ABSTRACT 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 p38β 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 p38β 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 p38β and supports exploring p38β 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.
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.
SUMMARY SARS-CoV-2 has been found capable of inducing prolonged pathologies collectively referred to as Long-COVID. To better understand this biology, we compared the short- and long-term systemic responses in the golden hamster following either SARS-CoV-2 or influenza A virus (IAV) infection. While SARS-CoV-2 exceeded IAV in its capacity to cause injury to the lung and kidney, the most significant changes were observed in the olfactory bulb (OB) and olfactory epithelium (OE) where inflammation was visible beyond one month post SARS-CoV-2 infection. Despite a lack of detectable virus, OB/OE demonstrated microglial and T cell activation, proinflammatory cytokine production, and interferon responses that correlated with behavioral changes. These findings could be corroborated through sequencing of individuals who recovered from COVID-19, as sustained inflammation in OB/OE tissue remained evident months beyond disease resolution. These data highlight a molecular mechanism for persistent COVID-19 symptomology and characterize a small animal model to develop future therapeutics.
SARS-CoV-2 has caused significant morbidity and mortality across the globe. As the virus spreads, new variants are arising that show enhanced capacity to bypass pre-existing immunity. To understand the memory response to SARS-CoV-2, here we monitored SARS-CoV-2-specific T and B cells in a longitudinal study of infected and recovered golden hamsters. We demonstrate that engagement of the innate immune system following 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-2-specific B cells demonstrating both lymphocyte populations contributed to the overall response. Reinfection of recovered animals with the SARS-CoV-2 beta variant showed SARS-CoV-2-specific memory T and B cells could effectively control the infection through neutralizing antibodies. 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 novel variants.
Influenza A viruses (IAV) have a remarkable tropism in their capacity to circulate in both mammalian and avian species. One major tropism determinant is their capacity to evade species-specific antiviral immune responses. 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, remains unknown. Here we use a novel approach to analyze the NS1 evolutionary landscape and host tropism through the use of a barcoded library of recombinant IAV. Results showed diverse and apparently random evolutionary pathways taken by IAV NS1 according to its multiple phylogenetic lineages. In summary, the high evolutionary plasticity of this viral protein underscores the ability of IAV to adapt to multiple hosts and aids in our understanding of its global prevalence.
