Effect of enzymes on rotavirus infectivity

Barnett, Bill B.; Spendlove, Rex S.; Clark, M L

doi:10.1128/jcm.10.1.111-113.1979

Cited by 42 publications

(13 citation statements)

References 11 publications

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“…Rotaviruses are, in general, difficult to isolate and to grow in cell cultures using conventional techniques. However, certain strains may be serially propagated in cell cultures if the virus inuculum is treated with proteolytic enzymes or if proteolytic enzymes are incorporated into the medium after infection (1,2,3,6,16,40,41,44,45,49,56). ENGI, A~D and POSTON (9) reported that rotavirus observed in feces by E?¢[ from a diarrheal dog grew in cell cultures with the aid of trypsin, but did not grow in cell cultures without trypsin treatment.…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of a canine rotavirus

Hoshino

Wyatt

Scott

et al. 1982

Archives of Virology

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Canine rotavirus particles were visualized by direct electron microscopy in the feces from a clinically normal dog. The virus was subsequently propagated in cell cultures; it was characterized and compared with rotaviruses from other species. Replication of the virus in cell culture was found to be less dependent upon trypsin than that of human, bovine and porcine rotaviruses. Reproducible, sharp-edged plaques of various sizes were produced by the canine rotavirus in an established cell line of fetal rhesus monkey kidney, MA 104, under overlays of carboxymethyl cellulose or agarose. Intracytoplasmic inclusion bodies of different sizes and shapes were produced in infected MA 104 cells. By plaque reduction neutralization assay, a two-way antigenic relationship was found between the canine (CU-1) and simian (rhesus MMU 18006 and SA-11) rotaviruses. The canine rotavirus had a one-way antigenic relationship with feline (Taka), bovine (NCDV), and porcine (OSU) rotaviruses.

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Section: Discussionmentioning

confidence: 99%

Isolation and characterization of a canine rotavirus

Hoshino

Wyatt

Scott

et al. 1982

Archives of Virology

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“…Inhibitors and antibody were present overnight. Positive controls for the effect of weak bases on intravesicular pH were provided by infection of treated MA104 cell monolayers with reovirus types 1 and 3 (MOI, 5). After 24 h the reovirus-infected monolayers were freezethawed three times, and viral titers were determined by plaque assay.…”

Section: Methodsmentioning

confidence: 99%

“…The effect of trypsin on viral infectivity is mediated by alteration of the virion rather than the cell surface (5,19). The outer shell of the rotavirus capsid is formed by two proteins, viral proteins 3 and 7 (VP3 and VP7, respectively), which are the products of genes 4 and 9, respectively (35).…”

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confidence: 99%

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

Kaljot

Shaw

Rubin

et al. 1988

J Virol

181

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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

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“…In vitro, this is simulated by controlled incubation with trypsin. Trypsin activation is necessary for rapid internalization and infection (6,18); uncleaved rotavirus is internalized slowly and is not infectious (17,25). Trypsin cleaves the VP4 spike protein at two highly conserved sites (Arg-241 and Arg-247) to yield two peptides, VP8* (28 kDa) and VP5* (65 kDa), which remain attached to the outer capsid (16,29).…”

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confidence: 99%

Rotavirus-induced fusion from without in tissue culture cells

Falconer

Gilbert

Röper

et al. 1995

J Virol

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We present the first evidence of fusion from without induced in tissue culture cells by a nonenveloped virus. Electron micrographs of two strains of rotavirus, bovine rotavirus C486 and rhesus rotavirus, show that virally mediated cell-cell fusion occurs within 1 h postinfection. Trypsin activation is necessary for rotavirus to mediate cell-cell fusion. The extent of fusion is relative to the amount of virus used, and maximum fusion occurs between pHs 6.5 and 7.5. Fusion does not require virus-induced protein synthesis, as virus from both an empty capsid preparation and from an EDTA-treated preparation, which is noninfectious, can induce fusion. Incubation of rotavirus with neutralizing and nonneutralizing monoclonal antibodies before addition to cells indicates that viral protein 4 (VP4; in the form of VP5* and VP8*) and VP7 are involved in fusion. Light and electron micrographs document this fusion, including the formation of pores or channels between adjacent fused cells. These data support direct membrane penetration as a possible route of infection. Moreover, the assay should be useful in determining the mechanisms of cell entry by rotavirus.

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Effect of enzymes on rotavirus infectivity

Cited by 42 publications

References 11 publications

Isolation and characterization of a canine rotavirus

Isolation and characterization of a canine rotavirus

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

Rotavirus-induced fusion from without in tissue culture cells

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