Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles - PubMed (original) (raw)
Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles
M N Teng et al. J Virol. 1998 Jul.
Abstract
We developed a system to identify the viral proteins required for the packaging and passage of human respiratory syncytial virus (RSV) by reconstructing these events with cDNA-encoded components. Plasmids encoding individual RSV proteins, each under the control of a T7 promoter, were cotransfected in various combinations together with a plasmid containing a minigenome into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Supernatants from these cells were passaged onto fresh cells which were then superinfected with RSV. Functional reconstitution of RSV-specific packaging and passage was detected by expression of the reporter gene carried on the minigenome. As expected, the four nucleocapsid proteins N, P, L, and M2-1 failed to direct packaging and passage of the minigenome. Passage was achieved by further addition of plasmids expressing three membrane-associated proteins, M, G, and F; inclusion of the fourth envelope- associated protein, SH, did not alter passage efficiency. Passage was reduced 10- to 20-fold by omission of G and was abrogated by omission of either M or F. Coexpression of the nonstructural NS1 or NS2 protein had little effect on packaging and passage except through indirect effects on RNA synthesis in the initial transfection. The M2-1 transcription elongation factor was not required for the generation of passage-competent particles. However, addition of increasing quantities of M2-1 to the transfection mediated a dose-dependent inhibition of passage which was alleviated by coexpression of the putative negative regulatory factor M2-2. Omission of the L plasmid reduced passage 10- to 20-fold, most likely due to reduced availability of encapsidated minigenomes for packaging. However, the residual level of passage indicated that neither L protein nor the process of RSV-specific RNA synthesis is required for the production and passage of particles. Omission of N or P from the transfection abrogated passage. Thus, the minimum RSV protein requirements for packaging and passaging a minigenome are N, P, M, and F, although the efficiency is greatly increased by addition of L and G.
Figures
FIG. 1
Reconstitution of packaging and passage from plasmid-encoded minigenome RNA and protein. Plasmids encoding RSV proteins and a minigenome analog (as indicated), each under the control of a T7 promoter, were cotransfected into HEp-2 cells which were concomitantly infected with vaccinia virus expressing T7 RNA polymerase (MVA-T7) (MOI, ∼3). The minigenome analog contains a reporter gene (encoding either luciferase [C2L] or GFP [C41-GFP]) whose expression is controlled by RSV transcription signals. At 72 h posttransfection, culture supernatants (sup) were harvested and passaged onto fresh cells which were subsequently infected with RSV (MOI, ∼3). Passage and expression of minigenomes were assessed by either luciferase assay or fluorescence microscopy (at 24 or 48 h, respectively, after passage).
FIG. 2
Passage of minigenomes detected by luciferase activity. HEp-2 cells were transfected as shown in Fig. 1 with the plasmids encoding the N, P, M2-1, and L proteins, the minigenome (C2L), and plasmids encoding the indicated additional structural proteins (0.1 μg/well). Luciferase assays were performed on passage cell extracts. Bars represent averages from 2 experiments and are normalized to the passage of samples transfected only with the minigenome and N, P, M2-1, and L proteins.
FIG. 3
Effect of titration of F, M, and G plasmids in transfection on the efficiency of passage. HEp-2 cells were transfected with the N, P, M2-1, L, and C2L plasmids as described for Fig. 2. (a) Luciferase activity after passage of supernatants from transfections with increasing amounts of M plasmid in the presence of a constant amount of F plasmid. (b) Effects of increasing G plasmid while keeping M and F constant. (c and d) Increasing amounts of F plasmid were cotransfected with constant amounts of M (c) or M and G (d). Bars represent averages for two samples, normalized within each group to passage of samples in which the transfections contained 0.1 μg of M and F plasmids per well (bars 3, 7, and 15) or 0.1 μg of M and G plasmids per well (bar 19). Note the difference in scale for samples with G (b and d) or without G (a and c). Panels a to c are derived from a single experiment; panel d shows the results of an independent experiment.
