requires two proteins encoded by the viral rep gene. Virology 2001,
279:429-438.
Figures
Figure 1. VP2 protein is localized to the nucleus of the mammalian cells. HeLa
(A) and CHO (B) cells both were transfected with GFP and VP2-GFP expressing
plasmids (green). At 48 hours post-transfection, the cells were fixed and stained
with DAPI (blue). The distribution of GFP and VP2-GFP in the cells were followed
by microscopy (phase) and fluorescence microscopy.
Figure 2. Analysis and predication of NLS and NES motifs present in the VP2
amino acid sequence. The various VP2 amino acid sequences (51 to 200) from
different CAV isolates were aligned as described in the Materials and Methods. The
putative NLS motifs (BiNLS1: under line and NLS2: bold words) and NES motifs
(under line, bold, shadow and Italic) are shown. The cysteine residues at positions
95 and 97 in the catalytic motif of VP2 are also indicated by an arrow and an arrow
head, respectively.
Figure 3. Mutant constructs of VP2-GFP that were used in this study. (A)
Truncated fragments of the VP2 (black bars) encoding constructs fused with GFP
(white bars) were used in this study. The right panels indicate the localization
patterns in cells. GFP distribution in the cytosol is indicated by C; GFP signal in
nucleus that is stronger than that in the cytosol is indicated by N; GFP detected in
both the cytosol and nucleus is indicated by N/C. The putative NLS motifs
(BiNLS1 and NLS2) and the weak NES motif are shown. The amino acids present
in the constructs are also indicated. (B) Subcellular localization of C-terminal
deleted VP2-GFP constructs (115dC, 132dC and 145dC) in HeLa and CHO cells
(green). (C) Subcellular localization of N-terminal deleted VP2-GFP constructs
(111dN, 141dN and 160dN) in HeLa and CHO cells (green). All cells were fixed
and stained with DAPI (blue).
Figure 4. Identification of the NLS motifs (BiNLS1 and NLS2) in VP2. (A)
Substitutive mutagenesis was used to create three constructs, VP2 150-152 (RKK
to AAA), VP2 136-138 (KRK to AAA), as well as double and triple mutations,
within the BiNLS1 region of VP2 (also saw in Table 2). The subcellular
localization of these mutants (green) were examined by fluorescent microscopy in
HeLa and CHO cells. (B) To further identify the functional NLS motif, K133 and
R134 were replaced by alanine. The mutants (VP2 136-138A/134A, VP2
136-138A/133A and VP2 136-138A/133A/134A) were then expressed and
examined by fluorescent microscopy (green). All cells were fixed and stained with
DAPI (blue). (C) Identification of NLS2 in VP2. The point mutations VP2 133A,
VP2 134A and VP2 133A/134A were transfected into HeLa cells. After 48 h
post-transfection, the distributions of GFPs (green) were monitored by fluorescence
microscopy. The cells were fixed and stained with DAPI (blue).
Figure 5. The distribution of VP2-GFP and mutants in cytoplasm and nucleus
of cells. VP2-GFP and various mutants (VP2 136-138A/134A, VP2
136-138A/133A and VP2 136-138A/133A/134A) were transfected into CHO cells.
After 48 h transfection, all cells were fixed and stained with DAPI. The distribution
of GFP was monitored by fluorescence microscopy. In VP2-GFP and mutants,
twenty cells were measured the distribution of GFP signals from cytoplasm and
nucleus using Alpha View® Software (Alpha Innotech Corporation).
Figure 6. VP2 binds to chromatin and interacts with MCM3 but this does not
require dual phosphatase activity. (A) The soluble (S) and chromatin (C)
fractions were prepared by using CSK buffer containing 0.5% Triton X-100 in
CHO cells of transfected with VP2-GFP plasmid. The fractions were treated with
0U (-) and 150 U (+) of MNase or the indicated concentrations of NaCl. Nuclear
extracts (N) containing VP2-GFP were as positive control and all fractions were
subjected to immunoblotting against with lamin B receptor, MCM3, and VP2
antibodies. (B) At 48 h post-transfection, the GFP (as Control) and Flag-VP2-GFP
(as wild-type; WT) were found in the CHO cells. Next the lysates were
immunoprecipitated using Flag M2 beads and immunoblotted against VP2 and
MCM3 antibodies. (C) The WT and mutants (C95S, C97S, and C95S/C97S) of the
Flag-VP2-GFP in CHO cells were also examined at 48 h post-transfection. The cell
lysates were immunoprecipitated using Flag M2 beads and immunoblotted against
with VP2 and MCM3 antibodies. The arrow head was indicated a none-specific
band. The nuclear extracts containing VP2-GFP were designated as N.
