Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are critical signaling adaptors downstream of many receptors in the TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Whereas TRAF2, 5, and 6 are activators of the canonical NF-kappaB signaling pathway, TRAF3 is an inhibitor of the noncanonical NF-kappaB pathway. The contribution of the different domains in TRAFs to their respective functions remains unclear. To elucidate the structural and functional specificities of TRAF3, we reconstituted TRAF3-deficient cells with a series of TRAF3 mutants and assessed their abilities to restore TRAF3-mediated inhibition of the noncanonical NF-kappaB pathway as measured by NF-kappaB-inducing kinase (NIK) protein levels and processing of p100 to p52. We found that a structurally intact RING finger domain of TRAF3 is required for inhibition of the noncanonical NF-kappaB pathway. In addition, the three N-terminal domains, but not the C-terminal TRAF domain, of the highly homologous TRAF5 can functionally replace the corresponding domains of TRAF3 in suppression of the noncanonical NF-kappaB pathway. This functional specificity correlates with the specific binding of TRAF3, but not TRAF5, to the previously reported TRAF3 binding motif in NIK. Our studies suggest that both the RING finger domain activity and the specific binding of the TRAF domain to NIK are two critical components of TRAF3 suppression of NIK protein levels and the processing of p100 to p52.

A recent paper published in Nature by Caroline Junqueira et al. (2022)1 reveals that FcγR and opsonizing antibodies can mediate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of monocytes/macrophages, which can then activate NLRP3 inflammasomes, caspase-1, and Gasdermin D (GSDMD), and thereby trigger pyroptosis and severe inflammatory reaction. Since the first description in late 2019, coronavirus disease 2019 (COVID-19), caused by RS-CoV-2, has emerged as one of the most significant global public health crises. In some patients, SARS-CoV-2 infection can induce a severe inflammatory cytokine storm that can lead to respiratory syndrome and multiorgan failure.1, 2 From a biological perspective, when myeloid cells sense invasive infection, they activate inflammasomes, which recruit apoptotic speck protein containing a caspase recruitment domain (ASC) adaptors and further activate downstream caspase-1, which cleaves the suppressant C-domain and releases the pore-forming N-domain of GSDMD (a pyroptosis execution protein that can be activated by caspase-1), leading to cell membrane breaks, pyroptosis, and the release of inflammatory cytokines3 (Figure 1). In this paper, Junqueira et al.1 reported that SARS-CoV-2 can also infect monocytes/macrophages, and this is through the Fcγ receptors-mediated uptake of antibody-opsonized virus. They then showed that SARS-CoV-2 triggers NLRP3 inflammasomes, caspase-1, and GSDMD-dependent pyroptosis, an inflammation-induced programmed cell death,4 exacerbating systemic inflammation and the symptoms of COVID-19. The pyroptosis blocks the infectious virus production despite the replication of the viral genome in monocytes. The study began with the detection of inflammasome activation and pyroptosis in blood samples obtained from SARS-CoV-2-infected patients. They found that approximately 6% of circulating monocytes from SARS-CoV-2 infected patients were stained by Zombie dye, a sign of membrane damage consistent with pyroptosis. A series of pyroptosis biomarkers, including GSDMD, interleukin 1β (IL-1β), IL-1RA, IL-18, and lactate dehydrogenase activity, were studied and found to be elevated. Through imaging flow cytometry, they found that about 4% of monocytes from COVID-19 patients exhibit activation of canonical inflammasomes, which form large micron-sized inflammasome-ASC-caspase-1 specks. ASC specks were colocalized with caspase-1, NLRP3 (a canonical inflammasome), and AIM2. Moreover, the lung-resident macrophages were also found to contain activated inflammasomes.1 Previous studies have proven that several SARS-CoV-2 proteins are involved in NLRP3 inflammasome activation. Nucleocapsid (N) protein directly interacts with NLRP3 and contributes to severe inflammatory responses in patients.5, 6 Spike (S) protein was also proven to activate NLRP3 inflammasome-induced pyroptosis in hematopoietic stem/progenitor cells.7 Given that ACE2, the viral entry receptor, is undetected in monocytes, they examine how SARS-CoV-2 activates inflammasome COVID-19 monocytes. They found that 10% of the monocytes could be stained with SARS-CoV-2 N protein and double-stranded RNA antibody, and the proportion was consistent with the proportion of monocytes expressing CD16 (FcγRIIIa) in blood monocytes (∼10%) (more details on this later).8 Moreover, all monocytes with ASC specks were infected; and all infected monocytes had ASC specks, indicating that SARS-CoV-2 directly infects monocytes and activates inflammasomes and pyroptosis. Using an engineered infectious clone (icSARS-CoV-2-mNG, which encodes a neon green (NG) fluorescent reporter of viral replication), the author found that preincubation with antispike monoclonal antibodies or COVID-19 patient plasma permits the viral entry into monocytes. The highest infection rate requires preincubation of the virus with patient plasma and pretreating of the healthy donor monocytes with lipopolysaccharides. Meanwhile, immunoglobulin G (IgG)-depletion of COVID-19 plasma, but not IgA depletion, abolished the viral infection. Purified IgG from COVID-19 plasma also facilitated viral infection, while IgG from healthy donor plasma barely affect viral entry, suggesting that the infection is mediated by antispike antibody-opsonized virus. Interestingly, patient plasma potentiated the healthy donor monocyte infection more than purified IgG from COVID-19 plasma, suggesting that other components might also be involved. Importantly, plasma from vaccinated individuals did not facilitate the viral infection. To identify the viral receptor, blocking antibodies to ACE2, CD147, CD16, CD32, and CD64, were utilized to block potential monocyte receptors. Blocking of monocyte FcγRs, CD16 or CD64, drastically inhibited infection, suggesting that CD16 or CD64 mediate the entry of opsonized SARS-CoV-2 into monocytes. Although blocking antibodies to CD64 could also block the infection, CD64 is more widely expressed than CD16, that is, more restricted and as all infected monocytes expressed CD16; the authors concluded that CD16 is probably the major Fc receptor. Despite neutrophils, cytotoxic T and NK cells also express CD16, SARS-CoV-2 infection was undetected in these cells of COVID-19 patients, implying that the antibody-dependent entry is confined to monocytes. Furthermore, despite quantitative polymerase chain reaction assays confirming the replication of the viral genome, no infectious virus was detected in SARS-CoV-2-infected healthy donor monocyte culture supernatants, indicating that pyroptosis and inflammatory responses terminate the productive viral cycle. This paper showed the following important points. First, they confirmed the antibody-mediated SARS-CoV-2 infection of monocytes. The antibody-mediated enhancement of infection has been reported in serval viruses, including influenza virus, respiratory syncytial virus, Middle East respiratory syndrome coronavirus, SARS-CoV, as well as dengue virus. Similarly, a recent study reported that the monoclonal antibodies of SARS-CoV-2 S protein facilitate the SARS-CoV-2 entry into lung-resident macrophages, driving inflammasome activation and pyroptosis. Blocking CD16 significantly restrained the SARS-CoV-2 entry despite macrophages also expressing ACE2. Caspase-1 and NLRP3 inhibitors ameliorate inflammation but lead to the release of infectious viruses from the infected macrophages.9 Another study showed that IgG receptors, FcγRIIA and FcγRIIIA, can contribute to antibody-dependent enhancement of SARS-CoV-2.10 Second, this study reveals that the infection of monocytes can induce pyroptosis of monocytes, releasing the inflammatory cytokines and contributing to the pathogenesis of COVID-19. Pyroptosis might act as a double-edged sword during SARS-CoV-2 infection, which on the one hand eliminates viral infection before producing infectious virions, and on the other, facilitates the release of inflammatory factors contributing to the cytokine storm. These findings may provide some insights into the pathogenetic mechanism involved that some patients had clinical deterioration, which coincides temporally with the detection of the SARS-CoV-2 antibody. Finally, and fortunately, the antibodies induced by the COVID-19 vaccines do not mediate the infection, probably related to the binding to different epitopes, or related to the change in a typology that did not facilitate the viral entry. Tong Yao and Shuai Wang wrote the manuscript; Long Zhang, Johnson Yiu-Nam Lau, and Fangfang Zhou provided valuable discussion. This work was supported by the National Science Fund for Distinguished Young Scholars (32125016), the Chinese National Natural Science Funds (31871405, 32070907 and 31870902), and the Major Natural Science Research Projects of Colleges and Universities in Jiangsu Province (19KJA550003). The authors declare no conflict of interest. Not applicable. Not applicable.

