Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles.
OBJECTIVE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.

BACKGROUND: Tumor-associated macrophages (TAMs) constitute a substantial part of human hepatocellular carcinoma (HCC). The present study was devised to explore TAM diversity and their roles in HCC progression.
METHODS: Through the integration of multiple 10 × single-cell transcriptomic data derived from HCC samples and the use of consensus nonnegative matrix factorization (an unsupervised clustering algorithm), TAM molecular subtypes and expression programs were evaluated in detail. The roles played by these TAM subtypes in HCC were further probed through pseudotime, enrichment, and intercellular communication analyses. Lastly, vitro experiments were performed to validate the relationship between CD63, which is an inflammatory TAM expression program marker, and tumor cell lines.
RESULTS: We found that the inflammatory expression program in TAMs had a more obvious interaction with HCC cells, and CD63, as a marker gene of the inflammatory expression program, was associated with poor prognosis of HCC patients. Both bulk RNA-seq and vitro experiments confirmed that higher TAM CD63 expression was associated with the growth of HCC cells as well as their epithelial-mesenchymal transition, metastasis, invasion, and the reprogramming of lipid metabolism.
CONCLUSIONS: These analyses revealed that the TAM inflammatory expression program in HCC is closely associated with malignant tumor cells, with the hub gene CD63 thus representing an ideal target for therapeutic intervention in this cancer type.

Periodontitis is an infection-driven inflammatory condition of the periodontium. Neutrophils are one of the most important first-line immune cells that protect against pathogen microorganisms in the saliva, but they may also mediate tissue death in inflammatory disorders. The aim of our study was to estimate salivary levels of azurocidin and extracellular azurophilic granules cluster of differentiation (CD63) as biomarkers of neutrophil activation in patients with periodontal diseases and to study the correlation between the levels of these two biomarkers and clinical periodontal parameters. The study included 60 patients with periodontal disease (30 patients with periodontitis and 30 with gingivitis) and 25 healthy controls. The assessed parameters were bleeding on probing, the plaque index, clinical attachment loss, and probing pocket depth. Saliva samples were taken from each study participant, and azurocidin and CD63 levels were measured using ELISA. Azurocidin and CD63 levels were significantly higher in patients with periodontitis and patients with gingivitis than in controls (P < 0.05), and significantly higher in patients with periodontitis than in patients with gingivitis (P < 0.05). Moreover, we found a significant positive correlation between the two biomarkers with clinical attachment loss in the periodontitis group. This study has shown that increased salivary azurocidin and extracellular CD63 levels are associated with enhanced innate response in periodontal disease and can be considered biomarkers of neutrophil activation.

Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents and drug delivery vehicles. Targeted modification of sEVs and their contents using genetic modification strategies is one of the most popular methods. This study investigated the effects of p53 fusion with arrestin domain-containing protein 1 (ARRDC1) and CD63 on the generation of sEVs, p53 loading efficiency, and therapeutic efficacy. Overexpression of either ARRDC1-p53 (ARP) or CD63-p53 (CDP) significantly elevated p53 mRNA and protein levels. The incorporation of ARRDC1 and CD63 significantly enhanced HEK293T-sEV biogenesis, evidenced by significant increases in sEV-associated proteins TSG101 and LAMP1, resulting in a boost in sEV production. Importantly, fusion with ARRDC1 or CD63 substantially increased the efficiency of loading both p53 fusion proteins and its mRNA into sEVs. sEVs equipped with ARP or CDP significantly enhanced the enrichment of p53 fusion proteins and mRNA in p53-null H1299 cells, resulting in a marked increase in apoptosis and a reduction in cell proliferation, with ARP-sEVs demonstrating greater effectiveness than CDP-sEVs. These findings underscore the enhanced functionality of ARRDC1- and CD63-modified sEVs, emphasizing the potential of genetic modifications in sEV-based therapies for targeted cancer treatment.

Dermatofibroma (DF) is a benign tumor that forms pedunculated lesions ranging in size from a few millimeters to 2 cm, usually affecting the extremities and trunks of young adults. Histopathologically, DF is characterized by the storiform proliferation of monomorphic fibroblast-like spindle cells. In addition to neoplastic cells, secondary elements such as foamy histiocytes, Touton-type giant cells, lymphoplasmacytes, and epidermal hyperplasia are characteristic histological features. Several histological variants, including atypical, cellular, aneurysmal, and lipidized variants, have been reported; cases with variant histologies are sometimes misdiagnosed as sarcomas. We present a case of metastasizing aneurysmal DF that was initially diagnosed as an angiosarcoma on biopsy. A 26-year-old woman was referred to our hospital with a gradually enlarging subcutaneous mass in her lower left leg. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose uptake not only in the tumor but also in the left inguinal region. On biopsy, ERG and CD31-positive atypical spindle cells proliferated in slit-like spaces with extravasation, leading to the diagnosis of angiosarcoma. Histology of the wide-resection specimen was consistent with DF, and lymph node metastasis was also observed. Nanopore DNA sequencing detected CD63::PRKCD fusion and copy number gain, although CD63 was not included in the target region of adaptive sampling. This report highlights the importance of recognizing the unusual clinical, radiological, and pathological features of DF to avoid misdiagnosis, and the potential diagnostic utility of nanopore sequencer.

The human-specific bacterial pathogen group A Streptococcus (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.

