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JCPyV has been shown to infect cells by both direct and indirect mechanisms, the latter involving extracellular vesicle (EV) mediated infection. While direct mechanisms of infection are well studied indirect EV mediated mechanisms are poorly understood. Using a combination of chemical and genetic approaches we show that several overlapping intracellular pathways are responsible for the biogenesis of virus containing EV. Here we show that targeting neutral sphingomyelinase 2 (nSMase2) with the drug cambinol decreased the spread of JCPyV over several viral life cycles. Genetic depletion of nSMase2 by either shRNA or CRISPR\/Cas9 reduced EV\u2010mediated infection. Individual knockdown of seven ESCRT\u2010related proteins including HGS, ALIX, TSG101, VPS25, VPS20, CHMP4A, and VPS4A did not significantly reduce JCPyV associated EV (JCPyV(+) EV) infectivity, whereas knockdown of the tetraspanins CD9 and CD81 or trafficking and\/or secretory autophagy\u2010related proteins RAB8A, RAB27A, and GRASP65 all significantly reduced the spread of JCPyV and decreased EV\u2010mediated infection. These findings point to a role for exosomes and secretory autophagosomes in the biogenesis of JCPyV associated EVs with specific roles for nSMase2, CD9, CD81, RAB8A, RAB27A, and GRASP65 proteins.<\/jats:p>","DOI":"10.1002\/jex2.43","type":"journal-article","created":{"date-parts":[[2022,5,3]],"date-time":"2022-05-03T06:41:07Z","timestamp":1651560067000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Biogenesis of JC polyomavirus associated extracellular vesicles"],"prefix":"10.1002","volume":"1","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4853-2175","authenticated-orcid":false,"given":"Jenna","family":"Morris\u2010Love","sequence":"first","affiliation":[{"name":"Graduate Program in Pathobiology Brown University  Providence RI USA"},{"name":"Department of Molecular biology Cellular Biology and Biochemistry Brown University  Providence RI 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