The endoplasmic reticulum represents the quality control site of the cell for folding and assembly of cargo proteins. A variety of conditions can alter the ability of the endoplasmic reticulum (ER) to properly fold proteins, thus resulting in ER stress. Cells respond to ER stress by activating different signal transduction pathways leading to increased transcription of chaperone genes, decreased protein synthesis, and eventually to apoptosis. In the present paper we analyzed the role that the adaptor protein tumor necrosis factor-receptor associated factor 2 (TRAF2) plays in regulating cellular responses to apoptotic stimuli from the endoplasmic reticulum. Mouse embryonic fibroblasts derived from TRAF2-/- mice were more susceptible to apoptosis induced by ER stress than the wild type counterpart. This increased susceptibility to ER stress-induced apoptosis was because of an increased accumulation of reactive oxygen species following ER stress, and was abolished by the use of antioxidant. In addition, we demonstrated that the NF-kappaB pathway protects cells from ER stress-induced apoptosis, controlling ROS accumulation. Our results underscore the involvement of TRAF2 in regulating ER stress responses and the role of NF-kappaB in protecting cells from ER stress-induced apoptosis.

Severe COVID-19, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is characterized by widespread inflammation and cytokine storm. A number of viral proteins are capable of inducing inflammatory responses, one of which is open reading frame 3a (ORF3a). ORF3a from SARS-CoV shares 73% homology with SARS-CoV-2 ORF3a and was shown to stimulate NF-κB signaling and inflammasome activation in a manner dependent on TNF receptor-associated factor 3 (TRAF3). TRAF family proteins regulate NF-κB activation and thereby influence inflammatory responses. Both ORF3a homologs contain a TRAF-binding consensus sequence, and SARS-CoV ORF3a has been shown to bind to human TRAFs 2, 3, and 6. We therefore investigated the binding of SARS-CoV-2 ORF3a to TRAF proteins as a potential mechanism through which SARS-CoV-2 may activate inflammatory signaling. TRAF-C domains from TRAFs 2 and 3 were purified and co-crystallized with a peptide from ORF3a containing the TRAF-binding sequence (PIQAS). Fluorescence polarization (FP) was used to assess binding affinity between TRAF-C domains and ORF3a peptide, and dual-luciferase assays were used to measure NF-κB activation in cells transfected with wildtype ORF3a or ORF3a in which the TRAF-binding sequence was mutated to alanines. X-ray crystallography revealed that the ORF3a peptide is able to bind to the TRAF-C domain of TRAFs 2 and 3, and FP indicated that the binding affinity for these interactions is low. When we compared NF-κB activation in HEK293T cells expressing WT vs mutant ORF3a, there was no significant difference. Therefore, while ORF3a has the potential to bind to TRAFs, this interaction does not significantly contribute to inflammatory signaling through NF-κB in our assay. This work is supported by NIH grants R01GM127609 and P01AI141350.

Diagnoses of coronavirus disease 2019 (COVID-19) from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) were first reported in December 2019. Since its emergence from the Chinese province of Wuhan, the World Health Organization (WHO) has announced 162 million confirmed cases of the SARS-CoV-2 infection worldwide, and reported roughly 3.3 million deaths as of May 16, 2021.1 Stratified by country, the United States leads with nearly 33 million confirmed COVID-19 cases, followed by India, Brazil, France, Turkey, and Russia.2 Structural firefighters perform essential public safety work and have continued that work despite the challenges of COVID-19. Career firefighters typically have long work schedules (24 or 48 hours on duty followed by multiple days off) and eat and sleep at the station as part of a team/shift. Firefighters respond to multiple hazards which include fires and rescues. In many localities, firefighters are dual trained as emergency medical service (EMS) personnel and provide emergency medical care. Because of their close living quarters and contact with the public, including rendering patient care and transporting patients, it is likely that firefighters are at an increased risk of infection with SARS-CoV-2. The fire service is aware of the risk of infection and has quickly adopted the increased use of personal protective equipment (PPE) and modified policies and procedures aimed at reducing the risk to firefighters.3,4 However, very little attention has been paid to occupational risks that may increase the severity of COVID-19 or to the potential long-term consequences of COVID-19 that may pose specific concerns for firefighters. The purpose of this review is to 1) outline the pathogeneses of COVID-19, 2) explore clinical and mechanistic links between COVID-19 and cardiovascular disease, 3) review known risk factors for COVID-19 complications and their prevalence among firefighters, and 4) consider steps that can be taken to better understand the long-term consequences of COVID-19 in the fire service. The review is limited to occupational factors for structural firefighters and does not cover wildland firefighters, although we acknowledge that COVID-19 may also present special concerns for wildland firefighters. PATHOGENESIS OF COVID-19 The SARS-CoV-2 virus enters the body predominantly via the lungs, and often results in pronounced respiratory symptoms. Thus COVID-19 was initially described as a respiratory disease. Indeed, respiratory failure from acute respiratory distress syndrome has been shown to be leading cause of COVID-19 induced mortality.5 A study by Guan et al6 reported that the majority of COVID-19 related consequences feature pneumonia and acute respiratory distress, which is consistent with other analyses suggesting that about 40% of COVID-19 patients develop acute respiratory distress syndrome, and 20% of these syndromes are severe.7 Wang et al8 showed that 61% of the individuals that required intensive care due to COVID-19 developed acute respiratory distress syndrome. COVID-19 not only lead to respiratory symptoms, but also underlying respiratory conditions increase the likelihood of experiencing severe symptoms. Meta-analyses revealed that the odds of severe COVID-19 infection were 5.69 times higher if individuals who have a history of chronic obstructive pulmonary disease.9 The virus requires the cooperation of two key proteins, TMPRSS2, and angiotensinogen converting enzyme 2 (ACE2) to infiltrate the body via the lung pneumocytes. TMPRSS2 is a key cellular regulator of coronavirus spike protein (S protein), with the S1 domain of the S protein responsible for receptor binding and the S2 domain controlling membrane fusion. Thus, coronavirus requires the binding of the S1 region to a cell surface receptor followed by the S2 subunit mediated fusion of the viral and cellular membranes in order to enter its host.10 This process requires S protein priming, or cleavage, by host proteases at the S1and S2 domains of the virus. This process has been described as a principle step for the cellular entry of SARS-CoV-2.11 Following S protein cleavage, Sars-CoV-2 binds to and enters lung cells via the enzyme ACE2, which is highly expressed in alveolar type 2 cells.12 Dissimilar to the original SARS-CoV, it has been suggested that SARS-CoV-2 may have a higher affinity to ACE2 positive cells in the upper respiratory tract, exacerbating its potent and detrimental effects.11 ACE2 is a membranous protein and importantly, an inactivator of angiotensin II (AngII). The binding of SARS-CoV-2 to ACE2 in lung cells promotes the endocytosis of the ACE2-SARS-CoV-2 complex, resulting in a reduction of membrane ACE2 abundance and an increase in serum AngII.12 Thus, SARS-CoV-2's affinity for ACE2 could explain its downstream