Tables
Table 1. The primers used to create the various truncated, single and multiple mutants by PCR in this study
Primer name Type Lengt Sequence (5’–3’)
VP2 111N del EcoRI Forward 26-mer TGGAATTCATGGAGGACCGATCAACC VP2 141N del EcoRI Forward 26-mer AGGAATTCATGCACTACTCCCAGCCG VP2 160N del EcoRI Forward 26-mer AGGAATTCATGGACGAGCTCGCAGAC VP2 115C del XhoI Reverse 24-mer TCCTCGAGTGATCGGTCCTCAAGT VP2 132C del XhoI Reverse 23-mer TCCTCGAGACCCTGTACTCGGAG VP2 145C del XhoI Reverse 26-mer TCCTCGAGCTGGGAGTAGTGGTAATC VP2 136-138A Forward 27-mer AAACGAGCTGCTGCTGCTCTTGATTAC VP2 136-138A Reverse 27-mer GTAATCAAGAGCAGCAGCAGCTCGTTT VP2 150-152A Forward 39-mer ACCCCGAACGCAGCAGCAGTGTATAAGACTG VP2 150-152A Reverse 39-mer CCATCTTACAGTCTTATACACTGCTGCTGCGTT VP2 136-138A/134/A Forward 27-mer GTACAGGGTAAAGCTGCTGCTGCTGCT VP2 136-138A/134/A Reverse 27-mer AGCAGCAGCAGCAGCTTTACCCTGTAC VP2 136-138A/133/A Forward 27-mer GTACAGGGTGCTCGAGCTGCTGCTGCT VP2 136-138A/133/A Reverse 27-mer AGCAGCAGCAGCTCGAGCACCCTGTAC VP2136-138A/133-134/A Forward 27-mer GTACAGGGTGCTGCTGCTGCTGCTGCT VP2 136-138A/133-134/A Reverse 27-mer AGCAGCAGCAGCAGCAGCACCCTGTAC VP2 133A Forward 28-mer GTACAGGGTGCTCGAGCTAAAAGAAAGC
VP2 C95S Forward 20-mer CGCTAAGATCAGCAACTGCG
VP2 C95S Reverse 22-mer CGCAGTTGCTGATCTTAGCGTG
VP2 C97S Forward 21-mer ATCTGCAACAGCGGACAATTC
VP2 C97S Reverse 24-mer ATTGTCCGCTGTTGCAGATCTTAG VP2 C95S/C97S Forward 28-mer CGCTAAGATCAGCAACAGCGGACAATTC VP2 C95S/C97S Reverse 28-mer ATTGTCCGCTGTTGCTGATCTTAGCGTG
Table 2. Intracellular localization of VP2 and various mutants of the NLS motif
Name of mutants Localizationa NLS motifs of amino acids sequenceb,
c
VP2-GFP N K133R134A135K136R137K138L139D140Y141H142
Y143S144Q145P146T147P148N149R150K151K152
VP2 150-152A N K133R134A135K136R137K138L139D140Y141H142
Y143S144Q145P146T147P148N149A150A151A152
VP2 136-138A N K133R134A135A136A137A138L139D140Y141H142
Y143S144Q145P146T147P148N149R150K151K152
VP2 136-138A/150-152A N K133R134A135A136A137A138L139D140Y141H142
Y143S144Q145P146T147P148N149A150A151A152
VP2 136-138A/133A C A133R134A135A136A137A138 VP2 136-138A/134A C K133A134A135A136A137A138 VP2 136-138A/133-134A C A133A134A135A136A137A138 a. The N and C indicate nuclear and cytoplasm, respectively.
b. The BiNLS1 and NLS2 motifs are indicated by underlining and bold, respectively.
c. The amino acids of BiNLS1 are shown from K136 to K152 and of NLS2 are shown from K133 to K138.
Additional files
Additional file 1. The effect of LMB treatments on the various mutants of VP2.
The truncated mutants of VP2 in Figure 3A were all treated with LMB (+) (20
ng/ml) and LMB (-) (PBS buffer) for 1 h at 37oC. VP3-GFP is LMB sensitive and
was used as a positive control. The distribution of GFP was monitored by
fluorescence microscopy.
Additional file 2. Identification of protein-protein interactions with VP2
surveyed by co-immunoprecipitation. At 48 h post-transfection with plasmids
encoding GFP (as the Control) or Flag-VP2-GFP (WT), cell lysates were
immunoprecipitated by Flag M2 beads and immunoblotted against VP2, MCM3,
CDC7, SMC2, PCNA, and H2B antibodies. The nuclear extracts containing
VP2-GFP are designated as N.
Additional files provided with this submission:
Additional file 1: Supplemental data Figure 2.tif, 3187K
http://www.biomedcentral.com/imedia/3388345706277160/supp1.tiff Additional file 2: Supplemental data Figure 3.tif, 3185K
http://www.biomedcentral.com/imedia/1808622325627716/supp2.tiff