JOURNAL OF VIROLOGY, Oct. 1978, p. 199-211 0022-538X/78/0028-0199$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 28, No. 1 Printed in U.S.A.

Initiation and Maintenance of Persistent Infection by Respiratory Syncytial Virus C. R. PRINGLE,* P. V. SHIRODARIA,t P. CASH, D. J. CHISWELL, AND P. MALLOY Medical Research Council Virology Unit, Institute of Virology, Glasgow Gll 5JR, Scotland Received for publication 23 March 1978

Propagation of cells infected with temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus at nonpermissive temperature (3900) resulted in cytolytic, abortive, or persistent infection, depending on the mutant used to initiate infection. Five mutants from complementation group B produced cytolytic or abortive infections, whereas a single mutant (tsl) from group D and a noncomplementing mutant produced persistent infections. The persistently infected culture initiated by mutant tsl (RS tsl/BS-C-1) has been maintained in serial culture for >100 transfers, and infectious-center assays and immunofluorescent staining indicated that all cells harbored the RS virus genome. RS tsl/BSC-1 cultures were resistant to superinfection by homologous and some heterologous viruses, and interferon-like activity against some heterologous viruses was present in the culture medium. Small amounts (0.002 to 0.2 PFU/cell) of infectious virus were present in the culture fluid, but autointerfering defective particles were not detected. This released virus formed small plaques and produced persistent infection of BS-C-1 cells at 370C. The RS tsl/BS-C-1 cells contained abundant RS virus antigen internally, but little at the surface, although the cells showed enhanced agglutinability by concanavalin A. Nucleocapsids and the 41,000-molecular-weight nucleoprotein were present in extracts of both nucleated and enucleated cells. No infectious RS virus was obtained by transfection of DNA from RS tsl/BS-C-1 cells to susceptible BS-C-1 or feline embryo cells under conditions allowing efficient transfection of a foamy virus proviral DNA. It was concluded that persistent infection was maintained in part by a non-ts variant of RS virus partially defective in maturation. The karyotype of the RS tsl/BS-C-1 culture differed from that of uninfected cells.

Infection of cultures of BS-C-1 cells by respiratory syncytial (RS) virus results in production and release of infectious virus, with the eventual disintegration and death of the host cell. In the course of infection, the surface of the cell becomes drastically modified by the appearance of a profusion of slender processes, which have been visualized both by scanning electron microscopy and by immunofluorescent staining. These processes are virus specific because they were not present when certain temperature-sensitive (ts) mutants of RS virus were incubated at the restrictive temperature (4). This communication describes the establishment of persistent infection of BS-C-1 cells by propagation of ts mutant-infected cells at the restrictive temperature. The establishment of persistent infection appeared to be mutant specific in that stable persistence was achieved with a mutant classified in complementation group D and not with t Department of Microbiology and Immunobiology, Queen's University, Belfast BT12 6BN, Northern Ireland. 199

any of five mutants from group B. All cells in the persistently infected cultures expressed RS virus antigen, but little infectious virus was released and the formation of surface processes was greatly diminished. Previously, persistent infection of HeLa cells (25, 30) and calf kidney cells (25) by RS virus has been described. The persistent infection of HeLa cells described by Simpson and Iinuma was established with wild-type virus, and the cultures continued to produce low yields of virus. On the other hand, the persistent infection of bovine cells was initiated with a ts mutant, and this culture did not release infectious virus, except after induction by iododeoxyuridine treatment or after cocultivation with susceptible cells. In both cases RS virus could be recovered from persistently infected cultures by transfection of DNA from these cultures to uninfected cells, which indicated that the persistent infection of these cells by RS virus (usually considered to be a conventional cytoplasmic RNA virus) was maintained by the formation of a



