1 Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Trogerstr. 9, D-81675 Munich, Germany
2 Institut de Genetique et de Biologie Moleculaire et Cellulaire (CNRS/INSERM/ULP), Illkirch, France
Correspondence
Georg Häcker
hacker{at}lrz.tum.de
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ABSTRACT |
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Published ahead of print on 22 August 2003 as DOI 10.1099/vir.0.19395-0.
Present address: Nordic Bioscience A/S, Herlev Hovedgade 207, 2730 Herlev, Denmark.
Present address: UMR 7100, CNRS-ULP, ESBS, Boulevard Sebastien Brant, BP 10413, 67412 Illkirch Cedex, France.
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INTRODUCTION |
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Interference with the host cell's apoptosis system is a common feature of virus infection (for example, see reviews by Barry & McFadden, 1998; Everett & McFadden, 1999
). The fact that a number of viruses carry genes whose products function to inhibit cell death strongly suggests that apoptosis in virus infection is favourable to the host. This strategy to target the apoptotic pathway is found in viruses from various organisms and classes. In baculoviruses, the mechanism of apoptosis inhibition has been worked out in some detail. A number of baculoviruses carry genes for anti-apoptosis proteins, which fall into two classes: inhibitor of apoptosis proteins (IAP) and P35 proteins (Clem & Miller, 1994
; Miller, 1997
). Both types of proteins can function as inhibitors of apoptosis: IAP by inhibiting the insect pro-apoptotic proteins HID, Grim and Reaper or by directly inhibiting caspases (Clem, 2001
), and P35 probably largely by inhibiting cellular effector caspases (LaCount et al., 2000
; Vier et al., 2000
). Two very similar P35 proteins and one structurally related protein, termed P49, from different baculoviruses are known (Clem et al., 1991
; Du et al., 1999
; Kamita et al., 1993
). P35 from Autographa californica nucleopolyhedrovirus (AcP35; AcNPV) inhibits apoptosis in virus-infected cells as shown by the finding that infection with a p35-deficient mutant virus induces apoptosis (Clem & Miller, 1993
). However, P35 from Bombyx mori NPV (BmP35) appears to play a far less important role in this respect: a virus deficient in BmP35 displays almost normal infectious behaviour (Kamita et al., 1993
) and BmP35 is comparatively poor at inhibiting caspase activity (Morishima et al., 1998
; Vier et al., 2000
). This could mean that P35 has additional functions during virus infection and indeed other molecular roles have been proposed. In one study, P35 has been found to have a direct anti-oxidant function (Sah et al., 1999
). Furthermore, a regulatory function of P35 has been implied in two reports. Studies employing a series of virus mutants and analysing protein synthesis during AcNPV infection suggest that AcP35 is involved in inducing protein synthesis shut-down (Du & Thiem, 1997
). The AcP35 was found to promote transformation of mouse fibroblasts, an effect for which mere inhibition of apoptosis was insufficient (Resnicoff et al., 1998
). It is therefore not unlikely that viral P35 proteins act in roles other than apoptosis inhibition during baculovirus infection.
A cellular homologue of viral P35 has still not been found. We noticed that AcP35 was able to dimerize by yeast-two-hybrid assay. Pursuing the idea that AcP35 might also form dimers with a hypothetical cellular P35, we performed a yeast-two-hybrid screen with AcP35 as a bait in human cDNA libraries. We did not find the sought-after protein but isolated multiple clones of the hRPB11a subunit of the human RNA-polymerase II as an interactor with AcP35. Affinity blotting, homology searches and luciferase-reporter assays were performed to validate this interaction.
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METHODS |
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Libraries used for yeast-two-hybrid screening were purchased from Invitrogen. The libraries used were constructed from Jurkat (human T cell leukaemia) or human placenta cDNA in the prey vector pYESTrp (yielding expressed cDNAs as fusion proteins with B42).
Yeast-two-hybrid screening.
The yeast strain used for two-hybrid screening, Saccharomyces cerevisiae EGY 48/pSH18-34, was obtained from Invitrogen. Yeast transformations, selection on selective media and the isolation of putative interactors from interaction-positive clones were performed according to the manufacturer's instructions (based on the interaction trap/two-hybrid system described by Gyuris et al., 1993). The specificity of the interactions observed was assayed by re-transforming the isolated interactor plasmids into the yeast strain carrying the bait plasmid.
