Tetherin antagonism by SARS-CoV-2 ORF3a and spike protein enhances virus release.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS-CoV-2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

Extracellular Vesicles Potentiate Medulloblastoma Metastasis in an EMMPRIN and MMP-2 Dependent Manner.

Extracellular vesicles (EVs) have emerged as pivotal mediators of communication in the tumour microenvironment. More specifically, nanosized extracellular vesicles termed exosomes have been shown to contribute to the establishment of a premetastatic niche. Here, we sought to determine what role exosomes play in medulloblastoma (MB) progression and elucidate the underlying mechanisms. Metastatic MB cells (D458 and CHLA-01R) were found to secrete markedly more exosomes compared to their nonmetastatic, primary counterparts (D425 and CHLA-01). In addition, metastatic cell-derived exosomes significantly enhanced the migration and invasiveness of primary MB cells in transwell migration assays. Protease microarray analysis identified that matrix metalloproteinase-2 (MMP-2) was enriched in metastatic cells, and zymography and flow cytometry assays of metastatic exosomes demonstrated higher levels of functionally active MMP-2 on their external surface. Stable genetic knockdown of MMP-2 or extracellular matrix metalloproteinase inducer (EMMPRIN) in metastatic MB cells resulted in the loss of this promigratory effect. Analysis of serial patient cerebrospinal fluid (CSF) samples showed an increase in MMP-2 activity in three out of four patients as the tumour progressed. This study demonstrates the importance of EMMPRIN and MMP-2-associated exosomes in creating a favourable environment to drive medulloblastoma metastasis via extracellular matrix signalling.

A role for ABCB1 in prognosis, invasion and drug resistance in ependymoma.

Three of the hallmarks of poor prognosis in paediatric ependymoma are drug resistance, local invasion and recurrence. We hypothesised that these hallmarks were due to the presence of a sub-population of cancer stem cells expressing the multi-drug efflux transporter ABCB1. ABCB1 gene expression was observed in 4 out of 5 paediatric ependymoma cell lines and increased in stem cell enriched neurospheres. Functional inhibition of ABCB1 using vardenafil or verapamil significantly (p ≤ 0.05-0.001) potentiated the response to three chemotherapeutic drugs (vincristine, etoposide and methotrexate). Both inhibitors were also able to significantly reduce migration (p ≤ 0.001) and invasion (p ≤ 0.001). We demonstrate that ABCB1 positive patients from an infant chemotherapy-led trial (CNS9204) had a shorter mean event free survival (EFS) (2.7 versus 8.6 years; p = 0.007 log-rank analysis) and overall survival (OS) (5.4 versus 12 years; p = 0.009 log-rank analysis). ABCB1 positivity also correlated with reduced event free survival in patients with incompletely resected tumours who received chemotherapy across CNS9204 and CNS9904 (a radiotherapy-led SIOP 1999-04 trial cohort; p = 0.03). ABCB1 is a predictive marker of chemotherapy response in ependymoma patients and vardenafil, currently used to treat paediatric pulmonary hypertension in children, could be repurposed to reduce chemoresistance, migration and invasion in paediatric ependymoma patients at non-toxic concentrations.