Addition of Exogenous Protease Facilitates Reovirus Infection in Many Restrictive Cells

Abstract

Virion uncoating is a critical step in the life cycle of mammalian orthoreoviruses. In cell culture, and probably in extraintestinal tissues in vivo, reovirus virions undergo partial proteolysis within endosomal or/or lysosomal compartments. This process converts the virion into a form referred to as an intermediate subvirion particle (ISVP). In natural enteric reovirus infections, proteolytic uncoating takes place extracellularly within the intestinal lumen. The resultant proteolyzed particles, unlike intact virions, have the capacity to penetrate cell membranes and thereby gain access to cytoplasmic components required for viral gene expression. We hypothesized that the capacity of reovirus outer capsid proteins to be proteolyzed is a determinant of cellular host range. To investigate this hypothesis, we asked if the addition of protease to cell culture medium would expand the range of cultured mammalian cell lines that can be productively infected by reoviruses. We identified many transformed and nontransformed cell lines, as well as primary cells, that restrict viral infection. In several of these restrictive cells, virion uncoating is inefficient or blocked. Addition of proteases to the cell culture medium generates ISVP-like particles and promotes viral growth in nearly all cell lines tested. Interestingly, we found that some cell lines that restrict reovirus uncoating still express mature cathepsin L, a lysosomal protease required for virion disassembly in murine L929 cells. This finding suggests that factors in addition to cathepsin L are required for efficient intracellular proteolysis of reovirus virions. Our results demonstrate that virion uncoating is a critical determinant of reovirus cellular host range and that many cells which otherwise support productive reovirus infection cannot efficiently mediate this essential early step in the virus life cycle.Mammalian reoviruses (reoviruses) are prototypic members of the Reoviridae family, which includes the pathogenic rotaviruses, coltiviruses, and orbiviruses. Whereas reovirus causes infections that are generally asymptomatic in humans, it can induce respiratory, enteric, and nervous system diseases in animal models (reviewed in reference 67). Reovirus virions comprise a multilayered protein capsid that surrounds a segmented, double-stranded RNA genome (reviewed in reference 51). The outermost capsid layer consists of protein 3. The presence of 3 imparts environmental stability to the virion (54) but also appears to negatively regulate critical virion functions such as membrane penetration. The ability of reovirus to establish a productive infection requires proteolysis of the outer capsid (13,62,65). More recent data point toward 3 as the critical target for degradation (19,20).The first step in reovirus infection is attachment to cellular receptors through interactions with the viral receptor protein 1 (46, 75). Following attachment, virions are internalized by receptor-mediated endocytosis and delivered to endosomal compartments (12,13,65). In cell c...

Addition of exogenous polypeptides on the mammalian reovirus outer capsid using reverse genetics

“…This facilitated the recovery of the plaques that were readily visualized on these cells by direct visual inspection or by phase contrast microscopy. However, the addition of chymotrypsin was necessary to obtain visible plaques (data not shown), consistent with previous report that proteolytic uncoating is a limiting step for reovirus infection in these cells (Golden et al, 2002). The Vero cells could eventually be an useful alternative to L929 cells to recover certain virus mutants since these cells are defective in interferon production (Desmyter et al, 1968;Emeny and Morgan, 1979).…”

The oncolytic effect in vivo of reovirus on tumour cells that have survived reovirus cell killing in vitro

“…Our studies on the Raji cured cells have led us to suggest that proteases present within these tumours are responsible for the enhanced reovirus infection in vivo, since these cells are only efficiently infected in vitro when reovirus is first proteolytically processed into ISVP before infection. Indeed, addition of exogenous proteases to the culture medium has been shown to facilitate reovirus infection of a variety of cells, which would not otherwise be susceptible (Golden et al, 2002). Studies have shown that protease-generated ISVPs are capable of directly penetrating the cell membrane and entering the cytoplasm where viral replication can take place (Borsa et al, 1979;Chandran et al, 2002).…”

“…Such ISVPs have been shown to be capable of infecting many restrictive cells that have somehow lost their ability to process the uncoating of the incoming virion (Dermody et al, 1993;Baer and Dermody, 1997;Wetzel et al, 1997;Baer et al, 1999;Golden et al, 2002).…”

Combination of dextran sulfate and recombinant trypsin on aggregation of Chinese hamster ovary cells

“…Nowadays, there are still some commercial trypsins derived from animal pancreas, which may be toxic and cause infectious contamination to cultured cells (Golden et al 2002). Recombinant trypsin expressed in Escherichia coli eliminates the risks mentioned above.…”