INFECTION OF B5 CELLS WITH CELL-FREE HTLV-1 AND AN HTLV-1 INFECTIOUS MOLECULAR CLONE

Abstract

Studies of in vitro human T-cell leukemia virus, type 1 (HTLV-1) infection have been limited by the lack of reproducible infections using cell free HTLV-1 virus. A cell free viral infectivity system is needed to study viral kinetics without the involvement of virus donor cells as in the current cocultivation systems. To develop an HTLV-1 infectivity assay using cell free virus, a variety of cell lines were screened for their susceptibility to be infected with 0.2 uM filtered, cell free HTLV-1 (SGDOWTSP, a primary HTLV-1 T cell isolate). The cells most reproducibly infected with cell free supernatant were DBS-FRhL-2, rhesus monkey lung fibroblasts. DBS-FRhL-2 cells were cloned and clone BS was selected as the most susceptible clone for infection with cell free HTLV-1. The cell free infectivity titer of SGDOWTSP was determined to be 4510 pg/ml p24. Two other HTLV-1 viral isolates, MT-2 and F8953, could also infect BS by cell free methods. BS cells could also be productively transfected with a full length HTLV-1 molecular clone, pCS-HTLV. By day 18 post transfection, 75 pg/ml p24 was detected and infection persisted for more than a year. Virus from these transfected B5 cells resulted in a productive infection when passed cell free to other B5 cells. A nucleocapsid mutant was constructed with a second protease cleavage site inserted in the pCS-HTLV p15 (p15P). Attempts to transfect B5 with this mutant resulted in no virus production. 293 cells transfected with both wild type and mutant virus were transiently infected. The cell free wild type virus from these 293 cells could productively infect B5 cells. The cell free mutant p15P virus could not productively infect B5 cells. This alteration of the nucleocapsid protein results in a replication defective virus.