Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2.

SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus' evolution.

CCDC50 promotes tumor growth through regulation of lysosome homeostasis.

The maintenance of lysosome homeostasis is crucial for cell growth. Lysosome-dependent degradation and metabolism sustain tumor cell survival. Here, we demonstrate that CCDC50 serves as a lysophagy receptor, promoting tumor progression and invasion by controlling lysosomal integrity and renewal. CCDC50 monitors lysosomal damage, recognizes galectin-3 and K63-linked polyubiquitination on damaged lysosomes, and specifically targets them for autophagy-dependent degradation. CCDC50 deficiency causes the accumulation of ruptured lysosomes, impaired autophagic flux, and superfluous reactive oxygen species, consequently leading to cell death and tumor suppression. CCDC50 expression is associated with malignancy, progression to metastasis, and poor overall survival in human melanoma. Targeting CCDC50 suppresses tumor growth and lung metastasis, and enhances the effect of BRAFV600E inhibition. Thus, we demonstrate critical roles of CCDC50-mediated clearance of damaged lysosomes in supporting tumor growth, hereby identifying a potential therapeutic target of melanoma.

TRIM21 Ubiquitylates SQSTM1/p62 and Suppresses Protein Sequestration to Regulate Redox Homeostasis.

TRIM21 is a RING finger domain-containing ubiquitin E3 ligase whose expression is elevated in autoimmune disease. While TRIM21 plays an important role in immune activation during pathogen infection, little is known about its inherent cellular function. Here we show that TRIM21 plays an essential role in redox regulation by directly interacting with SQSTM1/p62 and ubiquitylating p62 at lysine 7 (K7) via K63-linkage. As p62 oligomerizes and sequesters client proteins in inclusions, the TRIM21-mediated p62 ubiquitylation abrogates p62 oligomerization and sequestration of proteins including Keap1, a negative regulator of antioxidant response. TRIM21-deficient cells display an enhanced antioxidant response and reduced cell death in response to oxidative stress. Genetic ablation of TRIM21 in mice confers protection from oxidative damages caused by arsenic-induced liver insult and pressure overload heart injury. Therefore, TRIM21 plays an essential role in p62-regulated redox homeostasis and may be a viable target for treating pathological conditions resulting from oxidative damage.

PACT inhibits the replication of SARS-CoV-2 through the blockage of GSK-3ß-N-nsp3 cascade.

The protein activator of protein kinase R (PKR) (PACT) has been shown to play a crucial role in stimulating the host antiviral response through the activation of PKR, retinoic acid-inducible gene I, and melanoma differentiation-associated protein 5. Whether PACT can inhibit viral replication independent of known mechanisms is still unrevealed. In this study, we show that, like many viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks GSK-3ß to facilitate its replication. GSK-3ß-induced phosphorylation on N protein increased the interaction between N protein and nsp3. Thus, GSK-3ß-N-nsp3 cascade promotes viral replication. Although SARS-CoV-2 can sabotage the activation of AKT, the upstream proteins suppressing the activation of GSK-3ß, we found that the host can use PACT, another protein kinase, instead of AKT to decrease the activity of GSK-3ß and the interaction between PACT and GSK-3ß is enhanced upon viral infection. Moreover, PACT inhibited the activity of GSK-3ß independent of its well-studied double-stranded RNA binding and PKR activating ability. In summary, this study identified an unknown function of PACT in inhibiting SARS-CoV-2 replication through the blockage of GSK-3ß-N-nsp3 cascade.

