The cluster cell adhesion was also relatively highly enriched when looking at all proteins, but less so in ProK treated virions as would be expected. Enrichment of proteins involved in vesicles and vesicle mediated transport, protein localization and cytoskeletal organization is consistent with known features of VSV assembly and budding. More than one third of the proteins identified are represented in at least one of these 4 clusters, indicating many virion-incorporated host proteins are likely involved in these functions. Transport of the VSV nucleocapsid to the site of budding has been shown to be dependent on microtubules and is an important step in VSV assembly. Furthermore, as seen for many enveloped viruses, VSV budding appears to use the host proteins involved in multivesicular body formation which are relocated from endosomal membranes to the OTX015 202590-98-5 plasma membrane in an M protein dependent manner. However, the budding of VSV and related viruses may still have someuniquefeatures. While the specific members of the MVB pathway utilized by VSV for its budding are not clear, it is known that, unlike what has been observed for many other viruses, Tsg101 is not essential for the budding of VSV and rabies virus, while contradictory reports exist regarding the importance of Vps4a. In contrast, the host ubiquitinproteasome system does appear to be essential. The lipid composition of the VSV envelope is consistent with that of its host cell, although levels of cholesterol and sphingomyelin are elevated, indicating that unlike some other viruses such as influenza virus and HIV-1, VSV does not bud through host membrane regions enriched in lipid rafts. Instead, VSV G and M proteins appear to initially localize to separate microdomains of the host plasma membrane but then either merge or cluster together with each other and host protein containing microdomains at the site of virus budding. Therefore, VSV readily incorporates the membrane proteins of its host. In pseudotyping experiments using both mixed virus infections and expression of viral or host proteins from VSV recombinants, non-VSV proteins can make up a significant portion of the protein in the VSV envelope, with incorporation levels up to 31% of that seen for VSV G protein reported. Consistent with these observations, 46% of proteins identified in our study are associated with the cellular component GO term “plasma membrane”, suggesting they were acquired along with the envelope. The tendency of VSV to indiscriminately acquire relatively large numbers of host.