effects on vascular parameters, including alterations in systolic and diastolic blood pressures, as elevated plasma AngII can increase blood pressure via aldosterone-mediated vasoconstriction and sodium and water retention on the kidneys.13 Furthermore, increased plasma AngII is associated with increased risks of myocardial infarction and left ventricular hypertrophy.13 In addition, SARS-CoV-2 promotes inflammation via the AT1R.12 The AngII-AT1R axis activates pro-inflammatory transcription factors NF-kB and STAT3, upregulating pro-inflammatory cytokines such as TNFa and IL-6 family cytokines,12,14 possibly leading to vascular inflammation and disease. Furthermore, recent studies suggest that the Sars-CoV-2 protein ORF3a encourages an aggressive inflammatory response via NF-κB activation, chemokine secretion, Golgi fragmentation, ER stress, and cell death.15 ORF3a can also inhibit type I interferon (type I IFN) signaling, downregulate major histocompatibility complex (MHC) class I expression, and reduce CD8+ cytotoxic T cell activity. Specifically, Siu et al15 demonstrated that ORF3a encourages the binding of TRAF3 to cytoplasmic portions of TNF receptors, promoting ubiquitination, and processing of p105 to p50. P50 is generated by TRAF3 ubiquitin-ligase ubiquitination of p150 and 26S proteasome-mediated removal of p105C terminal sequences. P50 then binds to RelA, RelB, or C-Rel subunits to produce functional NF-κB, a transcription factor essential for pro-IL-1β expression. The prevalence of pro-IL-1β transcripts is a requirement for NLRP3 inflammasome activation. Therefore, ORF3a-mediated p105 processing into p50 can help activate the NLRP3 inflammasome and lead to a robust inflammatory response.15 Siu et al further demonstrated ORF3a's ability to induce ASC polyubiquitination via a TRAF3 ubiquitin-ligase.15 ASC is the adapter complex of the NLRP3 inflammasome, and polyubiquitination of ASC provides a nondegradative signal necessary for ASC activation, caspase-1 activation, and mature IL-1β protein formation.15 Ultimately, the studies mentioned above illustrate how COVID-19 can target the cardiovascular system through its mode of entry and lead to vascular inflammation and dysfunction via upregulation of pro-inflammatory signaling. COVID-19 AND CARDIOVASCULAR DISEASE Although SARS-CoV-2 was first described as a respiratory disease, cardiac tissue and blood vessels express ACE2 receptors and appear to be particularly prone to COVID-19 infection.14 The heart, an ACE2 expressing tissue, was studied during the Toronto SARS outbreak (SARS-CoV), and investigators found evidence of SARS-CoV RNA in 35% of autopsied hearts.16 COVID-19 acts in a similar manner to the previous SARS-CoV, indicating that individuals with cardiovascular disease (CVD) are more prone to severe complications of SARS-CoV-2 compared to healthy individuals. Initial research on CVD-induced complications of COVID-19 was conducted in China. Wang et al investigated the association between biomarkers of CVD and the exacerbation of COVID-19 in hospitalized patients and found that cardiac injury, defined as either elevated high-sensitivity cardiac troponin I (hs-cTnI) or ECG/echocardiographic abnormalities, was present in 7.2% of the patients.8,17,19 The study also found that 22% of COVID-19 patients in ICU had biomarkers of cardiac injury.8,17 Zhou et al reported that hs-cTnI levels were at or greater than the 99th percentile upper reference limit in 46% of non-survivors, compared to only 1% of survivors who had levels this high.17,18 Thus, it has become apparent that COVID-19 can have severe cardiovascular consequences. Ultimately, it is also becoming clear that the presence of CVD, or CVD risk factors, can increase the likelihood of severe complications of COVID-19. The observational study by Zhou et al described above, also reported that 8% of patients (13% of non-survivors) had been diagnosed with CVD and 38% (48% of non-survivors) had been diagnosed with hypertension.17,18 Furthermore, Wang et al found that comorbidity of COVID-19 and CVD was prevalent in 15% (25% requiring ICU care) of patients analyzed, and Guan et al reported that 2.5% (9% among those with intubation or death) of COVID-19 patients also suffered from coronary artery disease.10,11,14 Chen et al demonstrated that in a cohort of 99 COVID-19 infected individuals at the Wuhan Jinyintan Hospital, 40% had some manifestation of cardiovascular or cerebrovascular disease.19 Other researchers have also reported on the higher prevalence of hypertension among COVID-19 patients; one study that although reports 15% of COVID patients had hypertension, 36% of those who needed intubation or suffered death had hypertension. Another study reported 31% of patients with COVID-19 had hypertension; however, 58% of patients requiring ICU care had hypertension.6,8 These findings demonstrate a clinical link between COVID-19 and CVD. EFFECT OF COVID-19 ON CARDIOVASCULAR SYSTEM Following the COVID-19 outbreak, researchers have begun to investigate the mechanisms associating COVID-19 and CVD. Emerging evidence strongly suggests the SARS-CoV-2 infection decreases myocardial functioning. Previous research has demonstrated that SARS-CoV, resembling both the structure and function of SARS-CoV-2, perturbates myocardial functioning.20 Recent research analyzing the cardiac manifestations of the SARS-CoV-2 infection found that the most common cardiac abnormality (39% of patients at baseline) was right ventricular dilation and dysfunction, followed by left ventricular diastolic and systolic dysfunction (16% and 10% of patients at baseline, respectively).21 In this study, 20% of these patients had clinical deterioration, with 60% of them having right ventricle deterioration and 25% having left ventricle systolic and diastolic deterioration.20 Thus, it appears that COVID-19, similar to other severe hypoxic respiratory illnesses, impairs cardiac function mostly by a right ventricular pressure overload state. Myocardial injury involves a pronounced escalation in pro-inflammatory cytokine secretions, which is commonly seen in COVID-19 patients. Specifically, research has found that patients suffering from COVID-19 had an upregulation of the pro-inflammatory cytokines IL1B, IFNγ, IP10, and MCP1. Individuals in ICU admission for COVID-19 had higher concentrations of the cytokines GCSF, IP10, MCP1, MIP1A, and TNFα than those not in ICU.22 An increase in these molecules due to COVID-19 severity can lead to an activation and dysregulation of T helper cells.22 Imbalances in (type 1 and type 2) T helper cells can lead to respiratory dysfunction, hypoxemia, and myocardial injury.20 Interestingly, Huang et al noticed that type 2 T-helper cell cytokines (IL4 and IL10), that suppress inflammation, were upregulated during infection of SARS-CoV-2.20 A study of competitive athletes recovering from COVID-19 found that 15% (4/26) had cardiovascular magnetic resonance findings suggestive of myocarditis despite only 2 of the 4 participants with findings suggestive of myocarditis having had COVID-19 symptoms.23 Acute thrombotic events are another major complication in individuals fighting the SARS-CoV-2 infection. Blood hypercoagulability has been shown to be common among hospitalized COVID-19 patients.24 Elevated D-Dimer levels, associated with thrombus formation and breakdown, are also reported in COVID-19 patients, worsening over the course of the disease.24 A review by Terpos et al elegantly describes how thrombus degradation products including PT and aPT are consistently upregulated in individuals requiring ICU admission.24 COVID-19 has also been shown to induce acute pulmonary embolisms in certain individuals,24–27 and one study found that 30% of COVID-19 patients had acute pulmonary embolus, measured by a CT coronary angiogram.28 This rate of pulmonary embolus is higher than what is usually seen