On the entry of semliki forest virus into BHK-21 cells

Abstract

The pathway by which Semliki Forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically . After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes . Direct penetration of viruses through the plasma membrane was never observed .To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV . Thus, the inhibitory effect was concluded to be either on penetration of the nucleocapsid into the cytoplasm or on uncoating of the viral RNA .Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipid-cholesterol liposomes and isolated SFV . When the pH was 6 .0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes . Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked .The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them . The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration . KEY WORDS coated vesicleslysosomes " from the extracellular space to the cytosol . This endocytosis -membrane fusion process requires transport through one or more lysosomotropic agents membrane barriers . For animal viruses the mechanisms involved are poorly understood . Data from To infect a cell, a virus must transfer its genome electron microscopy suggest that penetration oc-404 J . CELL BIOLOGY

A Morphological Study of the Replication of Breda Virus (Proposed Family Toroviridae) in Bovine Intestinal Cells

“…Since infection in this calf had already progressed to the large intestine, it is probable that a second wave of infection had begun and cells of the mid-jejunum were newly infected, lacking mature, assembled virions but possessing viral antigen, nonetheless. The presence of Breda virions in close association with tips and sides of microvilli and within coated pits suggests that the virus may enter cells by receptor-mediated endocytosis (RME), a common mechanism of virus uptake (Goldstein et al, 1979;Helenius et al, 1980). This is supported further by the restricted association of virus with microvilli of absorptive enterocytes only.…”

Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. I. Morphological evidence.

“…Others have also observed the endocytosis of probes and liga~ds into vacuoles morphologically similar to those described here (12,45,48). In particular Van Deurs et al (42) reported that cationized ferritin initially entered electronlucent endocytic vacuoles and "light" multivesicular bodies containing some vesicular profiles, and only later could been seen in "dark" multivesicular bodies and dense bodies.…”

“…3, a and a') the fluorescence appeared mainly as dots at the apical surface. This is the pattern generally seen when viruses are added to MDCK cells (22) or to BHK cells (12), and probably represents viruses bound or fused mainly to microvilli on the Fig. 3 were incubated at 31 °C after implantation and EDTA treatment at 0°C and then processed for differential immunofluorescence staining as described in Fig.…”

Stepwise dismantling of adenovirus 2 during entry into cells

“…Our previous studies on virus entry and uncoating have shown that incoming viruses receive signals from the host cell that trigger appropriate molecular changes in structural proteins required for penetration and uncoating (Helenius et al, 1980;Helenius, 1992). For example, independent signals for membrane fusion and for uncoating of influenza virus are mediated by the low pH in endosomes (Martin and Helenius, 1991).…”

“…These processes are rather well understood for several enveloped animal viruses that use refined strategies of membrane fusion and uncoating (for reviews see Helenius, 1992;Kielian and Jungerwirth, 1990;Marsh and Helenius, 1989). Some penetrate directly through the plasma membrane while others enter from endosomes (Helenius et al, 1980;Pauza, 1991;Rigaut et al, 1991;Wittels and Spear, 1991). For nonenveloped viruses, the early events in the replication cycle are far less understood.…”