Affinity blotting.
Bacteria were transformed with constructs encoding either full-length AcP35 or, as a control, a deletion mutant of human FADD (Newton et al., 1998) both fused onto an N-terminal 6x His tag and the Tat translocation signal [the sequence from HIV-Tat allows a protein to cross into intact cells (Schwarze et al., 1999
); however, this is irrelevant for the procedure here]. Single colonies were picked, grown overnight in liquid culture and induced with IPTG. Cells were collected, boiled in Laemmli buffer and run on SDS-polyacrylamide gels (approximately the equivalent of 30 µl culture per lane). Gels were either Coomassie-stained or transferred onto nitrocellulose membrane and probed with cytosol from COS7 cells (monkey kidney fibroblast cells) which had been either mock-transfected or transfected with an expression construct encoding FLAG-hRPB11a as described (Blanar & Rutter, 1992
). Blots were then probed with anti-FLAG and secondary peroxidase-labelled antibodies and developed using an enhanced chemiluminescence system.
GST-pull-down.
293T cells were transfected by electroporation with either the empty expression vector or the construct encoding FLAG-AcP35. Cells were collected and frozen 48 h later. Lysates were thawed in hypotonic buffer (20 mM HEPES/KOH pH 7·5, 10 mM KCl, 1 mM EDTA, 1 mM EGTA), and protein was measured by UV absorption. 293T protein extract (600 µg) was then incubated with approximately 100 µg hypotonic extract from Sf9-insect cells infected with a recombinant hRPB11a baculovirus (Acker et al., 1997). GSTproteins were collected and washed three times in buffer (20 mM HEPES/KOH pH 7·5, 150 mM NaCl, 0·1 % Triton X-100, 10 % glycerol). Resulting pellets were boiled in Laemmli buffer and subjected to Western blotting with anti-FLAG-M2 and anti-GST mAb (provided by H. Flaswinkel, Institut for Medical Microbiology, Technical University, Munich).
Homology searches.
Amino acid sequences of P35 proteins from the baculoviruses AcNPV and BmNPV were aligned to those of the RPB3 proteins from Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Homo sapiens. Sequence comparisons and homology searches were done with the alignment programs CLUSTAL and MACAW.
Transfection and immunostaining.
HeLa human epitheloid cells were transfected by electroporation (2·5x106 cells per transfection): 1020 µg of the plasmids pEF-p35-FLAG (P35 with a C-terminal FLAG-tag under the control of the elongation factor 1 promoter) and pCMV-hRPB11a-GFP (hRPB11a-GFP protein under the control of the CMV promoter) were used per transfection. Transfected cells were placed on coverslips in 12-well plates, and after culture for 1 or 2 days, the cells were fixed with 2 % Formalin and stained for FLAG-expression [anti-FLAG M2 (Sigma), followed by Cy3-conjugated anti-mouse antibody (Jackson)]. Cells were analysed for Cy3- and GFP-fluorescence using a laser scanning confocal microscope (Zeiss).
COS7 cells were transfected with either the empty pEF vector or with pEF-AcP35. Cells were harvested 24 h later, extracted with detergent-containing buffer and extracts were analysed for AcP35 by immunoblotting using a rabbit polyclonal antiserum raised against AcP35 expressed in E. coli.
Luciferase reporter assays.
Cells from the human cell line LoVo (colon adenocarcinoma, ATCC) were cultured to about 5070 % confluence in 150 mm dishes, harvested and transfected by electroporation in a Bio-Rad gene pulser (about 5x106 cells per sample, 230 or 260 V, 960 µF, 400 µl complete medium). The reporter plasmids used were pGL2-Ecad-Luc (a gift from E. R. Fearon, University of Michigan) and pGL3--actin-Luc (a gift from R. M. Vabulas, Technical University Munich). In these constructs, the firefly luciferase (Luc) gene is placed under the control of the constitutively active promoters from the human E-cadherin and
-actin genes. Two µg of pGL3-
-actin-Luc or 8 µg of pGL2-Ecad-Luc were co-transfected with various concentrations of pEF-p35 and or pEF-hRPB11a. In some experiments, the plasmid pEF-bcl-2 (a gift from D. Huang, WEHI, Melbourne) encoding human Bcl-2 protein was used as an additional control. After transfection cells were cultured for 24 or 48 h under normal culture conditions. Cell extracts were then prepared and luciferase activity was measured in a LB9507 luminometer (EG & G Berthold) using substrate and buffers from Promega.