in critically ill patients without COVID-19 (1.3%).28 Ultimately, COVID-19 patients are at higher risk for thromboembolic events, leading to adverse cardiovascular health risks. The endothelium plays key roles in regulating blood flow, maintaining hemostatic balance, and in immune response. Emerging evidence suggests that a vascular disease process contributes to COVID-19 pathogenesis.29 Several studies have begun to elucidate the role of endothelial dysfunction with COVID-19. Epithelial dysfunction, specifically pulmonary endothelial damage, is a common manifestation observed in patients infected with SARS Cov-2 virus and other coronaviruses.26 Endothelium damage due to COVID-19 is thought to occur by multiple mechanisms, including: a dysregulated immune response, enhanced vascular permeability, and exacerbated presence of pulmonary edemas.26,30 Varga et al31 demonstrated endothelial cell dysfunction in vital organs of individuals after becoming infected with COVID-19. These authors presented convincing evidence to indicate that the SARS CoV-2 virus has direct effects on endothelial cells, possibly due to the fact that ACE2 is also widely expressed on endothelial cells in multiple organs.14 Thus, it appears that recruitment of immune cells and pro-inflammatory cytokines due to ubiquitous expression of ACE2 can result in extensive endothelial dysfunction and cellular apoptosis. THE EFFECTS OF OBESITY ON CARDIOVASCULAR HEALTH AND COVID-19 Obesity has been recognized as an important predictor of CVD risk and adverse cardiorespiratory outcomes. Genetic and clinical experiments have found that that obesity is causally related to many disease states including hypertension, diabetes mellitus type 2, coronary heart disease, stroke, atrial fibrillation, renal disease, and heart failure.32 Others have reported that around 75% of hypertension can be attributed to obesity.33 It is clear that this obesity-induced hypertension leads to renal dysfunction due to an increased sympathetic nervous state and upregulated renin–angiotensin system.33 Obesity has effects on the infection and exacerbation of the SARS-CoV-2 infection. Sattar et al propose that obesity and ectopic fat deposition might reduce both optimal cardiorespiratory and immune response mechanisms, two major factors that can lead to severe manifestations of COVID-19.32 Several studies have reported on an association between obesity and COVID-19. Hamer et al reported a two-fold risk ratio of being infected with COVID-19 for obese individuals compared to normal weight individuals.34 These risk ratios were adjusted for age, sex, and mutually for each lifestyle, and physical inactivity. Furthermore, obesity was identified as the risk factor that contributed greatly to the prediction of COVID-19 infection risk. Finally, Hamer et al calculated a Population Attributable Fraction (PAF), which corresponds to the prevalence of risk factors in a population and the strength of its association with an outcome (COVID-19).34 The PAF used adjusted effect estimates on lifestyle factors (smoking, physical inactivity, overweight, and obesity) and COVID-19 and found that the total PAF for the three unhealthy lifestyle factors was 51.4%.34 Specifically, overweight and obesity had a PAF of 29.5%, smoking had a PAF of 13.3%, and physical inactivity had a PAF of 8.6%. Overall, it has become quite clear through both mechanistic and clinical research that there is a powerful effect of obesity on COVID-19 infection and severity. POTENTIAL RELATIONSHIPS BETWEEN FIREFIGHTERS AND COVID-19 As discussed through this paper, there is a strong relationship between both pulmonary disease, CVD and COVID-19. While initial research has focused on risk factors that place individuals at increased risk for COVID-19 complications, this section details ways that occupational exposures and cardiovascular risk factors that are known to be prevalent among firefighters, might make firefighters an occupational group that is at high risk of developing COVID-19 complications and for whom the long-term effects of COVID-19 infection might be particularly problematic. As summarized in Tables 1 and 2 and discussed in the following section, there are multiple factors that are known to exacerbate the rate of infection or severity of infection with SARS-CoV-2 and that are occupationally associated with firefighting. TABLE 1 - Association Between Medical Conditions of COVID-19 and Firefighting Medical Conditions COVID-19 Research Fire Service Research 1. Pulmonary disease • Significantly associated with a severe COVID-19 infection (OR 5.69, 95% CI: 2.49–13.00)9• 30% of studied COVID-19 patients developed acute respiratory distress syndrome,28 61% of studied COVID-19 patients developed acute respiratory distress syndrome,7,8,28 with approximately 20% of these cases being severe8 • Decrements in respiratory function were two-to-four-times greater in firefighters than general population35• Pulmonary function is associated with frequency of fire exposure36• Those who transitioned to less active assignments might not be protected from pulmonary disease88 2. Cardiovascular disease • 15–40% of patients had some manifestation of cardiovascular or cerebrovascular disease7,8,19 • Firefighters with other comorbidities demonstrated unfavorable CVD and cardiorespiratory fitness profiles70 COVID-19, coronavirus disease 2019; CVD, cardiovascular disease. TABLE 2 - Association Between Risk Factors of COVID-19 and Firefighting Risk Factors COVID-19 Research Fire Service Research 1. Age • Significant association of older age (≥65 years) and risk of COVID-19 mortality• Ranging from an OR of 3.76 (95% CI: 1.15–17.39; P = 0.023) to 4.59 (95% CI: 2.61–8.04; P < 0.001)57,58 • 9% of the entire US firefighting cohort is 60 years of age or older55 2. Sex • Males have made up as much as 60.3–70% of patients hospitalized with the SARS-CoV-2 infection• Prostatic diseases are associated with elevations in COVID-19 induced cardiac injury (OR 1.505, 95% CI; P = 0.046)60• In males, each standard deviation increase in free androgen escalates risk of severe COVID-19 manifestations (OR 1.22, 95% CI: 1.03–1.45; P = 0.024)60 • 96% of the US fire service is comprised of men, and more than half of US metropolitan departments have no women firefighters55,62 3. Hypertension • 56.6% of New York City area COVID-19 patients had hypertension59• Significant associate of COVID-19 mortality (pooled OR 2.70, 95% CI: 1.40–5.24; P = 0.003)57 • Up to 30% of the entire fire service have hypertension63,72• 46% of males and 29% of females firefighters had blood pressure the of 1 or 2 58% of firefighters and of firefighters have Obesity • been as the one of COVID-19 obese individuals are at greater risk for severe COVID-19 • of firefighters = were as either overweight or of overweight and obese firefighters may the US Cardiovascular • troponin is associated with COVID-19 mortality risk (OR 95% CI: P < cardiac injury in 7.2% of patients, and in 22% of ICU of patients had right ventricular dilation and dysfunction, had left ventricular diastolic dysfunction, 10% had systolic • Acute of decreases can induce ventricular and of myocardial and blood and • increased ACE2 and TMPRSS2 in alveolar type 2 cells and • Decrements in were more than the rate in were related to frequency of fire but not to age, smoking or Firefighters who a during fire a times greater rate of compared to COVID-19, coronavirus disease 2019; vital Pulmonary from recent study indicate that in the respiratory function of firefighters years) was two-to-four-times greater than the in the general reports with findings and also that the of pulmonary function in firefighters is associated with the frequency of fire of fires are more potent of than previous on their occupational firefighters appear to be at an increased risk of pulmonary is less evidence that firefighting leads to