SARS-CoV-2 NSP7 inhibits type I and III IFN production by targeting the RIG-I/MDA5, TRIF, and STING signaling pathways.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a poor inducer of innate antiviral immunity, and the underlying mechanism still needs further investigation. Here, we reported that SARS-CoV-2 NSP7 inhibited the production of type I and III interferons (IFNs) by targeting the RIG-I/MDA5, Toll-like receptor (TLR3)-TRIF, and cGAS-STING signaling pathways. SARS-CoV-2 NSP7 suppressed the expression of IFNs and IFN-stimulated genes induced by poly (I:C) transfection and infection with Sendai virus or SARS-CoV-2 virus-like particles. NSP7 impaired type I and III IFN production activated by components of the cytosolic dsRNA-sensing pathway, including RIG-I, MDA5, and MAVS, but not TBK1, IKKε, and IRF3-5D, an active form of IRF3. In addition, NSP7 also suppressed TRIF- and STING-induced IFN responses. Mechanistically, NSP7 associated with RIG-I and MDA5 prevented the formation of the RIG-I/MDA5-MAVS signalosome and interacted with TRIF and STING to inhibit TRIF-TBK1 and STING-TBK1 complex formation, thus reducing the subsequent IRF3 phosphorylation and nuclear translocation that are essential for IFN induction. In addition, ectopic expression of NSP7 impeded innate immune activation and facilitated virus replication. Taken together, SARS-CoV-2 NSP7 dampens type I and III IFN responses via disruption of the signal transduction of the RIG-I/MDA5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways, thus providing novel insights into the interactions between SARS-CoV-2 and innate antiviral immunity.

Inhibition of ALG3 stimulates cancer cell immunogenic ferroptosis to potentiate immunotherapy.

Immune checkpoint blockade therapy has drastically improved the prognosis of certain advanced-stage cancers. However, low response rates and immune-related adverse events remain important limitations. Here, we report that inhibiting ALG3, an a-1,3-mannosyltransferase involved in protein glycosylation in the endoplasmic reticulum (ER), can boost the response of tumors to immune checkpoint blockade therapy. Deleting N-linked glycosylation gene ALG3 in mouse cancer cells substantially attenuates their growth in mice in a manner depending on cytotoxic T cells. Furthermore, ALG3 inhibition or N-linked glycosylation inhibitor tunicamycin treatment synergizes with anti-PD1 therapy in suppressing tumor growth in mouse models of cancer. Mechanistically, we found that inhibiting ALG3 induced deficiencies of post-translational N-linked glycosylation modification and led to excessive lipid accumulation through sterol-regulated element-binding protein (SREBP1)-dependent lipogenesis in cancer cells. N-linked glycosylation deficiency-mediated lipid hyperperoxidation induced immunogenic ferroptosis of cancer cells and promoted a pro-inflammatory microenvironment, which boosted anti-tumor immune responses. In human subjects with cancer, elevated levels of ALG3 expression in tumor tissues are associated with poor patient survival. Taken together, we reveal an unappreciated role of ALG3 in regulating tumor immunogenicity and propose a potential therapeutic strategy for enhancing cancer immunotherapy.

Reverse genetics systems for SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) has caused severe public health crises and heavy economic losses. Limited knowledge about this deadly virus impairs our capacity to set up a toolkit against it. Thus, more studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology are urgently needed. Reverse genetics systems, including viral infectious clones and replicons, are powerful platforms for viral research projects, spanning many aspects such as the rescues of wild-type or mutant viral particles, the investigation of viral replication mechanism, the characterization of viral protein functions, and the studies on viral pathogenesis and antiviral drug development. The operations on viral infectious clones are strictly limited in the Biosafety Level 3 (BSL3) facilities, which are insufficient, especially during the pandemic. In contrast, the operation on the noninfectious replicon can be performed in Biosafety Level 2 (BSL2) facilities, which are widely available. After the outbreak of COVID-19, many reverse genetics systems for SARS-CoV-2, including infectious clones and replicons are developed and given plenty of options for researchers to pick up according to the requirement of their research works. In this review, we summarize the available reverse genetics systems for SARS-CoV-2, by highlighting the features of these systems, and provide a quick guide for researchers, especially those without ample experience in operating viral reverse genetics systems.

ORF8 protein of SARS-CoV-2 reduces male fertility in mice.

