Productive Penetration of Rotavirus in Cultured Cells Induces Coentry of the Translation Inhibitor α-Sarcin

Abstract

Internalization of rotavirus in MA104 cells was found to induce coentry of alpha-sarcin, a toxin that inhibits translation in cell-free systems and to which cells are normally impermeable. Entry of the toxin, measured by inhibition of protein synthesis at early times after infection, correlated with virus penetration leading to expression of infectivity, since toxin entry (1) was induced only by trypsin-treated triple-layered virions, to a degree dependent on the toxin and the virus concentration; (2) correlated with the degree of permissivity of different cell lines to rotavirus infection; (3) was inhibited to a similar extent as infectivity by treatment of cells with neuraminidase; and (4) was inhibited by pre- or postadsorption incubation of the virus with neutralizing monoclonal antibodies to VP7 and VP4 (VP8*). Neither the virus infectivity nor the toxin coentry was significantly affected by treatment of cells with bafilomycin A1, an inhibitor of the vacuolar proton ATPase, indicating that both events are independent of the endosomal acid pH. Virus-like particles (VLP), composed of rotavirus proteins 2/6/7/4, but not 2/6/7 or 2/6, were able to induce toxin entry as efficiently as virions. Use of genetically modified VLP in combination with the toxin coentry assay, which measures entry through a productive pathway, should allow identification of the regions of the outer capsid proteins essential for rotavirus penetration.

Human and most animal rotavirus strains do not require the presence of sialic acid on the cell surface for efficient infectivity.

“…ndez et al, 1993 ;Rolsma et al, 1994 ;Liprandi et al, 1997), we confirmed that both the adsorption and the infectivity of simian SA11 and RRV, bovine NCDV and porcine OSU rotavirus strains are reduced dramatically by treatment of MA104 cells with neuraminidase ( Fig. 2 A, i).…”

“…Among ARVs, only four strains are reported to be SA-dependent, including simian SA11 (P[2], G3) and RRV (P5B[3], G3) ; bovine NCDV (P6[1], G6) ; and porcine OSU (P9 [7], G5) (Yolken et al, 1987 ;Keljo & Smith, 1988 ;Fukudome et al, 1989 ;Superti & Donelli, 1991 ;Me! ndez et al, 1993 ;Rolsma et al, 1994 ;Liprandi et al, 1997). Recently, Ludert et al (1996) reported that the infectivity of murine rotavirus strains EW (P[16], G3) and EHP (P[20], G3) remained unaffected following treatment of cells with neuraminidase.…”

Requirements for the Formation of Membrane Pores by the Reovirus Myristoylated μ1N Peptide

“…Both are trimeric proteins with similarly folded subunits-a radially oriented, ␤-jelly-roll domain flanked in the polypeptide chain by N-and C-terminal extensions, which form a largely ␣-helical "pedestal" (35). VP6 is not a penetration effector, however, and it probably has a purely structural role as a connecting element between the actual penetration effectors of the outer layer (VP4 and VP7) and the RNA-enclosing VP2 shell (32,57,59). The pedestal of 1 is substantially larger than that of VP6: the additional structure is primarily the part that sequesters 1N and that unfolds in order to release it (31).…”

Individual Rotavirus-like Particles Containing 120 Molecules of Fluorescent Protein Are Visible in Living Cells

“…Depending on the set of expressed proteins several types of VLP are obtained (5). VLP are stable, and when they include the spike protein, they share several properties with infectious virus including cell binding, ␣ sarcine coentry, activation of NF-B, and cell fusion from without (5)(6)(7)(8).…”