increased pulmonary disease, but this is a pulmonary disease is associated with increased risk for developing a severe COVID-19 infection. Pulmonary Risk and the acute and long-term effects of and is a in the fire service. have that and can reduce firefighters in 1 by lung function often to Furthermore, et demonstrated an of in firefighters following a of with 30% of the cohort having a in of Other studies have shown that the in which firefighters for and of can cause decreases in and vital as as in serum cell protein and serum studies the effects of long-term and on health have been the results are A study conducted on firefighters from the fire showed that in the and were not associated with of firefighting in active and that the protective respiratory equipment used by the fire service to be the detrimental effects of enhanced and In addition, a review of studies from to that the of and on health is and limited by of and that firefighters in pulmonary However, a study by et found that the in were more than the rate and was related to frequency of fire but not to age, smoking or et further showed that active firefighters a greater in compared to those who had or firefighters who a during fire a times greater rate of compared to Other studies have shown that with respiratory use in et showed that after years of there was a 10% in the of firefighters who to the World Thus, there is but not evidence that to and can both and pulmonary function in firefighters, use of respiratory protective equipment in the fire service. As pulmonary function is a robust to COVID-19 infection and severe COVID-19 Recent evidence also demonstrated that to can increase both ACE2 and TMPRSS2 in alveolar type 2 cells and due to firefighting might have a direct effect on COVID-19 but further research is Cardiovascular Risk Age cardiovascular health is by the prevalence of cardiovascular risk factors which can include age, sex, hypertension, and from that 9% of US firefighters are 60 years of age or Although a this of the fire service might have a more pronounced risk of COVID-19 infection than the general A recent observational study reported that age is one of the leading risk factors for infection and death due to Other studies have confirmed this that older individuals (≥65 years) have from to times higher risk of COVID-19 Cardiovascular Risk Sex suggests that males are more to a COVID-19 infection than with one study from the New York City area that males made up of the patients hospitalized with the SARS-CoV-2 A study in found that males made up of the patients on in the males were more in COVID-19 patients than in The in and COVID-19 infection is thought to be due to levels of between males and Specifically, TMPRSS2 expression has been shown to be by and androgen receptor which is a requirement for the transcription of et reported that related to androgen increased the odds of having troponin T levels induced cardiac by the et also found that free androgen associated with COVID-19 and severity in males, but not in among males who were for COVID-19, each standard deviation increase in free androgen increased the odds of a positive COVID-19 as as severe COVID-19 by The fire service is et reported that to of the US fire service is comprised of and more than half of US metropolitan departments have no women firefighters. Other that of firefighters and of firefighters are an occupational group by is most likely to be by the SARS-CoV-2, as higher androgen levels are found in Cardiovascular Risk Hypertension Hypertension is a risk factor of COVID-19 and CVD that is known to have a high prevalence the US fire service. Hypertension is reported to be one of the most common comorbidities related to COVID-19 infection. In et al found that hypertension was present in 56.6% of hospitalized COVID-19 patients the New York City A these that chronic hypertension, with other cardiovascular were more among patients than survivors (48% also suggests that hypertension is associated with COVID-19 and that individuals as have higher odds of from COVID-19 than a Research that approximately 20% to 30% of the entire fire service have recent study found that 46% of firefighters and 29% of females had blood pressure the of 1 or 2 Cardiovascular Risk Obesity As discussed obesity has been found to increase the risk of a COVID-19 infection. there is a high prevalence of obesity in the US fire service. have shown that obesity was present in of COVID-19 hospitalized Interestingly, work by et al a between age and body Thus, with pronounced obesity are at an increased risk of being infected with SARS-CoV-2. This is for the US fire as obesity is a major CVD risk factor found in firefighters. Obesity has also been found to increase the risk of coronary heart disease and links the mechanisms of vascular alterations to cardiac suggests that firefighters with high have vascular function and are at a greater risk for

Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM-1, also named TIR domain-containing adaptor-inducing interferon (IFN)-beta or TRIF)) is a signaling adaptor of Toll-like receptor (TLR) 3/4 that activates the transcription factors, interferon regulatory factor-3 (IRF-3) and NF-kappaB leading to inducing IFN-beta production. The mechanisms by which TICAM-1 is activated by TLR3/4 to serve as a signaling platform are unknown. In this study, we show that homo-oligomerization of TICAM-1 is critical for TICAM-1-mediated activation of NF-kappaB and IRF-3. Both TIR and C-terminal domain of TICAM-1 mediated TICAM-1 oligomerization. Pro(434) located in the TIR domain and the C-terminal region, with the exception of the RIP homotypic-interacting motif, were determinants of TICAM-1 oligomerization. Mutation of TIR domain (P434H) or deletion of C-terminal domain greatly reduced TICAM-1-mediated NF-kappaB and IFN-beta promoter activation. TICAM-1 oligomerization at either the TIR domain or the C-terminal region resulted in recruitment of tumor necrosis factor receptor-associated factor 3, a downstream signaling molecule essential for TICAM-1-mediated IRF-3 activation, but not recruitment of the IRF-3 kinase complex, NF-kappaB-activating kinase-associated protein 1 and TANK-binding kinase 1. In addition, RIP homotypic-interacting motif mutant, which possesses two oligomerization motifs but not the RIP1 binding motif, also failed to recruit NF-kappaB-activating kinase-associated protein 1 and TANK-binding kinase 1. Thus, full activation and formation of TICAM-1 signalosomes requires oligomerization induced at two different sites and RIP1 binding.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor/scaffold protein that mediates several important signaling pathways, including the tumor necrosis factor-R:NF-kappaB pathway, involved in immune surveillance, inflammation, etc. Because most studies of TRAF6 function have focused primarily on its role as an adaptor molecule in signaling pathways in the cytoplasm, the potential functions of TRAF6 in other cellular compartments has not been previously investigated. Here, we demonstrate that TRAF6 resides not only in the cellular cytoplasm but is also found in the nuclei of both normal and malignant B lymphocytes. TRAF6 does not possess a nuclear localization signal but enters the nucleus through the nuclear pore complex containing RanGap1. Chromatin immunoprecipitation cloning experiments demonstrated that nuclear TRAF6 associates with c-Myb within the 5'-end of the c-Myb promoter. Further analysis showed that nuclear TRAF6 is modified by small ubiquitin-related modifier-1, interacts with histone deacetylase 1, and represses c-Myb-mediated transactivation. Thus, TRAF6 negatively regulates c-Myb through a novel repressor function in the nuclei of both normal and malignant B-lymphocytes that could represent a novel control mechanism that maintains cell homeostasis and immune surveillance.

