Antinuclear antibodies in individuals with COVID-19 reflect underlying disease: Identification of new autoantibodies in systemic sclerosis (CDK9) and malignancy (RNF20, RCC1, TRIP13).

A high prevalence of antinuclear antibodies (ANA) in COVID-19 has been insinuated, but the nature of the target antigens is poorly understood. We studied ANA by indirect immunofluorescence in 229 individuals with COVID-19. The target antigens of high titer ANA (≥1:320) were determined by immunoprecipitation (IP) combined with liquid-chromatography-mass spectrometry (MS). High titer ANA (≥1:320) were found in 14 (6%) of the individuals with COVID-19. Of the 14 COVID-19 cases with high titer ANA, 6 had an underlying autoimmune disease and 5 a malignancy. IP-MS revealed known target antigens associated with autoimmune disease as well as novel autoantigens, including CDK9 (in systemic sclerosis) and RNF20, RCC1 and TRIP13 (in malignancy). The novel autoantigens were confirmed by IP-Western blotting. In conclusion, in depth analysis of the targets of high titer ANA revealed novel autoantigens in systemic sclerosis and in malignant disease.

Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality.

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins as viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal (SL) partners of cancer-specific mutations. Similarly, SL interactions of viral-induced hypomorphs can potentially be targeted as host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for SL partners of GBF1 revealed ARF1 as the top hit, disruption of which selectively killed cells that synthesize 3A alone or in the context of a poliovirus replicon. Thus, viral protein interactions can induce hypomorphs that render host cells selectively vulnerable to perturbations that leave uninfected cells otherwise unscathed. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2.

MIS-C: A COVID-19-as sociated condition between hypoimmunity and hyperimmunity.

Multisystem inflammatory syndrome in children (MIS-C) is a rare, severe complication of COVID-19. A better knowledge of immunological, cellular, and genetic characteristics of MIS-C could help better understand the pathogenesis of the disease and contribute to identifying specific diagnostic biomarkers and develop targeted therapies. We studied 37 MIS-C children at hospital admission and 24 healthy controls analyzing serum cytokines (IFN-α, IFN-ß, IFN-γ, IL-6, IL-10, IL-17A, IL-12p70 and TNF), lymphocyte populations by flow cytometry and 386 genes related to autoimmune diseases, autoinflammation and primary immunodeficiencies by NGS. MIS-C patients showed a significant increase of serum IFNγ (despite a significant reduction of activated Th1) and ILs, even if with a great heterogeneity among patients, revealing different pathways involved in MIS-C pathogenesis and suggesting that serum cytokines at admission may help to select the inflammatory pathways to target in each patient. Flow cytometry demonstrated a relevant reduction of T populations while the percentage of B cell was increased in agreement with an autoimmune pathogenesis of MIS-C. Genetic analysis identified variants in 34 genes and 83.3% of patients had at least one gene variant. Among these, 9 were mutated in more patients. Most genes are related to autoimmune diseases like ATM, NCF1, MCM4, FCN3, and DOCK8 or to autoinflammatory diseases associated to the release of IFNγ like PRF1, NOD2, and MEF. Thus, an incomplete clearance of the Sars-CoV2 during the acute phase may induce tissue damage and self-antigen exposure and genetic variants can predispose to hyper-reactive immune dysregulation events of MIS-C-syndrome. Type II IFN activation and cytokine responses (mainly IL-6 and IL-10) may cause a cytokine storm in some patients with a more severe acute phase of the disease, lymphopenia and multisystemic organ involvement. The timely identification of such patients with an immunocytometric panel might be critical for targeted therapeutic management.

DOCK2 is involved in the host genetics and biology of severe COVID-19.

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.

Intracellular receptor EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K-/eNOS-dependent manner during inflammation.

Coagulopathy is associated with both inflammation and infection, including infections with novel severe acute respiratory syndrome coronavirus-2, the causative agent Coagulopathy is associated with both inflammation and infection, including infection with novel severe acute respiratory syndrome coronavirus-2, the causative agent of COVID-19. Clot formation is promoted via cAMP-mediated secretion of von Willebrand factor (vWF), which fine-tunes the process of hemostasis. The exchange protein directly activated by cAMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a regulatory role in suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1-null mouse model and revealed increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1-/- phenotype. In addition, EPAC1 regulated tumor necrosis factor-α-triggered vWF secretion from human umbilical vein endothelial cells in a manner dependent upon inflammatory effector molecules PI3K and endothelial nitric oxide synthase. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro. Our data delineate a novel regulatory role for EPAC1 in vWF secretion and shed light on the potential development of new strategies to control thrombosis during inflammation.