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RESULTS |
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Isolation of hRPB11a as an interaction partner of P35
A number of screens (five separate transformations, eight screens) were performed with libraries from Jurkat (human T cell line) or human placenta cDNA. A large number of lacZ-positive clones was isolated and further characterized. PCR screening of about 200 yeast clones gave one predominant size for the interactor insert (PCR products from over 80 % of the clones had the same size of about 500 bp). Interactor plasmids were recovered from seven of these clones and re-transformed into LexA-P35-expressing yeast; in all cases, strong reporter activity signifying interaction was seen. These plasmids were sequenced and all of them were found to carry the full-length open reading frame of the subunit of the human RNA polymerase II 11a (hRPB11a; Grandemange et al., 2001) together with seven triplets of 5' UTR; all were in-frame with LexA. The inserts from the remaining two plasmids were confirmed as the same gene by PCR with primers specific for hRPB11a (data not shown). An example of the interaction test is shown in Fig. 3
. The interaction between P35 and hRPB11a was mediated via the P35 N-terminal part since co-transformation of yeast cells of hRPB11a with N-P35 but not C-P35 led to the activation of the reporters. In order to confirm the interaction and to exclude the possibility that factors other than the interaction were the reason for the positive signal in the yeast-two-hybrid assay, the expression vectors were swapped. The fusion protein LexA-hRPB11a together with the fusion protein B42-P35 showed the same level of interaction as did the original combination (i.e. LexA-P35 and B42-hRPB11a, Fig. 3
). This excludes the possibilities of artefacts, such as detection of a transactivating activity of P35 alone (visible upon DNA-binding through LexA), and indicates that indeed an interaction between the two proteins occurred. A by-product of these experiments is the observation that hRPB11a was also capable of homomeric interaction (Fig. 3
).
To obtain further independent evidence for the specificity of this interaction, hRPB11a binding to P35 was investigated by far-Western (affinity) blotting. P35 and a truncated version of human FADD as a negative control (both with the same N-terminal tag, see Methods) were expressed in bacteria. Total bacterial cell preparations were run on SDS-polyacrylamide gels and Coomassie staining showed the presence of the two proteins (Fig. 4a, left panel, arrows). Bacterial extracts were then blotted onto nitrocellulose membranes and probed with cytosol from COS7 cells transfected with an expression vector for FLAG-hRPB11a (Fig. 4
, right panel) or with cytosol from control (vector)-transfected COS7 cells (middle panel); membrane-bound hRPB11a was consecutively visualized by anti-FLAG immunodetection. As shown in Fig. 4a
, FLAG-hRPB11a was detected by this assay when cytosol from COS7 cells transfected with this expression construct was loaded directly (middle and right panels, first lane, open arrows); as a control, cytosol from control-transfected cells was loaded (middle and right panels, second lane). A number of background bands were visible in the lanes where the bacteria had been loaded and a strong FLAG-positive band was seen in the position where bacterially expressed AcP35 ran (Fig. 4a
, right panel, closed arrow); this band was absent when the membrane was probed with cytosol not containing FLAG-hRPB11a (middle panel), indicating that hRPB11a had been specifically retained on the membrane by AcP35. To confirm the interaction between hRPB11a and AcP35 in solution, pull-down experiments were performed. Recombinant (baculovirus-expressed) GSThRPB11a was co-incubated with extracts from 293T cells transfected to express FLAG-AcP35. When GSThRPB11a was collected onto glutathione beads, FLAG-AcP35 was efficiently co-precipitated (Fig. 4b
). These experiments provide independent evidence for the interaction between AcP35 and hRPB11a, confirming the yeast-two-hybrid results.