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

Toll-like receptors and COVID-19: a two-faced story with an exciting ending.

Open AccessCCS ChemistryCOMMUNICATION1 Jan 2022Potential Antiviral Target for SARS-CoV-2: A Key Early Responsive Kinase during Viral Entry Siwen Liu†, Lin Zhu†, Guangshan Xie†, Bobo Wing-Yee Mok, Zhu Yang, Shaofeng Deng, Siu-Ying Lau, Pin Chen, Pui Wang, Honglin Chen and Zongwei Cai Siwen Liu† State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Lin Zhu† State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 HKBU Shenzhen Institute of Research and Continuing Education, Shenzhen 518000 , Guangshan Xie† State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 , Bobo Wing-Yee Mok State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Zhu Yang State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 , Shaofeng Deng State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Siu-Ying Lau State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Pin Chen State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Pui Wang State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Honglin Chen *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 and Zongwei Cai *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 Beijing Normal University-Hong Kong Baptist University United International College, Zhuhai 519087 https://doi.org/10.31635/ccschem.021.202000603 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Currently, there is no effective antiviral medication for coronavirus disease 2019 (COVID-19) and the knowledge on the potential therapeutic target is in great need. Guided by a time-course transmission electron microscope (TEM) imaging, we analyzed early phosphorylation dynamics within the first 15 min during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry. Based on alterations in the phosphorylation events, we found that kinase activities such as protein kinase C (PKC), interleukin-1 receptor-associated kinase 4 (IRAK4), MAP/microtubule affinity-regulating kinase 3 (MARK3), and TANK-binding kinase 1 (TBK1) were affected within 15 min of infection. Application of the corresponding kinase inhibitors of PKC, IRAK4, and p38 showed significant inhibition of SARS-CoV-2 replication. Additionally, proinflammatory cytokine production was reduced by applying PKC and p38 inhibitors. By an acquisition of a combined image data using positive- and negative-sense RNA probes, as well as pseudovirus entry assay, we demonstrated that PKC contributed to viral entry into the host cell, and therefore, could be a potential COVID-19 therapeutic target. Download figure Download PowerPoint Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has been identified to be the cause of coronavirus disease 2019 (COVID-19) since December 2019,1–3 posing huge challenges on health, social, and economic systems globally. SARS-CoV-2 is a zoonotic betacoronavirus; its exact origin or reservoir has not been defined.4–6 Besides, several betacoronaviruses have infected humans, causing respiratory diseases.7 SARS-CoV-2 infection could induce asymptomatic, mild to severe disease, characterized by a range of symptoms, including fever, dry cough, extreme tiredness associated with acute respiratory distress syndrome (ARDS), and lung injury.3,8 Presently, there are no clinically approved antiviral drugs that could effectively inhibit the replication of the SARS-CoV-2. It has been shown that SARS-CoV-2 has a structure similar to receptor-binding domain (RBD) like SARS-CoV or cellular receptor angiotensin-converting enzyme 2 (ACE2), critical for its entry into the host cell.9 Therefore, targeting the viral entry process could be a useful approach to antiviral strategy for COVID-19.10 Enfuvirtide was the first approved viral entry inhibitor that obstructed HIV fusion to host cells.11 Fusion inhibitors, blocking the fusion process of multiple viruses have been developed and shown antiviral activity in vivo.12,13 Consequently, gaining knowledge on SARS-CoV-2 viral entry stage is an urgent requirement for valuable drug development. It is widely reported that coordinated kinase activities are crucial during viral entry.14,15 For example, an activation of protein kinase C (PKC) contributed to influenza viral entry through late endosomes.14,15 Profiling of kinase activity post influenza virus infection showed G protein-coupled receptor kinase 2 was activated within 5 min of influenza infection.16 A comprehensive analysis on SARS-CoV-2 phosphorylation networks during viral uncoating to replication phase (2–24 h postinfection [PI]) was performed by Bouhaddou et al.,17 which demonstrated that SARS-CoV-2 infection promoted multiple kinases' activation, including casein kinase II and p38. However, information on how phosphorylation dynamics changes during the SARS-CoV-2 entry process is still limited. Results and Discussion To determine the appropriate time points to examine changes in the host phosphorylation network, transmission electron microscopy (TEM) was employed to monitor the cell entry process of SARS-CoV-2 within the first 30 min of host infection (Figure 1a). A fetal rhesus monkey kidney Vero E6 cell line was used as a model of infection, as it is highly susceptible to SARS-CoV-2. A high multiplicity of infection (MOI) of 25 was used to ensure universal infection of the Vero E6 cells. Viral particles were found to attach onto the cell surface at 5 min post viral infection, while a thickened membrane was observed at 15 min PI (Figure 1a), denoting the areas where the viral envelope was fusing with the plasma membrane. Within the first 30 min, whole virion was no longer observed. Instead, viral cores of consistent size (40–50 nm in diameter) without envelope were observed in large vacuoles (Figure 1a, 30 min), indicative of the endosome, as coronavirus entered the cells by endocytosis.18 Hence, we selected 5 and 15 min PI as time points to examine the phosphorylation dynamics during viral infection. Figure 1 | Phosphorylation network response during the early phase of SARS-CoV-2 infection. (a) TEM analysis of Vero E6 infected with SARS-CoV-2 for 5, 15, 30 min. Viral particles attaching and fusing with cell membrane (5 and 15 min, black arrow). Some viral particles were observed in the large vacuoles (30 min, white arrowhead). (b) Workflow of phosphopeptides' enrichment. (c and d) Regulation of phosphopeptides identified in 5 (c) and 15 min (d) PI with SARS-CoV-2. Download figure Download PowerPoint Vero E6 cells were harvested at 5 or 15 min PI after SARS-CoV-2 or mock infection (Figure 1b) in four independent biological experiments. At 5 min PI, 115 phosphorylation sites were upregulated and 106 were downregulated, while 37 were upregulated and 65 were downregulated at 15 min PI (Figures 1c and 1d). The altered phosphopeptides were then used for further functional analysis and kinase prediction. At both study time-points, PKC activity was the highest enriched molecular function by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis ( Supporting Information Figure S1a). Enrichment map analysis also showed significant overlaps in PKC related pathways ( Supporting Information Figure S1c), suggesting that kinase activities were perturbed during the viral entry