Identification of mutations in the rotavirus protein VP4 that alter sialic-acid-dependent infection.

To explore further the role of VP4 as the rotavirus cell attachment protein, VP7 monoreassortants derived from the sialic-acid-dependent simian strain RRV and from the sialic-acid-independent human strains D, DS-1 and ST-3 were tested for susceptibility of infectivity of neuraminidase-treated MA-104 cells. Infectivity of RRV x D VP7 and RRV x ST-3 VP7 monoreassortants decreased when sialic acid was removed from the cell surface. However, of three separate RRV x DS-1 VP7 monoreassortants tested, only one was sialic-acid-dependent. Sequence analysis showed that both sialic-acid-independent strains contained a single amino acid change, Lys to Arg, at position 187. In addition, sialic-acid-independent infectivity was seen in one of 14 RRV VP4 neutralization escape mutants tested, and this strain was found to have a Gly to Glu change at amino acid position 150. These results indicate that positions 150 and 187 of VP4 play an important role in early rotavirus-cell interactions.

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

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 Ma104 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 Ma104 and HT29 cells when rotavirus infection was mediated by transfection of single- or double-shelled rotavirus particles with cationic liposomes (Lipofectin). 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.