Download Herpesvirus alters splicing of PML

Herpesvirus protein ICP27 switches PML isoform
by altering mRNA splicing
Takayuki Nojima1,2, Takako Oshiro-Ideue1, Hiroto Nakanoya1, Hidenobu Kawamura1,
Tomomi Morimoto4, Yasushi Kawaguchi4, Naoyuki Kataoka3 and Masatoshi Hagiwara1,2,*
From 1Laboratory of Gene Expression, School of Biomedical Science, 2Department of
Functional Genomics, and 3Medical Top Track (MTT) Program, Medical Research Institute,
Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8510, Japan;
4
Department of Infectious Disease Control, International Research Center for Infectious
Diseases, The Institute of Medical Science, The University of Tokyo, Shirokanedai 4-6-1,
Minato-ku, Tokyo 108-8639, Japan
Running title: Herpesvirus alters splicing of PML
*To
whom correspondence should be addressed.
Tel: +81-3-5803-5836; Fax: +81-3-5803-5853;
E-mail: [email protected]
Supplementary Figure Legends
Fig. S1. Effects of HSV-2 infection and ICP27 expression on cellular pre-mRNA splicing
(A) RT-PCR analysis of uninfected (U) HEK293 cells, HEK293 cells infected (I) with HSV-2 at
MOI 10, 3 hpi (left panel), and HEK293 cells transfected with either myc-vector or myc-ICP27
(right panel). PML was examined for the removal of intron 8 and introns 1 to 3 with primer sets exon
8/exon 9 and exon 1/exon 4, respectively. GAPDH mRNA was analyzed by RT-PCR as a control.
Western blot analysis of whole HEK293 cell extracts with anti-ICP27 antibody (left panel) and
anti-myc antibody (right panel).
(B) RT-PCR analysis of uninfected (U) HEK293 cells and
HEK293 cells infected (I) with HSV-2 at MOI 10, 3 hpi with the several primers sets indicated. (C)
RT-PCR analysis of T-REx293 cells expressing Flag-vector and Flag-ICP27, which were induced by
20 hours of tetracycline treatment. Primer sets for Aly/REF exon 4-5, and Lamin A exon 6-7 detected
the complete removal of the GT-AG intron. Although the Lamin A exon 9-11 primer set is capable of
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detecting an alternative splicing event, only splicing isoform ex9/10/11 was detected. HPS1 and the
P120 gene contain a minor (AT-AC) intron.
Fig. S2. The reduction of the foci of PML-II isoform by HSV-2 infection
Immunofluorescence of HeLa cells infected with HSV-2 at MOI 0.01 for 24 hours. HSV-2-infected
cells were stained with anti-ICP27 antibody and are indicated by yellow arrows. DNAs were stained
with DAPI. Scale bar, 10 m.
Fig. S3. The reduction of the RFP expression of PML splicing reporter in HSV-2 infected cells.
(A) HeLa cells were uninfected (lanes 1 and 2) or infected with Venus-HSV-2 at MOI 0.01 (lane 3)
or MOI 0.05 (lane 4). After 3 hours infection, the reporter E6-7b-RFP was transfected into the cells
(lanes 1-3). After 24 hours infection, whole cell extracts were analyzed by western blot using
anti-RFP and anti-tubulin  antibody. (B) The fold of RFP expression was determined by dividing
the amount of each RFP by the amount of each tubulin  protein. (C) Microscopy analysis of
Venus-HSV-2-infected cells. HeLa cells transfected by E6-7b-RFP were indicated by red.
Venus-HSV-2-infected cells were indicated by green.
Fig. S4. The reduction of the RFP expression of PML splicing reporter in ICP27-transfected
cells.
Western blot analysis of HeLa cells transfected with the combinations of E6-7b-RFP constructs and
plasmids containing either myc-tagged vector or myc-tagged HSV-2 cDNAs. Whole cell extracts of
HeLa cells transfected with these plasmids were subjected to Western blot analysis for RFP (upper
panel) or GAPDH (lower panel). The fold of RFP expression was determined by dividing the amount
of each RFP by the amount of each GAPDH protein.
Fig. S5. The importance of ICP27-mediated splicing regulation in herpesvirus family.
(A) HEK293 cells were transfected with the PML E6-7b construct and the expression plasmid for
either HSV-2 ICP27 or HSV-1 ICP27 (upper panels), and then analyzed by RT-PCR. GAPDH is
shown as a control (middle). ICP27 and GAPDH expression was confirmed with anti-myc and
anti-GAPDH antibodies (lower panels). (B) Alignment of ICP27 homologues of herpesvirus family.
M15 mutation sites are conserved in ICP27 homologues of the herpesvirus family.
Fig. S6. Immunofluorescence analysis of HSV-2-infected HeLa cells in the presence (upper
panel) or absence (lower panel) of PML
HeLa cells were exposed to either scramble siRNA (siCtrl) or siPML4. HSV-2-infected cells were
detected with anti-ICP27 antibody.
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Fig. S7. Transfection efficiencies of the rescue plasmids of PML-II and PML-V
siRNAs against LacZ (siLacZ) and PML (siPML) were transfected into HeLa cells. After 48 hours of
transfection, the expression plasmids of siRNA-resistant PML-II (PML-IIsiR) and PML-V
(PML-VsiR) were further transfected for 24 hours. DNAs were stained with DAPI at the left panels.
Endogenous PML and exogenous PML were stained with the anti-PML antibody H-238 at the
middle panels. The expressions of exogenous RFP, RFP-PML-IIsiR and RFP-PML-VsiR were
described at the right panels. The numbers of exogenous RFP and RFP-PML expression were
divided by those of DNAs to obtain transfection efficiency.
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