DNA provirus, possibly integrated into or replicating in unison with the chromatin of the host cell. Yankevich and Dreizin (30) established a persistent infection of HeLa cells by passage of infected cells in the presence of specific antiserum. Infectious virus was not released after passage 3 and, unlike the previous case, could not be induced subsequently by iododeoxyuridine treatment or cocultivation. However, RS virus antigen persisted in a constant proportion of the cells during 18 subsequent transfers, even after cell cloning. Moreover, the proportion of cells expressing viral antigen was increased after iododeoxyuridine treatment. It was concluded that the persistent infection reflected integration of at least part of the viral genome. In our study of persistent infection of BS-C-1 cells by RS virus, we looked for evidence of integration by using a sensitive DNA transfection method developed for assay of the proviral DNA of a feline foamy virus (3). Contrary to these previous results, we conclude that the fornation of proviral DNA is not essential for establishment or maintenance of persistent infection. MATERLALS AND METHODS Virus and cells. The origin and characteristics of the RSN-2 strain of human RS virus and its ts mutants have been described (4). The other pneumoviruses used were the BRS-127 strain of bovine RS virus and murine pneumonia virus (PVM), both of which had been cloned by several serial isolations from single plaques (2). Batai and Bunyamwera viruses were obtained from N. Karabatsos, Center for Disease Control, Fort Collins, Colo., and cloned by three sequential isolations from individual plaques. Vaccinia virus was provided by T. H. Pennington, Virology Department, University of Glasgow, and the other virusespoliovirus type 3, measles-SSPE (Halle strain), Sindbis, pseudorabies, and vesicular stomatitis-were all cloned laboratory strains as previously described (6). The infectivity of these viruses was assayed under 0.9% agar overlay (or 0.6% agarose overlay in the case of Bunyamwera and Batai viruses). Feline syncytiumforming virus (FSFV) was grown and assayed in subconfluent monolayers of feline embryo (FEA) cells as described by Chiswell and Pringle (3). The BS-C-1 and HeLa cell lines were obtained from J. F. Williams, Institute of Virology; XC cells were obtained from W. P. Rowe, National Institutes of Health, Bethesda, Md.; NIH/3T3 cells were obtained from S. A. Aaronson, Hazelton Laboratories, Maryland; and FEA cells were obtained from 0. Jarrett, Veterinary Pathology Department, University of Glasgow. All these cells were propagated in roller bottles in Eagle minimal essential medium supplemented with 5% fetal calf serum. Polyacrylamide gel electrophoresis of intracellular polypeptides. Confluent monolayers of RS


tsl/BS-C-1 or RS tsl8/BS-C-1 cells were incubated for 2 h in Eagle medium containing 2.5 ytg of actinomycin D (Cosmegen; Merck Sharp & Dohme, Rahway, N.J.) per ml and low serum (2% fetal calf serum; Biocult Laboratories, Glasgow, Scotland). This medium was replaced with fresh Eagle medium with the same additives and containing 5 uCi of L-[355]methionine (200 to 300 Ci/mmol; Radiochemical Centre, Amersham, England) per ml. The cultures were incubated at 370C for 24 h, and then the cells were scraped into suspension, chilled, and sedimented by centrifugation at 3,000 rpm for 30 min in an M.S.E. Mistral centrifuge at 40C. The cell pellet was washed twice with high-salt buffer (0.18 M NaCl-0.01 M Tris-hydrochloride, pH 7.4) and then lysed in 6 ml of 0.5% Nonidet P-40 in 0.01 M NaCl (pH 7.4)-0.1 M EDTA on ice for 10 min. The cell extract was treated with 2% (wt/vol) sodium dodecyl sulfate (SDS)-5% (vol/vol) 2-mercaptoethanol in 0.14 M Tris-hydrochloride (pH 6.7)-10% (vol/vol) glycerol (final concentration), and bromophenol blue was added as a tracking dye. Discontinuous SDS-polyacrylamide gel electrophoresis was carried out by using gradient slab gels of 6 to 15% total acrylamide which contained 0.1% (wt/vol) SDS and a constant proportion (5%, wt/wt) of N,N'-methylenebisacrylamide. Fluorographs were prepared as described previously (2). Isolation of RS virus nucleocapsid. L-['S]methionine-labeled RS tsl/BS-C-1 cells were scraped into high-salt buffer (10 mM Tris-hydrochloride, pH 7.4, 0.18 M NaCl). The cells were washed twice in high-salt buffer and then once in hypotonic buffer (10 mM Tris-hydrochloride, pH 7.4, 0.1 mM EDTA) and allowed to swell for 20 min at 40C in 6 ml of hypotonic buffer containing 0.5% Triton X-100. The cells were then disrupted by gentle homogenization with a handoperated Teflon pestle. NaCl was added to 0.1 M, and nuclei and cell debris were sedimented at 600 x g for 3 min. This post-nuclear supernatant was applied to a 15 to 45% metrizamide gradient containing 0.5% Triton X-100 in 0.1 M NaCl and centrifuged for 6 h in a Beckman SW65 titanium rotor at 145,000 x g (40,000 rpm) and 40C. Fractions were collected from the bottom of the gradient, and radioactivity and density were measured as described previously (29). UW inactivation. The culture fluid from RS virus cultures was clarified by low-speed centrifugation (1,000 rpm for 5 min at 40C in an M.S.E. Mistral centrifuge) and then irradiated. Amounts of 1 ml were exposed in 50-mm petri dishes at 40 cm from a Hanovia germicidal lamp. The total incident radiation was approximately 3,000 ergs/cm2, a dose sufficient to reduce RS virus infectivity by >5 log1o units. Infectious-center assay. A single cell suspension of RS tsl/BS-C-1 cells was prepared by trypsinization of newly confluent monolayers, and the cells were suspended in Eagle medium containing 10% fetal calf serum and anti-RS virus serum. The antiserum was bovine anti-bovine RS virus at a final dilution of 1:100, a gift from J. Stott, Agricultural Research Council, Animal Diseases Research Institute, Compton, England. The cell suspension was held at room temperature for 30 min with gentle agitation, a treatment which was sufficient to reduce cell-free RS virus infec-

