MOISES MALLO, JOSE MARTINEZ-COSTAS, AND JAVIER BENAVENTE*. Departamento de Bioquimica y Biologia Molecular, Faciultad de Farmacia, 15706 ...
Vol. 65, No. 10
JOURNAL OF VIROLOGY, Oct. 1991, p. 5499-5505
0022-538X/91/105499-07$02.00/0 Copyright © 1991, American Society for Microbiology
Avian Reovirus S1133 Can Replicate in Mouse L Cells: Effect of pH and Cell Attachment Status on Viral Infection MOISES MALLO, JOSE MARTINEZ-COSTAS, AND JAVIER BENAVENTE* Departamento de Bioquimica y Biologia Molecular, Faciultad de Farmacia, 15706 Santiago de Compostela, Spain Received 29 January 1991/Accepted 3 July 1991
Previous reports have suggested that avian reovirus S1133 fails to replicate in mouse L cells. In this article, report that replication does occur under certain culture conditions. The avian reovirus was found to grow in mouse L cells at pH 6.4 and 7.2 but not at pH 8.2. Culture medium with a basic pH directly inhibited viral transcription and genome replication. As a result, viral protein synthesis was also affected. At permissive pH levels, avian reovirus grew better in monolayers than in suspension cultures of L cells because of the influence of cell attachment status on viral macromolecular synthesis. Our results not only show that avian reovirus can replicate in mouse L cells but also help to explain why it did not in previous studies. we
The members of the Orthoreovirus genus have been classified into two groups: those of mammalian origin and those of avian origin (13). Both groups of viruses have a genome consisting of 10 segments of double-stranded RNA (dsRNA), separable into three size classes, which are enclosed within a double protein capsid shell 70 to 80 nm in diameter with a similar protein composition and distribution (12, 21, 23). Their virions also contain the enzymes that catalyze the synthesis of capped and methylated viral transcripts (17). However, avian reoviruses differ from their well-studied mammalian counterparts by their lack of hemagglutinating activity (9), their ability to induce cell fusion (27), their possession of a different group-specific antigen (18, 20), and their different host range (20). Avian reoviruses can be readily grown in the laboratory in primary cultures of cells of avian origin (1, 11), but they fail to replicate in most of the established mammalian cell lines (1). Only Vero cells have been reported to be permissive hosts for the replication of certain strains of avian reovirus (1, 28; unpublished data). The nonpermissive infection of mouse L cells by the strain S1133 of avian reovirus was first investigated by Spandidos and Graham (24), who found that L cells cultured in suspension allow viral penetration and uncoating but no viral genome replication or progeny particle formation (24). Moreover, viral transcription in this heterologous system was limited to 4 of the 10 genomic segments, suggesting the existence of a cellular factor that inhibited the transcription of the other 6 avian reovirus RNA segments (24). Later, Benavente and Shatkin (2) showed that all viral transcripts are produced in the cytoplasm of avian reovirus-infected L cells. Therefore, the suggestion was made that viral replication is prevented at the initiation of translation (2), which is consistent with the absence of in vivo viral protein synthesis. We were interested in obtaining avian reovirus replication in L cells. Spandidos and Graham (24) referred to the existence of mutant viruses capable of growing in L cells, but to our knowledge, no study on these mutants has ever been reported. Since viable mutants were not available, we attempted an alternative approach by varying culture conditions of the infected cells and analyzing their influence on viral growth and macromolecular synthesis. Here, we present evidence that avian reovirus S1133 is able to pro*
Corresponding author.
ductively infect L cells and show the effects that the pH of the incubation medium and the attachment status of the cell have on this infection. MATERIALS AND METHODS
Cells and viruses. Mouse L cells were maintained in suspension in Joklik's modified minimal essential medium containing 5% calf serum. L-cell monolayers were obtained by plating suspension cells and replacing the suspension medium with Dulbecco's modified Eagle medium containing 5% calf serum. Primary cultures of chicken embryo fibroblasts (CEF) were prepared from 9- to 10-day-old chicken embryos and grown in medium 199 supplemented with 10% tryptose phosphate broth and 7.5% calf serum. Strain S1133 of avian reovirus (26) was grown in confluent monolayers of primary CEF. Conditions for growing, purifying, and titering the virus have been described previously (23). Virus infections. Monolayer cultures of L cells (106 cells per 35-mm plate) were washed with warm Puck's saline and incubated with 100 PFU of avian reovirus per cell at 37°C for 2 h. Then, virus input was removed, and after being washed with warm Puck's saline, the cells were incubated in the appropriate medium at 37°C. To infect suspension cultures, L cells were collected by low-speed centrifugation, washed with warm Puck's saline, and resuspended to 2 x 106 cells per ml in a viral suspension containing 100 PFU per cell. After 2 h of virus adsorption at 37°C, the cells were centrifuged, washed with warm Puck's saline, resuspended to 5 x 105 cells per ml in the corresponding incubation medium, and incubated at 37°C. We considered this moment to be time zero of infection. To keep the pH of the incubation media constant during the experiments and to use similar environmental conditions for suspension and monolayer cell cultures, we incubated infected cells maintained in either type of culture in Joklik's modified minimal essential medium without NaCO3H and buffered with 20 mM HEPES (N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid). Media were adjusted to the desired pH with NaOH and were supplemented with 1% calf serum. Infected monolayer cultures were incubated in an atmosphere lacking CO2. No differences in cell viability were observed when L cells were incubated in this medium or in Dulbecco's modified Eagle medium for at least 96 h. The pH of the media was always checked both at the 5499
