Jan 4, 1988 - The time course of defective interfering (DI) particleand B particle release from vesicular stomatitis virus-infected BHK-21 cells was studied at ...
JOURNAL OF VIROLOGY, Apr. 1988, p. 1323-1329 0022-538X/88/041323-07$02.00/0 Copyright © 1988, American Society for Microbiology
Vol. 62, No. 4
Delayed Formation of Defective Interfering Particles in Vesicular Stomatitis Virus-Infected Cells: Kinetic Studies of Viral Protein and RNA Synthesis during Autointerference DOROTHEE M. VON LAER, DIETRICH MACK, AND JOACHIM KRUPPA* Department of Molecular Biology, Institute of Physiological Chemistry, University of Hamburg, D 2000 Hamburg 13,
Federal Republic
of Germany
Received 20 May 1987/Accepted 4 January 1988
The time course of defective interfering (DI) particle and B particle release from vesicular stomatitis virus-infected BHK-21 cells was studied at different multiplicities of defective and infective particles. Particle release was progressively delayed in cells infected with an increasing DI-to-B particle ratio. The delayed particle release during interference was found to be connected with a reduced but prolonged synthesis of viral proteins, a slower accumulation of viral proteins, and a delayed shutoff of cellular protein synthesis. The relative synthesis of M and G proteins was reduced during interference, whereas the relative synthesis of N and NS proteins was increased. On the level of genomic RNA replication, we found that DI RNA was replicated more slowly during interference than the standard genomic RNA was during acute infection. The ratio of DI particles to B particles which were released increased throughout the infectious cycle. At a given time in the infectious cycle, this ratio was independent of the multiplicity of infecting DI and B particles. On the basis of the kinetic studies, we argue that cells infected with higher amounts of DI particles compared with B particles synthesize a higher DI-to-B particle ratio and release these progeny particles later than cells infected with a low DI-to-B particle ratio.
Serial passages of many viruses at high multiplicity lead to the enrichment of defective or incomplete virus particles which interfere with the replication of the homologous virus (B particle). These defective interfering (DI) particles are unable to replicate in a host cell by themselves and require the presence of homologous complete virus (17, 23). DI particles of vesicular stomatitis virus (VSV) are generally smaller than the complete virus and normally do not code for any viral protein (4, 24). VSV DI particles inhibit the amplification of the complete virus on the level of RNA replication (12). Like many other DI particles of normally cytocidal viruses, DI particles of VSV facilitate the establishment of persistent virus infections in cultured cells at high levels of interference (7, 8). At low levels of interference, the infectious cycle of VSV is merely prolonged with a selective 6- to 8-h delay of DI particle release (15). B particles are not released significantly more slowly during low levels of interference (13). To explain these data Lazzarini et al. (17) proposed that the assembly of DI particles is selectively inhibited. In this report, we attempted to elucidate the steps in macromolecular synthesis of viral constituents which cause the selective delay of DI particle release. We therefore compared the time courses of DI and B particle release and studied the kinetics of protein synthesis, as well as the kinetics of genomic RNA replication during acute infection and during autointerference at various multiplicities of DI particles. Viral RNA and protein synthesis were retarded during interference. As the multiplicities of DI particles were increased, the cycle of VSV infection was progressively
*
Corresponding author.
prolonged. These conditions may facilitate virus persistence at a very high level of interference. MATERIALS AND METHODS
Cellular and viral growth. BHK-21 cells were grown as monolayers in Joklik minimal essential medium (Flow Laboratories, Inc., Bonn, Federal Republic of Germany) supplemented with 5% (vol/vol) calf serum (Seromed, Berlin, Federal Republic of Germany). The San Juan strain of VSV (serotype Indiana, obtained from J. K. Rose, San Diego, Calif.) was grown and purified as described previously (6). The seed stock of defective particles (DI 0.52) was obtained from a fourth high multiplicity passage of VSV in BHK-21 cells. The supernatants were harvested, and the DI particles were separated from the infective particles by velocity sedimentation in sucrose gradients as described earlier (9). The DI particle band was collected and diluted with 2 volumes of water, and the defective particles were recovered by sedimentation at 120,000 x g for 2 h at 4°C. The pellet was suspended in 20 mM Tris hydrochloride (pH 7.5) and stored in small portions at -70°C. The concentration of DI particles was determined by a plaque reduction assay, as described earlier (25). Metabolic labeling of VSV-infected cells. Confluent monolayers of BHK-21 cells (1.5 x 107 cells) were infected with the indicated multiplicities of defective and infective particles in serum-free medium. After 30 min of adsorption at room temperature, the inoculum was removed, and medium containing 5% (vol/vol) calf serum was added. The infection was allowed to proceed at 37°C. At the times indicated, monolayers were washed once with prewarmed phosphatebuffered saline and labeled with 30 ,uCi of [35S]methionine in 2 ml of methionine-free medium for 15 min. The concentration of intracellular viral protein, which accumulated during infection, was determined by continu1323