As one of the most rapidly evolving proteins of the genus Betacoronavirus, open reading frames (ORF8's) function and potential pathological consequence in vivo are still obscure. In this study, we show that the secretion of ORF8 is dependent on its N-terminal signal peptide sequence and can be inhibited by reactive oxygen species scavenger and endoplasmic reticulum-Golgi transportation inhibitor in cultured cells. To trace the effect of its possible in vivo secretion, we examined the plasma samples of coronavirus disease 2019 (COVID-19) convalescent patients and found that the patients aged from 40 to 60 had higher antibody titers than those under 40. To explore ORF8's in vivo function, we administered the mice with ORF8 via tail-vein injection to simulate the circulating ORF8 in the patient. Although no apparent difference in body weight, food intake, and vitality was detected between vehicle- and ORF8-treated mice, the latter displayed morphological abnormalities of testes and epididymides, as indicated by the loss of the central ductal lumen accompanied by a decreased fertility in 5-week-old male mice. Furthermore, the analysis of gene expression in the testes between vehicle- and ORF8-treated mice identified a decreased expression of Col1a1, the loss of which is known to be associated with mice's infertility. Although whether our observation in mice could be translated to humans remains unclear, our study provides a potential mouse model that can be used to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the human reproductive system.

Optimal management of blood glucose, blood pressure and atrial fibrillation to reduce the risk of heart failure with preserved ejection fraction.

BACKGROUND: Type 2 diabetes mellitus (T2DM), hypertension and atrial fibrillation (AF) are risk factors for heart failure with preserved ejection fraction (HFpEF). AIM: To examine the effects of the simultaneous control of all three conditions on new-onset HFpEF in this population. METHODS: This prospective cohort study enrolled 552 patients with T2DM, hypertension and AF, but without clinical signs or symptoms of heart failure. The participants were followed up for 5 years to examine the effects of glycaemic control (haemoglobin A1c: <7.0%, 7.0%-8.0% and >8.0%), blood pressure (BP) control (systolic BP: <120, 120-140 and >140 mmHg) or rhythm versus rate control for AF on new-onset HFpEF. RESULTS: With a follow up of 5 years, the new-onset HFpEF occurred in 62 of 552 enrolled participants. Among the different control level for diabetes, hypertension and AF, the intensive blood glucose (BG) control, poor BP control and rate control of AF had the highest risk of new-onset HFpEF, and the conservative BG control, intensive BP control and rhythm control of AF had the lowest risk of new-onset HFpEF. Multivariable Cox regression analysis showed that both poor BP control (hazard ratio (HR): 1.421, 95% confidence interval (CI): 1.013-1.992, P = 0.042) and rate control of AF (HR: 1.362, 95% CI: 1.006-1.821, P = 0.033) were independently associated with the development of new-onset HFpEF. CONCLUSION: This study demonstrated that, besides intensive BP control, conservative BG control and rhythm control of AF were crucial factors to delay the progression of HFpEF among patients with T2DM, hypertension and AF.

Autophagy mediates degradation of nuclear lamina.

Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.

Class IA PI3K p110ß subunit promotes autophagy through Rab5 small GTPase in response to growth factor limitation.

Autophagy is an evolutionarily conserved membrane trafficking process. Induction of autophagy in response to nutrient limitation or cellular stress occurs by similar mechanisms in organisms from yeast to mammals. Unlike yeast, metazoan cells rely more on growth factor signaling for a wide variety of cellular activities including nutrient uptake. How growth factor availability regulates autophagy is poorly understood. Here we show that, upon growth factor limitation, the p110ß catalytic subunit of the class IA phosphoinositide 3-kinases (PI3Ks) dissociates from growth factor receptor complexes and increases its interaction with the small GTPase Rab5. This p110ß-Rab5 association maintains Rab5 in its guanosine triphosphate (GTP)-bound state and enhances the Rab5-Vps34 interaction that promotes autophagy. p110ß mutants that fail to interact with Rab5 are defective in autophagy promotion. Hence, in mammalian cells, p110ß acts as a molecular sensor for growth factor availability and induces autophagy by activating a Rab5-mediated signaling cascade.

Association between long-term prescription of febuxostat and the progression of heart failure with preserved ejection fraction in patients with hypertension and asymptomatic hyperuricemia.

