Biogenesis of the Semliki Forest virus RNA replication complex

Abstract

The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326-338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.

FIG. 1

Confocal fluorescence images of SFV-infected BHK cells stained for nsP2 at 6 h p.i. (A), nsP3 at 6 h p.i. (B), and nsP4 at 4 h p.i. (C), double-stained for nsP1 (green) and nsP3 (red) at 4 h p.i. (D), double-stained for nsP1 (green) and nsP2 (red) at 4 h p.i. (E), and stained for nsP1 (red) and nsP4 (green) at 4 h p.i. (F). (G and H) Mock-infected BHK cells treated with BrdUTP for 10 min at 4 h p.i., followed by detection with anti-BrdU antiserum (green) in the absence (G) and presence (H) of 5 μg of actinomycin D/ml. (I) SFV-infected cell labeled with BrdUTP for 10 min in the presence of actinomycin D at 4 h p.i. and stained for BrdU (green) and nsP1 (red). (J, K, and L) Time course of SFV infection of BHK cells double labeled with anti-nsP1 (red) and anti-nsP3 (green) at 2 (J), 4 (K), and 5 (L) h p.i. Bars, 10 μm. Infection and further incubation were done at 37°C.

FIG. 2

Immunolocalization of ns proteins in CPV structures. (A) Epon section of BSA-gold-labeled (asterisk) CPV at 5 h p.i. (B) Preembedded Epon section of double-labeled CPV: 10-nm-diameter gold particles detecting pulse-labeled BrdU-RNA and 5-nm-diameter gold particles detecting nsP3 better visualized in the enlarged inset. Cryoimmuno-EM images of gold-protein A labeling are shown. (C) Anti-nsP4 alone detected by 10-nm-diameter gold particles. (D) Double labeling with anti-nsP1 (5-nm-diameter gold particles) and anti-nsP3 (10-nm-diameter gold particles). The arrowheads point to some antibody-specific gold labeling. Representative detail is shown in the enlarged insets. Bars, 200 nm.

FIG. 3

Immunoprecipitation from P15 fraction of [³⁵S]methionine-labeled SFV-infected BHK cells. Shown is an analysis of immunoprecipitates formed by anti-nsP1 to -4 with denatured (A and B, lanes 1 to 4) and nondenatured (A and B, lanes 5 to 8) proteins. Antisera used for immunoprecipitations are indicated by the numbers of the corresponding nsPs above the lanes. SDS-PAGE was performed in a gel with 10% acrylamide and 0.8% bisacrylamide (A) and in a gel with 10% acrylamide and 0.1% bisacrylamide (B). (C) SDS-PAGE (10% acrylamide and 0.8% bisacrylamide) after two successive immunoprecipitations, first with anti-nsP1 (lane 1) and anti-nsP3 (lane 4) antisera, followed by second immunoprecipitations with anti-nsP1 plus anti-nsP4 (lanes 2 and 5) and anti-nsP2 plus anti-nsP3 (lanes 3 and 6). Molecular weight (Mw) markers are shown on the left. The positions of different ns proteins are indicated by arrowheads on the right.

FIG. 4

Confocal immunofluorescence images of SFV-infected BHK cells 3 (G and J), 4 (A to E), 5 (F, I, and L) and 6 (H and K) h p.i. The cells were stained with anti-nsP3 (green, except in panel I, where nsP3 is red) and different cellular markers (red). Merged images and enlarged insets thereof are shown. In panels I and L, SFV-infected BHK cells at 5 h p.i. were exposed for 30 min to fluorescent Tfn (green), and in panels F and L they were exposed for 30 min to LysoTracker (red). Bars, 10 μm.

FIG. 5

Early events in the biogenesis of CPV. BHK cells were exposed to BSA-gold during virus inoculation as described in Materials and Methods. EM images of SFV-infected cells at 1 (A), 2 (B), and 3 (C and D) h after infection are presented. The asterisks denote endocytosed BSA-gold particles, and the arrowheads point to some early spherule structures (C and D). Bars, 200 nm.

FIG. 6

Late events in the biogenesis of CPV. BSA-gold was administered as for Fig. 5. EM images of SFV-infected BHK cells at 4 (A) and 5 (B to E) h after infection are shown. The asterisks denote endocytosed BSA-gold particles (D and E). In panel E, CPV carrying BSA-gold is exocytosed into the medium. The arrowhead in panel D points to some budding virions at the plasma membrane. Bars, 200 nm.

FIG. 7

Ruthenium red-stained structures in SFV-infected BHK cells 5 h p.i. (50 PFU/cell). SFV-infected BHK cells at 5 h p.i. were fixed in the presence of ruthenium red for 60 min at RT, as described in Materials and Methods, to stain the cell surfaces. (A) CPV under intact plasma membrane; (B) remnants of a CPV after fusion with plasma membrane. Bars, 200 nm.