FIG. 4
Passage of a minigenome that expresses GFP, detected by fluorescence microscopy. HEp-2 cells were transfected as described for Fig. 2 except with C41-GFP as the minigenome. N, P, M2-1, and L plasmids were coexpressed in the transfection either alone (top panels) or in combination with the F, M, and G plasmids (middle panels) or the F, M, G, and SH plasmids (bottom panels). Photomicrographs (magnification, ×34) were taken at 3 days posttransfection (left panels) or 2 days postpassage (right panels). Shown at the middle and bottom left are syncytia resulting from coexpression of F and G.
FIG. 5
Effect of M2-1 on passage. HEp-2 cells were transfected with the N, P, L, F, M, G, and minigenome C2L plasmids plus the M2-1 (a and b) or M2(1+2) (c and d) plasmid as indicated. Luciferase activities at 72 h posttransfection (a and c) or 24 h postpassage (b and d) were measured as described for Fig. 2. Bars indicate averages for two samples, normalized within each group to samples derived from transfections containing no additional M2 plasmid (bars 3).
FIG. 6
Effect of M2-2 on passage. HEp-2 cells were transfected with the N, P, L, F, M, G, and minigenome C2L plasmids together with increasing amounts of M2-2 plasmid in the absence (a and b) or presence (c and d) of M2-1 plasmid (200 ng/well) as indicated. Luciferase activities at 72 h posttransfection (a and c) or 24 h postpassage (b and d) were measured as described for Fig. 2. Bars indicate averages for two samples, normalized to samples derived from transfections containing no M2-2 plasmid (bars 2 and 11). Luciferase activities in panel c (as measured in light units) were approximately 100-fold greater than those in panel a due to the presence of M2-1; luciferase expressions in panels b and d were comparable.
FIG. 7
Coexpression of M2-2 overcomes inhibition of passage by M2-1. HEp-2 cells were transfected with the N, P, L, F, M, G, and minigenome C2L plasmids together with increasing amounts of M2-1 plasmid in the presence of additional M2-2 plasmid (20 ng/well) as indicated. Luciferase activities at 72 h posttransfection (a) or 24 h postpassage (b) were measured as described for Fig. 2. Bars indicate averages for two samples, normalized to samples derived from transfections containing neither M2 plasmid (bars 1).
FIG. 8
Effect of NS1 and NS2 on passage. HEp-2 cells were transfected with the N, P, M2-1, L, F, M, G, and minigenome C2L plasmids together with the indicated amounts of NS1 (bars 4 to 8) or NS2 (bars 9 to 13) plasmid. Luciferase activity was measured 72 h after transfection (a) or 24 h after passage (b) as described for Fig. 2. Bars indicate averages for two samples, normalized to controls from transfections containing no NS1 or NS2 (bars 3).
FIG. 9
Effect of L on passage. HEp-2 cells were transfected with the N, P, F, M, G, and minigenome C2L plasmids (bars 2 to 15) as described for Fig. 2. The plasmid encoding the L (bar 3), NS1 (bars 4 to 6), NS2 (bars 7 to 9), M2-1 (bars 10 to 12), or M2-2 (bars 13 to 15) protein was added to the transfection mixture in the indicated amounts. A negative control lacked G, F, and M plasmids (bar 1). Luciferase activities at 24 h postpassage were measured. Bars indicate averages for two samples normalized to the passage of supernatants from transfections containing no additional plasmids (bar 2).
References
- Collins P L, McIntosh K, Chanock R M. Respiratory syncytial virus. In: Fields B N, Knipe D M, Howley P M, Chanock R M, Melnick J L, Monath T P, Roizman B, Straus S E, editors. Fields virology. 3rd ed. Vol. 2. Philadelphia, Pa: Lippincott-Raven; 1996. pp. 1313–1352.
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