VOL. 28, 1978

tivity by more than 3 logio units. The RS tsl/BS-C-1 cells were then diluted in 10-fold steps into tubes containing 106 uninfected BS-C-1 cells in Eagle medium and 10% fetal calf serum. The cell suspensions were carefully mixed and dispensed into 50-mm plastic petri dishes, which were incubated for 10 to 12 h to allow attachment to take place. The liquid medium was then replaced with an 0.9% agar-containing overlay, and the cultures were incubated at 310C for 7 days. The cultures were fixed with formol saline and stained with Giemsa stain. Plaques were counted under a low-power binocular microscope. Interferon assay. Interferon activity in RS tsl/ BS-C-1 culture fluids was assayed by first inactivating infectivity by exposure at 560C for 1 h or at pH 2.0 for 16 h. The inactivated medium was diluted into Eagle medium containing 2% fetal calf serum in threefold dilution steps and then added to uninfected monolayers of 106 BS-C-1 cells in 50-mm plastic petri dishes. Three plates at each dilution were incubated for 18 h at 31°C. The medium was then discarded, and approximately 50 to 100 PFU of the challenge virus was added. An overlay of 0.9% agar (or 0.6% agarose in the case of Bunyamwera virus) was added after a 30-min absorption at 310C. The plates were fixed and stained after various periods of incubation (see Table 2). Extraction and assay of infectious DNA. DNA was extracted from cells by a modification of the method of Marmur (16). Cells were suspended in NTE buffer (0.02 M Tris-hydrochloride, pH 7.4,0.2 M NaCl, 0.001 M EDTA), and boiled protease VI (Sigma-London, Ltd., London, England) and SDS were added to give final concentrations of 500 fg/ml and 0.5%, respectively. After 2 h at 370C, the digest was extracted by gentle mixing twice with phenol saturated with NTE buffer and twice with chloroform-isoamyl alcohol (24:1). The extract was dialyzed overnight against 0.1x SSC (0.15 M NaCl, 0.015 M sodium citrate, 0.015 M sodium acetate, pH 7.5), and then 50 jig of RNase A (Sigma-London, Ltd.; boiled for 3 min) per ml was added and the mixture was incubated for 1 to 3 h at 370C. A 1/20-volume of 20x NTE buffer was added, and the digest was extracted twice with NTE buffersaturated phenol, followed by two to four chloroformisoamyl extractions until protein was no longer visible at the interface. The extract was dialyzed against 0.lx SSC and stored at -20°C. DNA transfection was carried out by the modified calcium phosphate precipitation method as described by Chiswell and Pringle (3) for FSFV proviral DNA. DNA was diluted into a buffer containing 0.02 M N-2hydroxyethyl piperazine-N'-2-ethanesulfonic acid, 0.02 M NaCl, 0.005 M KCl, 0.0005 M Na2HPO4, and 0.006 M glucose and precipitated by addition of 2 M calcium chloride to give a final concentration of 125 mM. A 200-pl quantity was inoculated onto confluent (or subconfluent in the case of FSFV DNA) monolayers of BS-C-1 cells, and 2 ml of Eagle medium with 10% fetal calf serum was added after incubation for 40 min at 370C. After 4 h this medium was removed, and 1 ml of 15% dimethyl sulfoxide was added for 4 min at room temperature. The dimethyl sulfoxide was removed, and the monolayers were washed carefully with incubation medium and then incubated at 370C for 4 to 5 days. In some experiments an equal amount