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beginning and at the end of each experiment. Actinomycin D, at a concentration of 0.5 Fig/ml, was also added to the incubation media of infected cells to inhibit cellular RNA synthesis (22). Analysis of virus growth. At different times postinfection, cells and medium were collected (by scraping in the case of monolayer cultures) and were frozen and thawed three times. The suspension was clarified by low-speed centrifugation, and virus titer was determined by plaque assay on CEF (23). Analysis of protein synthesis. For suspension cultures, aliquots of 106 infected cells were collected by centrifugation, washed with warm Puck's saline, resuspended at the corresponding pH in 0.5 ml of methionine-free medium supplemented with 1% dialyzed calf serum and 100 ,uCi of L-[35S]methionine per ml, and incubated for 2 h at 37°C. Cells were then collected, washed with cold phosphatebuffered saline, and lysed with 80 [lI of cold lysis buffer (10 mM Tris-HCl [pH 8.6], 140 mM NaCl, 1.5 mM MgCl2, 0.5% Nonidet P-40) containing 1 mM phenylmethylsulfonyl fluoride. Nuclei were pelleted by centrifugation, and supernatants were analyzed by 13.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (14) and autoradiography. Monolayer cultures containing 106 infected cells in 35-mm plates were treated as described for suspension cultures except that the cells were lysed on the plate with 80 ,ul of lysis buffer, scraped off, and collected on ice. After clarification, cytoplasmic extracts were analyzed as described for suspension cultures. Analysis of RNA synthesis. Infected or mock-infected L cells incubated as above in either monolayer or suspension cultures were radiolabeled at different times postinfection by the addition of 20 ,uCi of [5,6-3H]uridine per ml and enough cold uridine to obtain a final uridine concentration of 2 puM. Infected CEF were labeled in a similar way except that Joklik's modified minimal essential medium was replaced by medium 199 and the actinomycin D concentration was lowered to 50 ng/ml. After the labeling period, cells were collected as described above and lysed with 80 pul (per 106 cells) of ice-cold lysis buffer containing 0.5% sodium deoxycholate. The extract was incubated at 0°C for 3 min with occasional vortexing. The postnuclear supernatant was extracted once with 1 volume of phenol and once with 1 volume of phenol-chloroform-isoamyl alcohol (25:24:1) (vol! vol). An aliquot of the aqueous phase was precipitated in 10% trichloroacetic acid (TCA), and the radioactivity was counted by liquid scintillation. To analyze dsRNA synthesis, we treated the aqueous phase with single-stranded RNases basically as described previously (15). Briefly, it was mixed with 10 volumes of RNase solution (40 pLg of RNase A per ml and 500 U of RNase T1 per ml in 10 mM Tris-HCl (pH 7.5)-5 mM EDTA-300 mM NaCl) and incubated at 30°C for 1 h. SDS and proteinase K were then added to a final concentration of 1% and 166 pug/ml, respectively, and further incubated for 10 min at 37°C. The digested sample was extracted once with 1 volume of phenol-chloroform-isoamyl alcohol (25:24:1) (vol/ vol), and the aqueous phase was made 0.3 M in sodium acetate (pH 5.5) and 80% in absolute ethanol and left at -20°C overnight. The precipitated RNA was collected by centrifugation, dried, and dissolved in water, and the TCAprecipitable radioactivity was counted. To obtain the pattern of the genomic dsRNA fragments, we labeled cells in vivo with [3H]uridine as described above for 12 h beginning at the onset of the infection. RNA was extracted, RNase treated,
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time postinfection (h) FIG. 1. One-step growth curves of avian reovirus S1133 in suspension and monolayer cultures of L cells at different pHs. Total virus production was determined at each time point by plaque assay on CEF. Symbols: 0, suspension cultures at pH 8.2; A, suspension cultures at pH 7.2; *, suspension cultures at pH 6.4; 0, monolayer cultures at pH 8.2; A, monolayer cultures at pH 7.2; O, monolayer cultures at pH 6.4.
and analyzed by electrophoresis on a 10% SDS-polyacrylamide gel (10) and fluorography (5). In vitro viral transcriptions. Avian reovirus cores were prepared and used for the synthesis of [3H]uridine-labeled viral mRNAs as described by Benavente and Shatkin (2). Incubation mixtures were buffered at different pHs with 70 mM Tris-HCl, and the incorporation of radioactivity into RNA was measured by TCA precipitation and liquid scintil-
lation counting. RESULTS
Effect of pH and cell attachment status on viral growth. In our effort to find suitable conditions for the replication of the avian reovirus S1133 in mouse L cells, we determined viral growth curves at pH 6.4, 7.2, and 8.2 for both suspension and monolayer cultures (Fig. 1). To our surprise, we found viral growth in both types of culture when incubations were done at pH 7.2 or 6.4 but not at pH 8.2. The shape of the curve and the final viral yield at pH 7.2 and 6.4 depended on cell attachment status. Viral growth was already detected by 8 h postinfection in monolayers but not in spinner cultures, and the maximum viral yield was 30 times higher in the
former. At pH 8.2, virus titer decreased with increasing incubation times in either type of culture. These results revealed that avian reovirus can grow in L cells and that viral growth is influenced by the pH and the attachment status of the cultured cells. Effect of pH and cell attachment status on viral protein synthesis. Benavente and Shatkin (2) were unable to detect in vivo viral protein synthesis in suspension cultures of avian reovirus-infected L cells; in that work, however, viral growth was not directly analyzed. Since viral growth without concomitant detection of viral proteins by in vivo metabolic labeling has been reported for another virus-cell system (19), we analyzed the pattern of protein synthesis in suspension and monolayer cultures at different pHs. Infected L cells were labeled with [35Slmethionine at 16 h postinfection,
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