Figure 1.
Three-dimensional model of the JCV Vp1 pentamer was constructed using data from an X-ray crystal structure of JCV Vp1 (PDB code: 3NXG) using the Discovery Studio 3.0 software package (Accelrys, San Diego, CA). Structure of the JCV Vp1 pentamer viewed on its top (A) and side (B) are shown as transparent surface and ribbon representation. Red spheres indicate the positions of cysteine residues on Vp1.
Figure 2.
HA titers and Vp1 expression levels of JCV Vp1 cysteine point mutants in mammalian cells.
(A) Schematic diagram of cysteine point mutant Vp1s. Each of the six cysteine residues in the 354-amino-acid JCV Vp1 is marked with a “C” for the WT Vp1 (WT), and the cysteine residue mutated into alanine is designated as “A” for each point mutant Vp1. (B) HA titers of extracts from Vp1-expressing cells. HA titers were determined from the last dilution showing hemagglutination for two-fold serially diluted extracts prepared from HeLa cells transfected with the expression plasmids for WT or individual point mutant Vp1s. The corresponding dilutions of extract from JCV-infected cells served as a positive control, whereas those of PBS or of HeLa extract from transfection with the empty plasmid (Mock) were used as negative controls. (C) Levels of Vp1 expression. Quantitation of the mutant Vp1s and actin in HeLa cells transfected with the Vp1 expression plasmids or with an empty vector (Mock) were analyzed by SDS-PAGE and immunoblotting for Vp1 (Vp1) and for actin (act).
Figure 3.
Stabilities of WT Vp1 and C80A and C247A mutant Vp1s expressed in HeLa cells.
(A) HeLa cells transfected with an empty plasmid (Mock) or plasmid encoding WT, C80A, or C247A Vp1 were pulse-labeled for 5 min and then either harvested immediately (lanes 1 to 3) or chased for 12 h (lanes 4 to 6) or 24 h (lanes 7 to 10). The anti-Vp1 immunocomplexes (IP) prepared from the lysates were analyzed by SDS-PAGE and autoradiography. (B) The intensities of the bands in A were quantified with an image analyzer and plotted as relative values compared to those obtained at 0 h of chase. Data are the means ± S.D. of values from three independent experiments. The solid line, dotted line, and broken line represent WT, C80A, and C247A samples, respectively.
Figure 4.
Structural environments of JCV Vp1 C80 and C247 in relation to Vp1 stability.
(A) Alignment of amino acid sequences of JCV, SV40, BKV, and MPyV Vp1s in the vicinity of C80. Amino acids homologous to C80 are in boldface. (B) C80T mutant Vp1, but not C80A or C80S Vp1, is stable. Lysates of HeLa cells were analyzed by SDS-PAGE and immunoblotting for Vp1. These cells were prepared 48 h after transfection with the expression plasmid encoding eithe WT Vp1 or each C80 mutant Vp1 or after transfection with empty expression plasmid (Mock). (C) Comparison of the structures of JCV and MPyV Vp1s surrounding C80. The JCV structure is superimposed on the MPyV structure. The sulfur atom of JCV's C80 and the methyl group of MPyV's T97 are circled in red. (D) Alignment of amino acid sequences of JCV, SV40, BKV, and MPyV Vp1s in the vicinity of C247. Amino acids homologous to C247 are in boldface. (E) All C247 substitution mutant Vp1s expressed in HeLa cells are unstable. Lysates of HeLa cells transfected with the expression plasmid for WT Vp1 or for each C247 mutant Vp1 or after transfection with empty expression plasmid (Mock) were analyzed for Vp1 by SDS-PAGE and immunoblotting for Vp1.
Figure 5.
Cysteine point-mutant Vp1s synthesized in vitro are stable.
The empty pURE2 plasmid (Mock) or pURE2-Vp1 plasmids encoding either WT Vp1 or cysteine point mutant Vp1s were subjected to cell-free transcription-coupled translation, and the translation products were examined for Vp1 by SDS-PAGE and immunoblotting.
Figure 6.
Oligomerization analysis of in vitro synthesized WT and mutant Vp1s.
(A) Electron micrographs of VLPs (VLP; scale bar, 10 nm) and pentamers (EGTA + DTT; scale bar, 10 nm) formed by WT JCV Vp1. (B) The mixtures resulting from the in vitro transcription-coupled-translation of the empty pURE2 plasmid (Mock) and of the Vp1-encoding pURE2-Vp1 (WT) were supplemented with DTT, boiled, separated by SDS-PAGE, and immunoblotted with a rabbit anti-Vp1 antibody. An arrowhead marks the position of monomeric Vp1. An arrow indicates nonspecific bands. (C) Sedimentation profiles of in vitro translated single cysteine substitution mutants. Monomeric Vp1s, pentameric Vp1s, and the in vitro translation products for WT Vp1 or cysteine point mutant Vp1s (C42A, C80A, C97A, C200A, C247A, C260A, or C80T) were separated by 5–20% sucrose gradient sedimentation under denaturing conditions, and the resulting fractions were examined for the presence of Vp1 by SDS-PAGE and immunoblotting.
Figure 7.
C80A and C247A mutations decrease infectivity.
(A) Expression of JCV-encoded agnoprotein and Vp1 after JCV genome transfection. SVG-A cells were transfected with WT JCV genome for 3 days, fixed, and examined for the presence of agnoprotein and Vp1 by immunofluorescence analysis. Cell nuclei were counterstained with DAPI. Merged images of DAPI (Blue), Agno (Red), and Vp1 (Green) are also presented. (B) Levels of Vp1 expression. SVG-A cells were transfected with JCV genomes encoding either WT Vp1 or cysteine point mutant Vp1s or transfected with the transfection reagent alone (vehicle) for 3 days, and cell lysates were analyzed by SDS-PAGE and immunoblotting for Vp1 (Vp1) and for actin (act). (C) Subcellular localization of C80A and C247A Vp1s. SVG-A cells at 3 days posttransfection with the WT or either mutant JCV genome were processed for immunofluorescence analysis with a mouse anti-Vp1 antibody (green). Cell nuclei were counterstained with DAPI (blue). Merged images of DAPI and Vp1 are also presented. (D) Infectivity of WT and mutant JCV genomes. JCV genomes encoding either WT or individual cysteine mutant Vp1s were transfected into SVG-A cells, and the effectiveness of infection initiation was determined by immunofluorescence analysis for the presence of agnoprotein. For each genome, the average percentage of cells positive for agnoprotein is presented in the bar graph as the mean ± SD of three microscopic fields. The data represent the mean ± SD of three independent experiments. The significance of the changes was analyzed by Student's t-test (*p<0.05).
Figure 8.
Structural difference around C80 between JCV Vp1 and SV40 Vp1.
Vp1 structures of JCV and SV40 are shown in green and red, respectively. The different residues within 4 Å of C80 of the Vp1s are shown as stick models. An arrow indicates C80 in JCV Vp1 and C87 in SV40 Vp1.