of calf thymus DNA (Sigma-London, Ltd.) was added as carrier DNA with the inoculum DNA. DNA concentrations were measured by the mithramycin-binding method of Hill and Whately (11). Enucleation. Monolayers of RS tsl/BS-C-1 cells and normal BS-C-1 cells were grown on 30-mm-diameter plastic petri dishes and enucleated in situ by the cytochalasin-centrifugation method described by Follett (5). After cytochalasin treatment and centrifugation, the enucleated monolayers were incubated at 310C for 1 h before addition of radioactive isotope as described above. The efficiency of enucleation was approximately 95%. Fluorescent-antibody technique. Bovine antibovine RS virus serum, a convalescent serum from a child with RS virus infection, control sera, and appropriate fluorescein-conjugated antiglobulins were used in the indirect immunofluorescence technique as described previously (4). Acetone-fixed cells and unfixed cells on cover slips were used to detect intracellular and surface antigens, respectively. The stained preparations were examined under UV light with a Leitz Ortholux microscope fitted with combined fluorescence and phase-contrast attachments. Karyotype analysis. Monolayers were exposed to 0.05 to 0.1 mg of colcemid (Ciba Laboratories Ltd., Horsham, Sussex, England) per ml for 1 to 1.5 h, and the cells were suspended in 0.075 M KCl for 5 min before fixation with methanol-acetic acid (3:1). After 24 h at 40C, the cell suspension was dropped from a height of 12 inches (ca. 30.5 cm) onto a 45°-inclined slide and dried before Giemsa staining. Electron microscopy. Cell lysates were prepared for electron microscopy by the method of Zakstelskaya et al. (34) and negative stained with 2% uranyl acetate.

RESULTS Establishment of persistent infection. Monolayers of BS-C-1 or HeLa cells were infected with seven different ts mutants of the RSN-2 strain of RS virus and incubated at 390C for up to 15 days. No cytopathic changes were observed; the monolayers were then trypsinized, and half the cells were transferred to a fresh culture flask. These cultures were in turn incubated at 390C for 3 to 4 days and again transferred. Serial transfers in this manner were continued as long as possible. The outcome of infection varied, and the results of 12 experiments with these seven mutants are summarized in Table 1. The seven mutants were chosen to represent the three types ofimmunofluorescence described by Faulkner et al. (4). Mutants belonging to types I and III exhibited a qualitative and/or quantitative reduction in antigen synthesis at 390C. Subsequently, it was established that the five type I and III mutants used in these experiments all belonged to complementation group B (6a). Infection with these mutants resulted in either abortive infection or complete cell lysis due to the appearance of wild-type virus by reversion.




TABLE 1. Characteristics of the mutants used in attempts to establish persistent infection Mutant (RSN-2 strain)



Complementation groupb

tsl ts9



BS-C-1 BS-C-1

Stable persistent infection




BS-C-1 BS-C-1 and HeLa BS-C-1 and HeLa

Abortive infection



BS-C-1 BS-C-1 BS-C-1 and HeLa BS-C-1

Lysis due to reversion

tsl5 tsl8 tsl5 tsl6 tsl7 tsl8 See reference 4. bSee reference 6a.

Host cel

Outcome of infection


Mutants belonging to type II, on the other hand, exhibited no reduction in antigen synthesis at 39°C, and persistent infections were established successfully with both of the mutants in this class. One mutant (tsl) was subsequently shown to belong to complementation group D, whereas the other (ts9) failed to complement with any of the seven complementation groups of RS virus (6a). The pattern of fluorescence in fixed and unfixed BS-C-1 cells at 48 h after infection with mutant tsl at both 31 and 390C is illustrated in Fig. la, b, c, and d. Cultures infected with mutants ts15 and tsl8 on two occasions were destroyed by the appearance of revertants, whereas on other occasions infection was apparently abortive and the cultures were cured. The characteristics of one of these abortively infected cultures (RS ts18/BSC-1) will be compared with those of one of the persistently infected cultures (RS tsl/BS-C-1). Culture RS ts18/BS-C-1 was initiated by infection with mutant tsl8 at a nominal multiplicity of 1.5 PFU/ml. Infectious virus disappeared rapidly from cultures maintained at 39°C, and by the second transfer virus was demonstrable only after shift-down to permissive temperature (31°C). Virus was not detectable (

Initiation and maintenance of persistent infection by respiratory syncytial virus.

JOURNAL OF VIROLOGY, Oct. 1978, p. 199-211 0022-538X/78/0028-0199$02.00/0 Copyright © 1978 American Society for Microbiology Vol. 28, No. 1 Printed i...
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