Both hypertension and hyperuricemia are closely associated with the morbidity and mortality of heart failure. This study was designed to evaluate the influences of long-term xanthine oxidase inhibitor (febuxostat) prescription on left ventricular hypertrophy (LVH), left ventricular (LV) diastolic function, and new-onset heart failure with preserved ejection fraction (HFpEF) in these patients. Using a propensity score matching of 1:2 ratio, this retrospective claims database study compared febuxosatat prescription (n = 96) and non-urate-lowering therapy (n = 192) in patients with hypertensive left ventricular hypertrophy (LVH) and asymptomatic hyperuricemia. With a follow-up of 36 months, febuxostat significantly decreased the level of serum uric acid as well as generated more prominent improvement in LVH and LV diastolic function. Besides, the new-onset symptomatic HFpEF occurred in 2 of 96 patients in febuxostat group and 13 of 192 patients in non-urate-lowering group (P = 0.091). No increased risk for major adverse cardiovascular events in patients prescribed with febuxostat was noted. In conclusion, long-term febuxostat exposure was associated with protective effects in terms of LVH or LV diastolic dysfunction in patients with hypertensive LVH and asymptomatic hyperuricemia. Febuxostat also displayed a trend for reduced risk of new-onset HFpEF in this population.

Prognostic impact of HbA1c variability on long-term outcomes in patients with heart failure and type 2 diabetes mellitus.

BACKGROUND: The prognostic impact of long-term glycemic variability on clinical outcomes in patients with heart failure (HF) and type 2 diabetes mellitus (T2DM) remains unclear. We determined and compared hemoglobin A1c (HbA1c) variability and clinical outcomes for patients with HF with preserved ejection fraction (HFpEF), HF with mid-range ejection fraction (HFmrEF) and HF with reduced ejection fraction (HFrEF) in a prospective longitudinal study. METHODS: Patients with HF and T2DM, undergone 3 or more HbA1c determinations during the first 18 months, were then followed for 42 months. The primary outcome was death from any cause. Secondary outcome was composite endpoints with death and HF hospitalization. Cox proportional hazards models were used to compare outcomes for patients with HFpEF, HFmrEF and HFrEF. RESULTS: Of 902 patients enrolled, 32.2% had HFpEF, 14.5% HFmrEF, and 53.3% HFrEF. During 42 months of follow-up, 270 (29.9%) patients died and 545 (60.4%) patients experienced composite endpoints of death and HF readmission. The risk of all-cause death or composite endpoints was lower for HFpEF than HFrEF. Moreover, higher HbA1c variability was associated with higher all-cause mortality or composite endpoints and HbA1c variability was an independent predictor of all-cause mortality or composite endpoints, regardless of EF. CONCLUSIONS: This prospective longitudinal study showed that the all-cause death and composite events was lower for HFpEF than HFrEF. HbA1c variability was independently and similarly predictive of death or combined endpoints in the three HF phenotypes.

Involvement of acid-sensing ion channel 1a in gastric carcinoma cell migration and invasion.

Acidic microenvironment, particularly acid-sensing ion channel 1a (ASIC1a), has been reported to promote carcinoma cell proliferation as well as migration. In this study, we explored the effect of ASIC1a on migration and invasion of gastric carcinoma (GC). ASIC1a expression levels were examined in paired GC and adjacent normal tissues from 16 patients by immunohistochemistry. Reverse transcription real-time PCR and immunoblotting were conducted to assess the ASIC1a expression levels in the GC cell line AGS after transfection with ASIC1a small hairpin RNA (shRNA). Wound healing and transwell invasion assays were utilized to detect metastasis and invasion following ASIC1a silencing. Tumor formation was used to detect the role of ASIC1a in tumorigenicity in vivo. It was found that ASIC1a expression level was significantly higher in GC tissues showing postoperative metastasis compared with non-metastasis and non-tumor tissues. Moreover, silencing of ASIC1a with shRNA significantly down-regulated ASIC1a expression and reduced GC cell migration and invasion. A moderately acidic extracellular environment inhibited GC cell viability. Furthermore, ASIC1a shRNA caused inhibition of tumorigenicity in vivo. Our study is the first report of attenuating the malignant phenotype of GC in vitro and in vivo by suppressing ASIC1a, and suggests a novel approach to study the relationship between ASICs and GC cell migration and invasion.

Hyperactivation of the mammalian degenerin MDEG promotes caspase-8 activation and apoptosis.

