Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

Abstract

Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 [VP3]; 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. Cleavage of VP3 does not alter viral binding to cell monolayers. In previous electron microscopic studies of infected cell cultures, it has been demonstrated that rotavirus particles enter cells by both endocytosis and direct cell membrane penetration. To determine whether trypsin treatment affected rotavirus internalization, we studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Endocytosis inhibitors (sodium azide, dinitrophenol) and lysosomotropic agents (ammonium chloride, chloroquine) had a limited effect on the entry of infectious virus into cells. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 medicated 51Cr, [14C]choline, and

Rotaviruses Reach Late Endosomes and Require the Cation-Dependent Mannose-6-Phosphate Receptor and the Activity of Cathepsin Proteases To Enter the Cell

“…In addition to proteins transported by CD-M6PR from TGN to LEs, it is likely that factors such as acidic pH or the low luminal calcium concentration of endosomes could be part of the requirement for E-P and L-P RVs to get uncoated and exit the endosomal compartment. In this regard, it has been reported that preventing endosomal acidification with NH 4 Cl and other weak bases does not block the infection of RRV (36,38,68,69), but it reduces the infectivity of RVs UK and Wa (36), as well as the infectivity of those UK ϫ RRV reassortants bearing BRV UK VP4 (M. A. Díaz-Salinas et al, unpublished results). These findings support the idea that, in contrast to RRV, the L-P RVs UK and Wa could require the low pH of LEs to initiate viral replication in the cell.…”

Interferon gamma and interleukin 1, but not interferon alfa, inhibit rotavirus entry into human intestinal cell lines

“…Infected cells were identified by immunoperoxidase staining as previously described. 11 Briefly, mono-monolayers were not warmed.…”

Three-dimensional visualization of the rotavirus hemagglutinin structure

“…The spike protein, VP4, has been implicated in several roles during rotavirus infection. VP4 is the rotaviral hemagglutinin (Kalica et al, 1983) is a determinant of virulence and growth in ceil culture (Offit et al, 1986), and appears to be involved in cell binding (Ruggeri and Greenberg, 1991;Bass et al, 1991) and penetration (Kaljot et al, 1988). In addition, VP4 is susceptible to trypsin, resulting in the cleavage products VP5' (-60 kd) and VP8' (-28 kd).…”