BACKGROUND: It is reported that CD123 + HLA-DR- cells in PBMC are basophils, and CD203c, CD63, and FcεRI molecules are activation markers of basophils. However, little is known of CD123 + HLA-DR-cells in blood granulocytes.
OBJECTIVE: To investigate the presence of CD123 + HLA-DR- cells in the blood granulocytes and peripheral PBMC of patients with allergic rhinitis (AR), as well as the impact of allergens on the cell membrane markers of basophils.
METHODS: Flow cytometry was used to detect the expression of the membrane molecules.
RESULTS: While CD123 + HLA-DR- PBMCs are representative of basophils, their presence did not significantly change in patients with AR. In contrast, both the percentage and number of CD123 + HLA-DR- granulocytes, which make up only up to 50% of basophils, were significantly increased in patients with seasonal (sAR) and perennial AR (pAR). CD63+, CD203c+, and FcεRIα+ cells within CD123 + HLA-DR- granulocytes also showed enhanced activity in patients with AR. Allergen extracts from house dust mite allergen extract (HDME) and Artemisia sieversiana wild extract further increased the number of CD123 + HLA-DR- cells in granulocytes of sAR and pAR patients, as well as in PBMCs of pAR patients.
CONCLUSIONS: The use of CD123 + HLA-DR- granulocytes and PBMC may not be sufficient for diagnosing AR. Allergens could potentially contribute to the development of AR by influencing the number of CD123 + HLA-DR- cells, as well as the expression of CD63, CD203c, and FcεRIαin these cells.

Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs.

OBJECTIVE: Tubal ectopic pregnancy (EP) is a leading cause of maternal morbidity and mortality. Studies have suggested that infection-induced inflammatory responses are major risk factors for EP. The aim of the present study was to find an association between MMP2 and CD63 gene variants and risk of EP during Chlamydia trachomatis infection in an Indian population.
METHODS: Fallopian tube samples of 120 EP and 120 tubal ligation women were collected. C. trachomatis was detected by PCR. The genotyping of MMP2 (rs17859882 G/T, rs7201A/C) and CD63(rs2231464 C/T, rs376086542 A/G) gene variants was done by qualitative real-time PCR using allelic discrimination method (VIC- and FAM-labeled).
RESULTS: The frequency of GG or GT genotype of MMP2 G/T polymorphism (rs17859882) was 66.6% in infected EP and 36.7% in uninfected EP and 22% in tubal ligation controls (P < 0.0001), while the frequency of AC or CC genotype of MMP2 A/C polymorphism (rs7201) was 66.6% in infected EP and 20.6% in uninfected EP and 13.5% in tubal ligation controls (P < 0.0001). The frequency of CT or TT genotype of CD63 C/T polymorphism (rs2231464) was 74% in infected EP and 21.8% in uninfected EP and 11.8% tubal ligation controls (P < 0.0001), while the frequency of AG or GG genotype of CD63 A/G polymorphism (rs376086542) was 48.1% in infected EP and 41.3% in uninfected EP and 18.6% tubal ligation controls (P < 0.0001).
CONCLUSIONS: The present study revealed a strong association between the presence of gene variants MMP2 (rs17859882 G/T, rs7201A/C) and CD63 (rs2231464 C/T, rs376086542 A/G) and risk of tubal EP during C. trachomatis infection.

The STimulator of INterferon Genes (STING) constitutes a major DNA-sensing pathway that restricts HSV-1 infection in different models by activating type I interferon and pro-inflammatory responses. To counteract STING, HSV-1 has evolved numerous strategies including mechanisms to interfere with its oligomerization, post-translational modifications, and downstream signaling. Previously, we demonstrated that STING is packaged in extracellular vesicles (EVs) produced from HSV-1-infected cells. These EVs activated antiviral responses in uninfected recipient cells and suppressed a subsequent HSV-1 infection in a STING-dependent manner. Here, we provide information on the packaging of STING in EVs and its exocytosis. We found that STING exocytosis did not occur in CD63 knockdown cells supporting that STING follows the CD63 exocytosis pathway. Consistently, we found that STING co-localized with CD63 in cytoplasmic globular structures and exosomal STING and CD63 co-fractionated. Both golgicide A and brefeldin A prevented STING exocytosis during HSV-1 infection suggesting that STING trafficking through the Golgi is required. A STING ligand was insufficient for STING exocytosis, and downstream signaling through TBK1 was not required. However, STING palmitoylation and tethering to the ER by STIM1 were required for STING exocytosis. Finally, we found that HSV-1 replication/late gene expression triggered CD63 exocytosis that was required for STING exocytosis. Surprisingly, HSV-2 strain G did not trigger CD63 or STING exocytosis as opposed to VZV and HCMV. Also, EVs from HSV-1(F)- and HSV-2(G)-infected cells displayed differences in their ability to restrict these viruses. Overall, STING exocytosis is induced by certain viruses and shapes the microenvironment of infection.IMPORTANCEExtracellular vesicles (EVs) are released by all types of cells as they constitute a major mechanism of intercellular communication. The packaging of specific cargo in EVs and the pathway of exocytosis are not fully understood. STING is a sensor of a broad spectrum of pathogens and a key component of innate immunity. STING exocytosis during HSV-1 infection has been an intriguing observation, raising questions of whether this is a virus-induced process, the purpose it serves, and whether it is observed after infection with other viruses. Here, we have provided insights into the pathway of STING exocytosis and determined factors involved. STING exocytosis is a virus-induced process and not a response of the host to the infection. Besides HSV-1, other herpes viruses triggered STING exocytosis, but HSV-2(G) did not. HSV-1 EVs displayed different restriction capabilities compared with HSV-2(G) EVs. Overall, STING exocytosis is triggered by viruses to shape the microenvironment of infection.