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ous labeling with [35S]methionine. Cells were infected as described above and incubated for 1 h at 37°C. [35S]methionine (30 ,uCi) was added, and the cells were labeled for various incubation periods. At the times indicated, cells were lysed and cytoplasmic extracts were prepared. For the preparation of [3H]uridine-labeled nucleocapsids, VSV-infected BHK-21 cells were incubated for 30 min with actinomycin D (1 ,ug/ml) and then labeled with [3H]uridine (10 ,uCi/ml) for 2 h before the cytoplasmic extract was prepared. Intracellular nucleocapsid species were analyzed in 10 to 30% sucrose gradients, as described previously (15). Preparation of cytoplasmic extracts. Infected and radioactively labeled monolayers (1.5 x 107 cells) were washed twice in ice-cold phosphate-buffered saline. Cells were scraped off, collected by low-speed centrifugation, and allowed to swell for 10 min in 500 pI of hypotonic buffer (0.01 M Tris hydrochloride, 0.01 M NaCl, 0.02 M EDTA [pH 7.3]). Triton X-100 was added to a final concentration of 1%, and the cytoplasmic extracts were freed of nuclei by centrifugation at 800 x g for 5 min at 4°C (5). Immunoprecipitation of viral proteins. Antisera against purified N protein and virus particles were raised in rabbits. For immunoprecipitation of cell extracts, the Staphylococcus aureus procedure (14) was used. The cytoplasmic extracts were adjusted to 1% sodium dodecyl sulfate (SDS) and heated to 95°C for 5 min. The samples were diluted with 10 volumes of TNET buffer (140 mM NaCl, 20 mM Tris hydrochloride [pH 7.5], 5 mM EDTA, 2 mM methionine, 1% Triton X-100). Saturating amounts of antisera were added, and the samples were kept on ice for 18 h. The antigenantibody complexes were recovered with 50 pul of a 10% (wt/vol) solution of S. aureus. Polyacrylamide gel electrophoresis. Protein compositions in cytoplasmic extracts were analyzed on 10% polyacrylamide gels by using the discontinuous buffer system of Laemmli (16). Electrophoretic separation was performed under reducing conditions at 15 mA (constant current) for 11 h. Gels were fixed in 10% (vol/vol) acetic acid-40% (vol/vol) methanol-50% (vol/vol) water, impregnated with 1 M sodium salicylate (3), vacuum dried, and exposed on Cronex 4 X-ray film (Dupont Co., Wilmington, Del.) at -70°C. Separation and estimation of released infective and defective particles. BHK-21 cells were coinfected with purified DI particles and standard virus, as described above. At the indicated times postinfection, the released particles were harvested and the culture medium was renewed. Cell debris was removed from the supernatants by centrifugation at 10,000 x g for 10 min, and the particles were recovered by centrifugation at 120,000 x g for 1 h. The pellet was suspended in 500 p.1 of TEN (100 mM NaCl, 10 mM Tris hydrochloride [pH 7.5], 1 mM EDTA) buffer and carefully layered on to a 5 to 40% (wt/vol) sucrose gradient in TEN. The infective and defective particles were separated by velocity sedimentation at 35,000 rpm for 35 min at 4°C in a Beckman SW41 rotor (Beckman Instruments, Inc., Fullerton, Calif.). Particle yields were determined spectrophotometrically at 254 nm by collecting the gradients continuously from the top with an ISCO absorbance monitor. DI and B particle yields were quantitated by weighing the peaks (10, 11). The amounts represent relative values whereby 10 relative units correspond to an optical density of 0.06. Materials. Proteinase K and RNase A and T1 were purchased from Boehringer (Mannheim, Federal Republic of Germany). [35S]methionine (specific activity, 1,440 Ci/mmol) and [3H]uridine (specific activity, 41 Ci/mmol) were obtained from Amersham-Buchler (Braunschweig, Federal Republic
of Germany). Actinomycin D was from Serva (Heidelberg, Federal Republic of Germany). RESULTS Time course of infective and defective particle release at different multiplicities of infection. Khan and Lazzarini (15) reported that during autointerference, BHK-21 cells release defective particles of VSV more slowly than standard virus. Since the time course of DI and B particle release was studied only at relatively low interference levels (approximately 3 IFU per cell [15]), we followed the kinetics of particle release after coinfection with various multiplicities of DI particles. Additionally, the effect of a comparatively high multiplicity of B particles on the kinetics of DI and B particle release was examined. Progeny particles were harvested at hourly intervals and then separated into DI and B particles on sucrose gradients and quantified as described in Materials and Methods (Fig. 1). Figure 1B and C shows that at low interference levels, B particles were released earlier than DI particles. At higher interference levels (Fig. 1D), B and DI particles were released almost simultaneously between 10 and 14 h postinfection.
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