process. We then established a kinase prediction pipeline using altered phosphosites (both up- and down-altered) identified. Kinase prediction was obtained initially by the Group-based Prediction System 5.0,19,20 followed by highly stringent cutoff adopted from previous publication.16 Four kinases were consistently predicted at both 5 and 15 min PI (Figure 2a), and consequently selected for further validation, as follows: PKC-gamma (PKCγ; p = 0.0109, which was also identified directly in phosphoproteomics analysis), interleukin-1 receptor-associated kinase 4 (IRAK4; p = 0.0203), MAP/microtubule affinity-regulating kinase 3 (MARK3; p < 0.0001), and TANK-binding kinase 1 (TBK1; p = 0.0003). Subsequently, amino acid sequences flanking hyperphosphorylation sites were retrieved to reveal the phosphorylation motifs (Figure 2b). Basophilic motif of arginine (R) at position-3 was significantly enriched, compared with background, a specific feature of conventional PKCs, including PKCγ,21 which was in agreement with our prediction. Intriguingly, recent studies showed that MARK3 and TBK1 could directly interact with SARS-CoV-2 viral proteins.22 Furthermore, TBK1 was targeted by SARS-CoV-2 proteins to antagonize type I interferon (IFN-I) response.23 Collectively, these lines of evidence confirmed our kinase prediction pipeline was able to identify crucial kinase for SARS-CoV-2 replication (Figure 2c). Figure 2 | Predicted early responsive kinase activity of SARS-CoV-2 infection. (a) Top kinases predicted to regulate differential phosphorylation at 5- and 15-min PI are marked in red. All enriched kinases passed the stringent score filter. (b) Enriched phosphorylation motifs from hyperphosphorylated peptides in both 5 and 15 min PI, phosphorylated sites (S/T) set as position 0. Size of the letter represents the enrichment degree. (c) Protein–protein interactions (PPI) map of SARS-CoV-2 viral protein with predicted host kinase-substrates network. Download figure Download PowerPoint Further, we used kinase inhibitors targeting the predicted kinases to evaluate their effects on viral replication. Inhibitors of PKC (Bisindolylmaleimide IX), TBK1 (Amlexanox & MRT67307 HCL), and IRAK (IRAK-1-4 Inhibitor I) were used. p38 kinase MAPK12 and cyclin-dependent kinase 6 (CDK6) were top-predicted kinases before a stringent filter was applied, and the hyperphosphorylated motifs suggested the involvement of CDK and MAPK kinases (Figure 2b). In addition, p38 was reported to affect SARS-CoV replication.24 Therefore, we included CDK inhibitor (Palbociclib HCI) and p38 inhibitor (SB203580) as well. Cytotoxicity of these inhibitors were determined ( Supporting Information Figure S2). Then inhibitors were used at concentrations without major cytotoxicity. Bafilomycin A1 (BafA1), reported to block SARS-CoV-2 viral entry,25,26 was used as positive control. As shown in Figure 3a, p38 inhibitor efficiently blocked virus replication in all three cell lines, as expected. Inhibition of either IRAK or PKC leads to suppression of viral replication and viral mRNA synthesis in a dose-dependent manner in both Calu3 (non-small-cell lung cancer) and Caco2 (human colorectal adenocarcinoma) cell lines (Figure 3b), confirming our kinase prediction. Importantly, PKC the inhibitor showed the most pronounced inhibition of viral replication and mRNA synthesis, consistent with both KEGG (Kyoto Encyclopedia of Genes and Genomes, a database resource for functional studies) analysis and kinase prediction. As kinase inhibitors might have off-target effect, two additional PKC inhibitors (Sotrastaurin and Enzastaumn) were used to evaluate their effects on viral replication ( Supporting Information Figure S5). All three PKC inhibitors demonstrated inhibitory effects on SARS-CoV-2 replication in a dose-dependent manner, confirming the critical role of PKC activity in viral replication. The discrepancy of inhibitory effects for kinase inhibitors were observed between Vero E6 and two human cell lines, which should be caused by the absence of IFN-I in cells ( Supporting Information Figure S3). The lack of IFN-I would only affect the overall viral replication but not the phosphodynamics we observed, as we focused on the viral entry process, which was before the participation of IFN-I. This observation also confirmed the role of interferon, as reported previously.27 Inhibition of CDK led to a slight increase in terms of viral replication, suggesting that alteration of CDK kinase activity might be an adversary for SARS-CoV-2 replication. Figure 3 | Effect of different inhibitor treatment on viral mRNA level and viral titer. Cells were pretreated with different inhibitors at the indicated dose, followed by SARS-CoV-2 infection. Indicated three different relative viral mRNA levels cells were measured by normalizing to control (a). Corresponding viral titers by plaque assay were shown in (b). For all panels, *p < 0.05, **p < 0.005, ***p < 0.0005, nonsignificant (ns) for two-tail Student's t-test. Error bars indicate SD (n = 3). Download figure Download PowerPoint Furthermore, we used RNA fluorescence in situ hybridization (RNA-FISH) to visualize the viral replication process. SARS-CoV-2 generates negative-strand RNA template to synthesize new genomic RNAs; therefore, the distribution of negative-strand RNA refers to the location of replicative-intermediate in replication-transcription complex.28 An A549 cell line expressing human ACE2 was generated (Figure 4c) and pretreated with inhibitors before SARS-CoV-2 infection. In the control group, viral genomic RNA and mRNA were widely distributed and accumulated in the perinuclear area. Replicative-intermediate RNAs, indicative of ongoing viral replication, were also clearly detected (Figure 4). In contrast, p38 or PKC inhibitors significantly repressed viral infection rate. Particularly, effect of PKC inhibitor was more significant than BafA1-positive control (Figure 4a). We found that inhibitor of p38 kinase did not change signal intensity of negative-sense viral RNA, suggesting the inhibition probably occurred in the late stage of viral cycle. However, perinuclear dots of negative-sense viral RNA were significantly decreased after the treatment of PKC inhibitor (Figure 4b), which signified the lack of ongoing replication events. The observation indicated that PKC activity was crucial during viral entry as we predicted. We then validated the role of PKC during the early stage of viral replication by SARS-CoV-2 pseudovirus entry assay. A home-made SARS-CoV-2 pseudovirus was constructed and used to infect 293T-ACE2 cells pretreated with PKC inhibitors. As shown in Figure 4d, an inhibition of PKC activity diminished pseudovirus signals, confirming the inhibitory role of PKC inhibitor during the early stage of SARS-CoV-2 replication. PKC is known to regulate PKC-dependent endocytosis and involve in influenza viral entry by regulating late endosomes.14,15 Alternatively, coronaviruses, including Middle East respiratory syndrome (MERS)- and SARS-CoV, are known to rely on endocytic pathway for entry.18,29 Consequently, we proposed that PKC was required by SARS-CoV-2 as an early responsive kinase for viral entry via an endocytic pathway, making it a potential therapeutic target for COVID-19. Figure 4 | Confocal