CHARACTERIZATION OF CHOLESTEROL-FREE INSECT CELLS INFECTIBLE BY BACULOVIRUSES - EFFECTS OF CHOLESTEROL ON VSV FUSION AND INFECTIVITY AND ONCYTOTOXICITY INDUCED BY INFLUENZA M2 PROTEIN

The patented cell line from the cabbage looper Trichoplusia ni (High F ive from Invitrogen) was found to grow readily under cholesterol-free (CF) culture conditions. Cellular cholesterol became undetectable by C F passage 4, while growth rate and overall cell morphology remained un affected for at least 59 CF passages. The Gels apparatus in CF cells w as significantly smaller than in control cells, and the CF cells also concentrated a ceramide-based fluorescent Golgi marker to a greater ex tent, but endoplasmic reticulum morphology appeared unaffected. Two pr oteins were expressed in High Five cells from recombinant baculoviruse s under CF and control conditions: the vesicular stomatitis virus (VSV ) fusion glycoprotein G and the influenza virus ion channel M2. Both p roteins were expressed in comparable amounts in CF and control cells, Both were properly assembled and transported to the plasma membrane in CF cells, indicating the presence of functional Golgi. Wild-type G pr otein expression resulted in extensive syncytia formation in both CF a nd control cells, showing that cholesterol is not required for VSV fus ion. However, a mutant G protein lacking six transmembrane domain resi dues was inactive in both CF and control cells, Influenza M2 protein w as functional in control cells, as indicated by its amantadine-inhibit able cytotoxicity, but cytotoxicity was absent in CF cells expressing this protein, indicating a cholesterol-dependence for the cytotoxic ac tion of this protein. CF and control cells were bath infectible with V SV, However, infected cell centers were modestly decreased (ca. 3.5-fo ld) in CF cells. CF cells offer a convenient and novel approach to the study of specific cholesterol functions, (C) 1997 Academic Press.