Broad Impact of Exchange Protein Directly Activated by cAMP 2 (EPAC2) on Respiratory Viral Infections.

The recently discovered exchange protein directly activated by cAMP (EPAC), compared with protein kinase A (PKA), is a fairly new family of cAMP effectors. Soon after the discovery, EPAC has shown its significance in many diseases including its emerging role in infectious diseases. In a recent study, we demonstrated that EPAC, but not PKA, is a promising therapeutic target to regulate respiratory syncytial virus (RSV) replication and its associated inflammation. In mammals, there are two isoforms of EPAC-EPAC1 and EPAC2. Unlike other viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) and Ebola virus, which use EPAC1 to regulate viral replication, RSV uses EPAC2 to control its replication and associated cytokine/chemokine responses. To determine whether EPAC2 protein has a broad impact on other respiratory viral infections, we used an EPAC2-specific inhibitor, MAY0132, to examine the functions of EPAC2 in human metapneumovirus (HMPV) and adenovirus (AdV) infections. HMPV is a negative-sense single-stranded RNA virus belonging to the family Pneumoviridae, which also includes RSV, while AdV is a double-stranded DNA virus. Treatment with an EPAC1-specific inhibitor was also included to investigate the impact of EPAC1 on these two viruses. We found that the replication of HMPV, AdV, and RSV and the viral-induced immune mediators are significantly impaired by MAY0132, while an EPAC1-specific inhibitor, CE3F4, does not impact or slightly impacts, demonstrating that EPAC2 could serve as a novel common therapeutic target to control these viruses, all of which do not have effective treatment and prevention strategies.

Nsp2 has the potential to be a drug target revealed by global identification of SARS-CoV-2 Nsp2-interacting proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health threat since December 2019, and there is still no highly effective drug to control the pandemic. To facilitate drug target identification for drug development, studies on molecular mechanisms, such as SARS-CoV-2 protein interactions, are urgently needed. In this study, we focused on Nsp2, a non-structural protein with largely unknown function and mechanism. The interactome of Nsp2 was revealed through the combination of affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), and 84 proteins of high-confidence were identified. Gene ontology analysis demonstrated that Nsp2-interacting proteins are involved in several biological processes such as endosome transport and translation. Network analysis generated two clusters, including ribosome assembly and vesicular transport. Bio-layer interferometry (BLI) assay confirmed the bindings between Nsp2- and 4-interacting proteins, i.e. STAU2 (Staufen2), HNRNPLL, ATP6V1B2, and RAP1GDS1 (SmgGDS), which were randomly selected from the list of 84 proteins. Our findings provide insights into the Nsp2-host interplay and indicate that Nsp2 may play important roles in SARS-CoV-2 infection and serve as a potential drug target for anti-SARS-CoV-2 drug development.

In silico comparative genomics of SARS-CoV-2 to determine the source and diversity of the pathogen in Bangladesh.

The COVID19 pandemic caused by SARS-CoV-2 virus has severely affected most countries of the world including Bangladesh. We conducted comparative analysis of publicly available whole-genome sequences of 64 SARS-CoV-2 isolates in Bangladesh and 371 isolates from another 27 countries to predict possible transmission routes of COVID19 to Bangladesh and genomic variations among the viruses. Phylogenetic analysis indicated that the pathogen was imported in Bangladesh from multiple countries. The viruses found in the southern district of Chattogram were closely related to strains from Saudi Arabia whereas those in Dhaka were similar to that of United Kingdom and France. The 64 SARS-CoV-2 sequences from Bangladesh belonged to three clusters. Compared to the ancestral SARS-CoV-2 sequence reported from China, the isolates in Bangladesh had a total of 180 mutations in the coding region of the genome, and 110 of these were missense. Among these, 99 missense mutations (90%) were predicted to destabilize protein structures. Remarkably, a mutation that leads to an I300F change in the nsp2 protein and a mutation leading to D614G change in the spike protein were prevalent in SARS-CoV-2 genomic sequences, and might have influenced the epidemiological properties of the virus in Bangladesh.