images suggested that PKC inhibitor block SARS-CoV-2 entry. A549-Ace2 cells were pretreated with the indicated inhibitor, followed by SARS-CoV-2 infection. (a) FISH and IFA imaging of infected cells using positive-sense RNA probe (purple) and antibody against viral N protein (green). (b) Fixed cells were processed for FISH assay using positive- (purple) and negative-sense RNA probe (green). Merge images also include 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). (c) Whole cell lysates were analyzed for Ace2 and tubulin expression by Western blot using their respective antibodies. (d) Pseudovirus entry assay showed that the inhibition of PKC activity prevented viral entry signals. Download figure Download PowerPoint It has been reported that poor prognosis outcomes of patients with COVID-19 were associated with cytokine storm, generated by innate immune response, while several cytokines have been reported as potential biomarkers for disease progression.30–32 Additionally, emerging pieces of evidence have shown that SARS-CoV-2 infection induces low types I and III IFNs' levels and limited interferon-stimulated genes' (ISG) response, but high level of chemokine expression.8,33 We showed that the inhibition of cytokine mRNA levels caused by kinase inhibitors correlated with that of viral titer. A significant and dose-dependent reduction of cytokine levels were observed when treated with PKC and p38 inhibitors across all three cell lines ( Supporting Information Figure S4). Also, these cytokines' expression were inhibited by IRAK inhibitor, but at a relatively moderate level. Finally, to validate the essential role of PKC activities in viral replication, three commercially available small interfering RNAs (siRNAs) targeting PKC-alpha (PKCα), PKC-beta (PKCβ), and PKC-epsilon (PKCɛ) were ordered to knock down the corresponding PKC isoforms. PKCα siRNA failed to achieve effective knock down, so only PKCβ and PKCɛ siRNAs were used in viral inhibition assay (Figure 5a). As shown in Figure 5b, only PCKβ knock down showed significant inhibitory effect on viral replication. A siRNA knock down of PKCɛ led to an increase in PKCβ activity, suggesting a potential compensation effect, which might explain the inefficiency of viral inhibition of PKCɛ siRNA. These results further confirmed with our earlier PKC inhibitors' data, as all three PKC inhibitors we tested in the study are efficient PKCβ inhibitors. Consequently, PKC activity, particularly PKCβ, might play a vital role in optimum replication of SARS-CoV-2. Figure 5 | siRNA knock down of PKCβ inhibits SARS-CoV-2 replication. (a) Transcriptional levels of PKC isoforms in siRNA knock down Calu-3 cells were examined using q-PCR. (b) Viral mRNA levels in siRNA knock down Calu-3 cells infected with SARS-CoV-2 were measured by normalizing to control. **p < 0.005, ***p < 0.0005, ****p < 0.00005, nonsignificant (ns) for two-tail Student's t-test. Error bars indicate SD (n = 3). Download figure Download PowerPoint Conclusion Using a time-course TEM imaging, we identified key time points of viral attachment and fusion of SARS-CoV-2 infection. By combining phosphoproteomics and kinase prediction pipeline, we found that PKC and IRAK4 activities were activated at the first 5–15 min of viral entry. We showed that the inhibition of PKC, IRAK4, and p38 could suppress optimal replication of the SARS-CoV-2 virus, among which IRAK4 activity initially associated with SARS-CoV-2 replication. We further demonstrated that inhibition of PKC activity, particularly PKCβ, would inhibit viral replication at early stage, probably via blockage of specific endocytosis phosphorylation events required for viral entry. Therefore, PKC might be required for SARS-CoV-2 entry, and thus, could serve as a potential therapeutic target for COVID-19. Data Availability The raw MS data from this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with accession number PXD021610. Supporting Information Supporting Information is available and includes detailed material and methods, as well as Figures S1–S5. Conflict of Interest There is no conflict of interest to report. Funding Information This research was made possible because of a generous grant from the National Key R&D Program, Ministry of Science and Technology, China (no. 2017YFC1600500), the National Natural Science Foundation of China (no. 21705137), the Theme-Based Research Scheme (no. T11/707/15) and General Research Fund (no. 17107019) of the Research Grants Council, Hong Kong Special Administrative Region, and the Sanming-Project of Medicine in Shenzhen, China (nos. SZSM201911014 and SZSM201811070).

Integrative Network Biology Framework Elucidates Molecular Mechanisms of SARS-CoV-2 Pathogenesis.

CD27 is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on T, B, and NK cells. The signal via CD27 plays pivotal roles in T-T and T-B cell interactions. Here we demonstrate that overexpression of CD27 activates NF-kappaB and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). Deletion analysis of the cytoplasmic domain of CD27 revealed that the C-terminal PIQEDYR motif was indispensable for both NF-kappaB and SAPK/JNK activation and was also required for the interaction with TNF receptor-associated factor (TRAF) 2 and TRAF5, both of which have been implicated in NF-kappaB activation by members of the TNF-R superfamily. Co-transfection of a dominant negative TRAF2 or TRAF5 blocked NF-kappaB and SAPK/JNK activation induced by CD27. Recently, a TRAF2-interacting kinase has been identified, termed NF-kappaB-inducing kinase (NIK). A kinase-inactive mutant NIK blocked CD27-, TRAF2-, and TRAF5-mediated NF-kappaB and SAPK/JNK activation. These results indicate that TRAF2 and TRAF5 are involved in NF-kappaB and SAPK/JNK activation by CD27, and NIK is a common downstream kinase of TRAF2 and TRAF5 for NF-kappaB and SAPK/JNK activation.

Multisystem Inflammatory Syndrome in Children and Kawasaki Disease: Two Different Illnesses with Overlapping Clinical Features

The coronavirus disease 2019 (COVID-19) pandemic constitutes a global health emergency. Currently, there are no completely effective therapeutic medications for the management of this outbreak. The cytokine storm is a hyperinflammatory medical condition due to excessive and uncontrolled release of pro-inflammatory cytokines in patients suffering from severe COVID-19, leading to the development of acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS) and even mortality. Understanding the pathophysiology of COVID-19 can be helpful for the treatment of patients. Evidence suggests that the levels of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1 and IL-6 are dramatically different between mild and severe patients, so they may be important contributors to the cytokine storm. Several serum markers can be predictors for the cytokine storm. This review discusses the cytokines involved in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, focusing on interferons (IFNs) and ILs, and whether they can be used in COVID-19 treatment. Moreover, we highlight several microRNAs that are involved in these cytokines and their role in the cytokine storm caused by COVID-19.

SARS-CoV-2-induced autophagy dysregulation may cause neuronal dysfunction in COVID-19.