Intracellular calcium overload plays a critical role in numerous pathological syndromes such as heart failure, brain ischemia, and stroke. Hyperactivation of the acid-sensing ion channels including degenerin/epithelial amiloride-sensitive sodium (DEG/ENaC) channels has been shown to elevate intracellular calcium and cause subsequent neuronal cell death that is independent of the canonical Egl-1/Ced-9/Ced-4/Ced-3 apoptotic pathway in Caenorhabditis elegans. In mammalian cells, hyperactivation of the DEG/ENaC channels can also lead to cell death, although the underlying mechanism remains largely unknown. Here, we use a tetracycline-inducible system to express the hyperactivation mutant of a mammalian DEG/ENaC channel protein, MDEG G430F, in murine kidney epithelial cells deficient in the key mitochondrial apoptotic proteins Bax and Bak. Remarkably, expression of MDEG G430F induces increased intracellular calcium, reactive oxygen species (ROS) production, and cell death. The MDEG G430F-induced cell death is blocked by the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester), ROS scavengers, and the caspase inhibitor z-VAD-fmk (where z and fmk are benzyloxycarbonyl and fluoromethyl ketone). Mechanistically, the intracellular calcium overload and ROS increase lead to the inhibition of proteasomal and autophagic protein degradation, which promotes the accumulation of protein aggregates containing caspase-8 and subsequent caspase-8 activation. As protein aggregation upon the inhibition of proteasomal and autophagic degradation pathways is mediated by the ubiquitin-binding protein SQSTM1/p62 and the autophagy-related protein LC3, silencing of p62 and LC3 protects cells from MDEG G430F-induced cell death. Our results uncover a new mechanism of caspase-8-mediated apoptosis induced by intracellular calcium overload that is dependent on the autophagy-related proteins LC3 and p62 upon hyperactivation of DEG/ENaC channels.

TolC-dependent modulation of host cell death by the Francisella tularensis live vaccine strain.

Francisella tularensis is a facultative intracellular, Gram-negative pathogen and the causative agent of tularemia. We previously identified TolC as a virulence factor of the F. tularensis live vaccine strain (LVS) and demonstrated that a ΔtolC mutant exhibits increased cytotoxicity toward host cells and elicits increased proinflammatory responses compared to those of the wild-type (WT) strain. TolC is the outer membrane channel component used by the type I secretion pathway to export toxins and other bacterial virulence factors. Here, we show that the LVS delays activation of the intrinsic apoptotic pathway in a TolC-dependent manner, both during infection of primary macrophages and during organ colonization in mice. The TolC-dependent delay in host cell death is required for F. tularensis to preserve its intracellular replicative niche. We demonstrate that TolC-mediated inhibition of apoptosis is an active process and not due to defects in the structural integrity of the ΔtolC mutant. These findings support a model wherein the immunomodulatory capacity of F. tularensis relies, at least in part, on TolC-secreted effectors. Finally, mice vaccinated with the ΔtolC LVS are protected from lethal challenge and clear challenge doses faster than WT-vaccinated mice, demonstrating that the altered host responses to primary infection with the ΔtolC mutant led to altered adaptive immune responses. Taken together, our data demonstrate that TolC is required for temporal modulation of host cell death during infection by F. tularensis and highlight how shifts in the magnitude and timing of host innate immune responses may lead to dramatic changes in the outcome of infection.

CCDC50 mediates the clearance of protein aggregates to prevent cellular proteotoxicity.

