Faculty Bibliography

BACKGROUND AND AIMS/OBJECTIVE:Immune dysregulation caused by SARS-CoV-2 infection is thought to play a pathogenic role in COVID-19. SARS-CoV-2 can infect a variety of host cells, including intestinal epithelial cells. We sought to characterize the role of the gastrointestinal immune system in the pathogenesis of the inflammatory response associated with COVID-19. METHODS:We measured cytokines, inflammatory markers, viral RNA, microbiome composition and antibody responses in stool and serum samples from a prospectively enrolled cohort of 44 hospitalized COVID-19 patients. RESULTS:SARS-CoV-2 RNA was detected in stool of 41% of patients and was found more frequently in patients with diarrhea than those without (16[44%] vs 5[19%], p=0.06). Patients who survived had lower median viral genome copies than those who did not (p=0.021). Compared to uninfected controls, COVID-19 patients had higher median fecal levels of IL-8 (166.5 vs 286.5 pg/mg; p=0.05) and lower levels of fecal IL-10 (678 vs 194 pg/mg; p<0.001) compared to uninfected controls. Stool IL-23 was higher in patients with more severe COVID-19 disease (223.8 vs 86.6 pg/mg; p=0.03) and we find evidence of intestinal virus-specific IgA responses, which was associated with more severe disease. Fecal cytokines and calprotectin levels were not correlated with gastrointestinal symptoms or with the level of virus detected. CONCLUSIONS:Although SARS-CoV-2 RNA was detectable in the stools of COVID-19 patients and select individuals had evidence for a specific mucosal IgA response, intestinal inflammation was limited, even in patients presenting with gastrointestinal symptoms.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

The outbreak of COVID-19 caused by the SARS-CoV-2 virus has created an unparalleled disruption of global behavior and a significant loss of human lives. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible viral entry routes is essential. As aerosol transmission is thought to be the primary route of spread, we sought to investigate whether the eyes are potential entry portals for SARS-CoV-2. While virus has been detected in the eye, in order for this mucosal membrane to be a bone fide entry source SARS-CoV-2 would need the capacity to productively infect ocular surface cells.&nbsp; As such, we conducted RNA sequencing in ocular cells isolated from adult human cadaver donor eyes as well as from a pluripotent stem cell-derived whole eye organoid model to evaluate the expression of ACE2 and TMPRSS2, essential proteins that mediate SARS-CoV-2 viral entry. We also infected eye organoids and adult human ocular cells with SARS-CoV-2 and evaluated virus replication and the host response to infection. We found the limbus was most susceptible to infection, whereas the central cornea exhibited only low levels of replication. Transcriptional profiling of the limbus upon SARS-CoV-2 infection, found that while type I or III interferons were not detected in the lung epithelium, a significant inflammatory response was mounted. Together these data suggest that the human eye can be directly infected by SARS-CoV-2 and thus is a route warranting protection. Funding: The National Eye Institute (NEI), Bethesda, MD, USA, extramural grant 1R21EY030215-01 and the Icahn School of Medicine at Mount Sinai supported this study.

The SARS-CoV-2 virus has caused already over 3.5 million COVID-19 cases and 250,000 deaths globally. There is an urgent need to create novel models to study SARS-CoV-2 using human disease-relevant cells to understand key features of virus biology and facilitate drug screening. As primary SARS-CoV-2 infection is respiratory-based, we developed a lung organoid model using human pluripotent stem cells (hPSCs) that could be adapted for drug screens. The lung organoids, particularly aveolar type II cells, express ACE2 and are permissive to SARS-CoV-2 infection. Transcriptomic analysis following SARS-CoV-2 infection revealed a robust induction of chemokines and cytokines with little type I/III interferon signaling, similar to that observed amongst human COVID-19 pulmonary infections. We performed a high throughput screen using hPSC-derived lung organoids and identified FDA-approved drug candidates, including imatinib and mycophenolic acid, as inhibitors of SARS-CoV-2 entry. Pre- or post-treatment with these drugs at physiologically relevant levels decreased SARS-CoV-2 infection of hPSC-derived lung organoids. Together, these data demonstrate that hPSC-derived lung cells infected by SARS-CoV-2 can model human COVID-19 disease and provide a valuable resource to screen for FDA-approved drugs that might be repurposed and should be considered for COVID-19 clinical trials.

Heart injury has been reported in up to 20% of COVID-19 patients, yet the cause of myocardial histopathology remains unknown. In order to study the cause of myocardial pathology in COVID-19 patients, we used a hamster model to determine whether following infection SARS-CoV-2, the causative agent of COVID-19, can be detected in heart tissues. Here, we clearly demonstrate that viral RNA and nucleocapsid protein is present in cardiomyocytes in the hearts of infected hamsters. Interestingly, functional cardiomyocyte associated gene expression was decreased in infected hamster hearts, corresponding to an increase in reactive oxygen species (ROS). This data using an animal model was further validated using autopsy heart samples of COVID-19 patients. Moreover, we show that both human pluripotent stem cell-derived cardiomyocytes (hPSC-derived CMs) and adult cardiomyocytes (CMs) can be infected by SARS-CoV-2 and that CCL2 is secreted upon SARS-CoV-2 infection, leading to monocyte recruitment. Increased CCL2 expression and macrophage infiltration was also observed in the hearts of infected hamsters. Using single cell RNA-seq, we also show that macrophages are able to decrease SARS-CoV-2 infection of CMs. Overall, our study provides direct evidence that SARS-CoV-2 infects CMs in vivo and proposes a mechanism of immune-cell infiltration and pathology in heart tissue of COVID-19 patients.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

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.

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.

Every living entity requires the capacity to defend against viruses in some form. From bacteria to plants to arthropods, cells retain the capacity to capture genetic material, process it in a variety of ways, and subsequently use it to generate pathogen-specific small RNAs. These small RNAs can then be used to provide specificity to an otherwise non-specific nuclease, generating a potent antiviral system. While small RNA-based defenses in chordates are less utilized, the protein-based antiviral invention in this phylum appears to have derived from components of the same ancestral small RNA machinery. Based on recent evidence, it would seem that RNase III nucleases have been reiteratively repurposed over billions of years to provide cells with the capacity to recognize and destroy unwanted genetic material. Here we describe an overview of what is known on this subject and provide a model for how these defenses may have evolved.

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.