Liposome-mediated transfection of intact viral particles reveals that plasma membrane penetration determines permissivity of tissue culture cells to rotavirus.

Abstract

Rotaviruses are an important cause of gastroenteritis in human infants. In vivo, rotavirus displays striking cell tropism with viral replication generally restricted to the villus tip enterocytes of the small intestine. We studied a panel of cell lines that vary significantly in their permissivity to rotavirus infection. L cells and HEp2 cells were relatively resistant to rotavirus infection compared with permissive MalO4 cells and HT29 cells. RNA transcription among the cell lines was proportional to antigen synthesis making a translational or posttranslational block an unlikely source of observed differences in susceptibility. All of the cell lines bound and internalized radiolabeled virus equally well, as measured by escape from surface protease treatment. Analysis of the escape of cell bound virus from neutralizing monoclonal antibody revealed that rotavirus did not immediately enter an eclipse phase in nonpermissive cells, but was internalized in an infectious form for several hours, possibly sequestered within endocytic vacuoles. L cells and HEp2 cells were as permissive as MalO4 and HT29 cells when rotavirus infection was mediated by transfection of single-or doubleshelled rotavirus particles with cationic liposomes (LipofectinTM). Rotavirus cell tropism in tissue culture cells is determined by the ability of infecting virions to traverse the plasma membrane of the cells into the cytoplasmic compartment. (J.

Functional Maturation of the Human Antibody Response to Rotavirus

“…To test antiviral activity, we used a previously established assay based on DLP neutralization inside cells (4). DLPs are rendered infectious if introduced into the cytoplasm of permissive cells by lipid-mediated introduction of particles (36). In this assay, VP6 Fabs can exhibit antiviral activity if introduced into the cytoplasm concurrently with DLPs.…”

Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6–specific IgA mAb

“…Classical focus reduction neutralization assay for rotavirus was carried out as described (29). Infection of MA104 cells with rotavirus DLPs using lipofectin-mediated transfection was carried out as described (30). The lipofection-mediated intracellular neutralization assay was based on the rotavirus DLP transfection method.…”

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

“…Lysates and cell supernatants were harvested at different time points for virus quantification. RV TLPs, DLPs, and VLPs were prepared as previously described (20).…”

“…Infection of WT and DBN1 −/− HEK293 cells with RRV DLPs using lipofectin-mediated transfection was carried out as described (20). Briefly, DLPs were diluted in DMEM and incubated with a mixture of Lipofectamine (Life Technologies) in DMEM (15% vol/vol) for 40 min at room temperature.…”

“…To mechanistically determine how DBN1 antagonizes RV infection, we took advantage of RV double-layered particles (DLPs), which are transcriptionally active virus subparticles lacking the VP4 and VP7 surface proteins and cannot directly infect cells unless transfected into the cytoplasm to bypass entry (20). Transfected DLPs resulted in comparable levels of virus replication in WT and DBN1 −/− cells (Fig.…”