Protein aggregation caused by the disruption of proteostasis will lead to cellular cytotoxicity and even cell death, which is implicated in multiple neurodegenerative diseases. The elimination of aggregated proteins is mediated by selective macroautophagy receptors, which is termed aggrephagy. However, the identity and redundancy of aggrephagy receptors in recognizing substrates remain largely unexplored. Here, we find that CCDC50, a highly expressed autophagy receptor in brain, is recruited to proteotoxic stresses-induced polyubiquitinated protein aggregates and ectopically expressed aggregation-prone proteins. CCDC50 recognizes and further clears these cytotoxic aggregates through autophagy. The ectopic expression of CCDC50 increases the tolerance to stress-induced proteotoxicity and hence improved cell survival in neuron cells, whereas CCDC50 deficiency caused accumulation of lipid deposits and polyubiquitinated protein conjugates in the brain of one-year-old mice. Our study illustrates how aggrephagy receptor CCDC50 combats proteotoxic stress for the benefit of neuronal cell survival, thus suggesting a protective role in neurotoxic proteinopathy.Abbreviations: AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; ATG5: autophagy related 5; BODIPY: boron-dipyrromethene; CASP3: caspase 3; CCDC50: coiled-coil domain containing 50; CCT2: chaperonin containing TCP1 subunit 2; CHX: cycloheximide; CQ: chloroquine; CRISPR: clustered regulatory interspaced short palindromic repeat; Cas9: CRISPR-associated system 9; DAPI: 4',6-diamidino-2-phenylindole; FK2: Anti-ubiquitinylated proteins antibody, clone FK2; FUS: FUS RNA binding protein; GFP: green fluorescent protein; HD: Huntington disease; HTT: huntingtin; KEGG: Kyoto Encyclopedia of Genes and Genomes; LDS: LIR-docking site; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPT/tau: microtubule associated protein tau; MIU: motif interacting with ubiquitin; NBR1: NBR1, autophagy cargo receptor; OPTN: optineurin; PD: Parkinson disease; PI: propidium iodide; ROS: reactive oxygen species; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; Ub: ubiquitin; UDS: UIM-docking site; UIM: ubiquitin interacting motif; UPS: ubiquitin-proteasome system.

Capsid structure of bacteriophage ΦKZ provides insights into assembly and stabilization of jumbo phages.

Jumbo phages are a group of tailed bacteriophages with large genomes and capsids. As a prototype of jumbo phage, ΦKZ infects Pseudomonas aeruginosa, a multi-drug-resistant (MDR) opportunistic pathogen leading to acute or chronic infection in immunocompromised individuals. It holds potential to be used as an antimicrobial agent and as a model for uncovering basic phage biology. Although previous low-resolution structural studies have indicated that jumbo phages may have more complicated capsid structures than smaller phages such as HK97, the detailed structures and the assembly mechanism of their capsids remain largely unknown. Here, we report a 3.5-Å-resolution cryo-EM structure of the ΦKZ capsid. The structure unveiled ten minor capsid proteins, with some decorating the outer surface of the capsid and the others forming a complex network attached to the capsid's inner surface. This network seems to play roles in driving capsid assembly and capsid stabilization. Similar mechanisms of capsid assembly and stabilization are probably employed by many other jumbo viruses.

TRIM21-mediated ubiquitination of SQSTM1/p62 abolishes its Ser403 phosphorylation and enhances palmitic acid cytotoxicity.

Long-chain free fatty acids (FFAs) accumulation and oxidative toxicity is a major cause for several pathological conditions. The mechanisms underlying FFA cytotoxicity remain elusive. Here we show that palmitic acid (PA), the most abundant FFA in the circulation, induces S403 phosphorylation of SQSTM1/p62 (sequestosome 1) and its aggregation, which sequesters KEAP1 and activates the non-canonical SQSTM1-KEAP1-NFE2L2 antioxidant pathway. The PA-induced SQSTM1 S403 phosphorylation and aggregation are dependent on SQSTM1 K7-D69 hydrogen bond formation and dimerization in the Phox and Bem1 (PB1) domain, which facilitates the recruitment of TBK1 that phosphorylates SQSTM1 S403. The ubiquitin E3 ligase TRIM21 ubiquitinates SQSTM1 at the K7 residue and abolishes the PB1 dimerization, S403 phosphorylation, and SQSTM1 aggregation. TRIM21 is oxidized at C92, C111, and C114 to form disulfide bonds that lead to its oligomerization and decreased E3 activity. Mutagenizing the three C residues to S (3CS) abolishes TRIM21 oligomerization and increases its E3 activity. TRIM21 ablation leads to decreased SQSTM1 K7 ubiquitination, hence elevated SQSTM1 S403 phosphorylation and aggregation, which confers protection against PA-induced oxidative stress and cytotoxicity. Therefore, TRIM21 is a negative regulator of SQSTM1 phosphorylation, aggregation, and the antioxidant sequestration function. TRIM21 is oxidized to reduce its E3 activity that helps enhance the SQSTM1-KEAP1-NFE2L2 antioxidant pathway. Inhibition of TRIM21 May be a viable strategy to protect tissues from lipotoxicity resulting from long-chain FFAs.