A diverse range of gene products are effectors of the type I interferon antiviral response.

The type I interferon response protects cells against invading viral pathogens. The cellular factors that mediate this defence are the products of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified since their discovery more than 25 years ago, only a few have been characterized with respect to antiviral activity. For most ISG products, little is known about their antiviral potential, their target specificity and their mechanisms of action. Using an overexpression screening approach, here we show that different viruses are targeted by unique sets of ISGs. We find that each viral species is susceptible to multiple antiviral genes, which together encompass a range of inhibitory activities. To conduct the screen, more than 380 human ISGs were tested for their ability to inhibit the replication of several important human and animal viruses, including hepatitis C virus, yellow fever virus, West Nile virus, chikungunya virus, Venezuelan equine encephalitis virus and human immunodeficiency virus type-1. Broadly acting effectors included IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (also known as DDX58), MDA5 (also known as IFIH1) and IFITM3, whereas more targeted antiviral specificity was observed with DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also known as PBEF1), OASL, RTP4, TREX1 and UNC84B (also known as SUN2). Combined expression of pairs of ISGs showed additive antiviral effects similar to those of moderate type I interferon doses. Mechanistic studies uncovered a common theme of translational inhibition for numerous effectors. Several ISGs, including ADAR, FAM46C, LY6E and MCOLN2, enhanced the replication of certain viruses, highlighting another layer of complexity in the highly pleiotropic type I interferon system.