Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment

Abstract

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.

An Update on Non-clathrin-coated Endocytosis

“…In the picornavirus family, polioviruses may enter by both coated-vesicles 24 and another endocytic pathway. 25,26 Human rhinovirus type 2 has also been reported to infect cells in clathrinindependent manner. 24 Until recently, it has not been possible to investigate the process of viral entry via non-clathrin coated vesicles, because of a lack of information about their basic biology.…”

Viral cell recognition and entry

“…Such displacement probably requires large conformational changes in the rhinovirus coat. For poliovirus or rhinovirus, acidification of endosomes may be required for an infection to proceed normally as measured by either progeny virus production or cytopathic effects (Madshus et al, 1984a(Madshus et al, , 1984bZeichhardt et al, 1985;Neubauer et al, 1987;Gromeier & Wetz, 1990), although Perez and Carrasco (1993) conclude that acidification is not essential.…”

Picornavirus inhibitors

“…Most evidence indicates that the majority of naked animal virus particles follow the receptor-mediated endocytosis pathway to infect cells (Marsh and Helenius, 1989). Whether some of these viruses need a low pH step to trigger the crossing of the endosomal membrane, or enter cells by a pH-independent mechanism, remains open to debate (Brown and Burlingham, 1973;Madshus et al, 1984c;Svensson, 1985;Sturzenbecker et al, 1987;Gromeier and Wetz, 1990;Kronenberger et al, 1991;Varga et al, 1991). Two major questions, therefore, remain to be answered with respect to the mechanism of picornavirus entry into cells: Is a given picornavirus species internalized through endosomes in a particular cell type?, or do endosomes need to be acidified for the virus to enter cells?…”

“…Once the viral particles are in endosomes, they must exit from this organeile in order to initiate translation of the genomic RNA in the cytoplasm. It is still a matter of debate as to whether poliovirus requires acidification of endosomes to allow the passage of the genome through the endosomal membrane (Madshus et al, 1984b, c;Zeichhardt et al, 1985), or if poliovirus accomplishes this task in a pH-independent fashion (Gromeier and Wetz, 1990;P~rez and Carrasco, 1993). Indirect experiments involving complexing of poliovirus particles with neutral red prior to incubation for several minutes with weak amines or monensin, followed by very long incubation (18-40hr), have led to the suggestion that poliovirus requires a low-pH step for infectivity (Madshus et al, 1984b, c).…”

“…Hence, it is not surprising that during the long incubation times, where several rounds of viral replication take place, there was inhibition of the virus (Madshus et al, 1984b, c). Shorter incubation times testing the inhibition of poliovirus by chloroquine or monensin during the uncoating period lead to the idea that poliovirus entry is pH-independent (Gromeier and Wetz, 1990;P6rez and Carrasco, 1993). The presence of lysosomotropic agents, such as chloroquine, amantadine, dansylcadaverine or monensin, during poliovirus entry did not inhibit infection (P~rez and Carrasco, 1993).…”