title: Measles Virus Enters Breast and Colon Cancer Cell Lines through a PVRL4-Mediated Macropinocytosis Pathway.
abstract: Measles virus (MeV) is a member of the familyParamixoviridaethat causes a highly contagious respiratory disease but has emerged as a promising oncolytic platform. Previous studies of MeV entry focused on the identification of cellular receptors. However, the endocytic and trafficking pathways utilized during MeV entry remain poorly described. The contribution of each endocytic pathway has been examined in cells that express the MeV receptors SLAM (signaling lymphocyte-activating molecule) and PVRL4 (poliovirus receptor-like 4) (nectin-4). Recombinant MeVs expressing either firefly luciferase or green fluorescent protein together with a variety of inhibitors were used. The results showed that MeV uptake was dynamin independent in the Vero.hPVRL4, Vero.hSLAM, and PVRL4-positive MCF7 breast cancer cell lines. However, MeV infection was blocked by 5-(N-ethyl-N-propyl)amiloride (EIPA), the hallmark inhibitor of macropinocytosis, as well as inhibitors of actin polymerization. By using phalloidin staining, MeV entry was shown to induce actin rearrangements and the formation of membrane ruffles accompanied by transient elevated fluid uptake. Small interfering RNA (siRNA) knockdown of p21-activated kinase 1 (PAK1) demonstrated that MeV enters both Vero.hPVRL4 and Vero.hSLAM cells in a PAK1-independent manner using a macropinocytosis-like pathway. In contrast, MeV entry into MCF7 human breast cancer cells relied upon Rac1 and its effector PAK1 through a PVRL4-mediated macropinocytosis pathway. MeV entry into DLD-1 colon and HTB-20 breast cancer cells also appeared to use the same pathway. Overall, these findings provide new insight into the life cycle of MeV, which could lead to therapies that block virus entry or methods that improve the uptake of MeV by cancer cells during oncolytic therapy.IMPORTANCEIn the past decades, measles virus (MeV) has emerged as a promising oncolytic platform. Previous studies concerning MeV entry focused mainly on the identification of putative receptors for MeV. Nectin-4 (PVRL4) was recently identified as the epithelial cell receptor for MeV. However, the specific endocytic and trafficking pathways utilized during MeV infections are poorly documented. In this study, we demonstrated that MeV enters host cells via a dynamin-independent and actin-dependent endocytic pathway. Moreover, we show that MeV gains entry into MCF7, DLD-1, and HTB-20 cancer cells through a PVRL4-mediated macropinocytosis pathway and identified the typical cellular GTPase and kinase involved. Our findings provide new insight into the life cycle of MeV, which may lead to the development of therapies that block the entry of the virus into the host cell or alternatively promote the uptake of oncolytic MeV into cancer cells.
authors.affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.|Canadian Centre for Vaccinology, IWK Health Centre, Goldbloom Pavilion, Halifax, Nova Scotia, Canada.
authors.forename: Sebastien
authors.initials: S
authors.lastname: Delpeut
authors.affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Gary
authors.initials: G
authors.lastname: Sisson
authors.affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Black
authors.affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada chris.richardson@dal.ca.|Canadian Centre for Vaccinology, IWK Health Centre, Goldbloom Pavilion, Halifax, Nova Scotia, Canada.|Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Richardson
mesh_terms: D000199:Actins / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001943:Breast Neoplasms; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D003110:Colonic Neoplasms; D004847:Epithelial Cells / Q000821:virology; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D008459:Measles virus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D050504:Oncolytic Viruses / Q000502:physiology; D010873:Pinocytosis / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D014709:Vero Cells; D053586:Virus Internalization; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism; D000075983:Nectins
chemical_list: D000199:Actins; D000584:Amiloride; D015815:Cell Adhesion Molecules; D034741:RNA, Small Interfering; D054462:p21-Activated Kinases; C442228:NECTIN4 protein, human; D000075983:Nectins; C515903:PAK1 protein, human; C039614:ethylisopropylamiloride
keywords: MCF7 cell; PAK1; PVRL4; Rac1; SLAM; breast cancer; dynamin; endocytosis; macropinocytosis; measles virus; morbillivirus; nectin-4; p21-activated kinase; receptor; virus entry