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P35 was found to enhance the activity of both the human -actin promoter and the E-cadherin promoter in this assay. In a series of 12 experiments using various amounts of the pEF-p35 expression construct, the range of enhancement was two- to fivefold (Fig. 7
a and data not shown); similar enhancement was seen when using a construct encoding P35 with a C-terminal FLAG-epitope (together with the E-cadherin-promoter, data not shown). Thus, the activity of constitutively active promoters can be enhanced by a factor of about two to five; this increase in already strong promoters might well be expected to affect a cell. Similar enhancing results were also seen with a reporter construct where luciferase was under the control of the elongation factor 1
(data not shown). To control for the possibility that the anti-apoptotic activity of P35 contributed to the enhanced luciferase expression, cells were control-transfected and incubated with the caspase-inhibitor z-VAD-fmk (which, like P35, blocks apoptosis at the level of caspase-activity). This inhibitor did not have any enhancing effect on promoter activity (Fig. 7b
). Also, co-transfection of cells with reporter and an expression plasmid of human Bcl-2 (which blocks apoptosis upstream of caspase-activation) had no effect on promoter activity (data not shown). These results indicate that it was not the anti-apoptotic function of P35 that produced the observed effect.
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DISCUSSION |
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RNA polymerase II is part of the large (2 MDa) so-called pre-initiation complex that binds to the promoter regions of a gene to start transcription (for a recent model see (Langelier et al., 2001). The components of this enzyme have been well conserved throughout evolution. The structure of the complex and the function of the subunits have been studied especially well in the yeast Saccharomyces cerevisiae and are believed to be similar in all eukaryotes (Cramer et al., 2000
). The specific functions of the individual subunits are not well characterized. While some of them form the core of the enzyme, others are associated at the periphery of the complex and might play regulatory roles. In this respect, integration of RPB4 into the complex appears to contribute to stress resistance in yeast (Choder & Young, 1993
). Moreover, the RPB3 subunit (which exhibits a structural similarity to eubacterial RNA polymerase
subunit) has been shown to be involved in transcriptional activation, as has its eubacterial homologue (Tan et al., 2000
).
We found by yeast-two-hybrid analysis that hRPB11a can form homodimers. This homodimer may form under certain conditions (perhaps cellular stress which can change yeast RNA polymerase II composition as mentioned), and this may modify the availability of this subunit for RNA polymerase II complex formation. In addition, a physiological role for partial RNA polymerase subcomplexes cannot be excluded. An example of a situation where a viral protein binds to a subunit of human RNA polymerase II is provided by hepatitis B virus infection. The HBx protein from this virus binds to hRPB5 and likely uses this interaction to activate transcription (Cheong et al., 1995). The strategy of AcNPV in targeting a subunit of human RNA polymerase may serve a similar purpose.
One important function of AcP35 is undoubtedly the inhibition of apoptosis in the cytosol of the cell, where it can inhibit caspases. However, it has also been found to some extent in the nuclear fraction of infected cells (Hershberger et al., 1994), and studies with mutant viruses have suggested that AcP35 is involved in the protein synthesis shut-down during virus infection: the DNA synthesis inhibitor aphidicolin blocked protein synthesis shut-down in Ld652Y insect cells infected with AcNPV lacking p35, but not in cells infected with wild-type virus (Du & Thiem, 1997
). Although we have investigated the effect of P35 only on transcription in human cells, it is likely (given the high level of conservation of the RNA polymerase subunits) that P35 can also affect transcription in insect cells.
When Sf21 cells are infected with mutant virus lacking P35, they undergo apoptosis (Clem & Miller, 1993). In the same study, it was noted that the transcription of early viral genes was delayed in the absence of P35, a finding that would fit well with a role of P35 in transcription. Eventually, transcription of cellular mRNA is shut off during infection with AcNPV (Ooi & Miller, 1988
). A role for P35 in activating cellular transcription would therefore have to be confined to the early stages of infection; alternatively, P35 might also be capable of activating viral transcription. In this context, it should also be remembered that BmNPV carries a p35 gene whose product is a very poor inhibitor of caspases (Kamita et al., 1993
; Morishima et al., 1998
).
In mouse fibroblasts, P35 has been found to promote malignant transformation: cells transfected with p35 formed colonies in soft agar and tumours in nude mice (Resnicoff et al., 1998). Although the molecular circumstances are undefined, this could be the result of an activating effect on transcription of cellular promoters, perhaps of oncogenes such as myc and bcl-2. We have consistently been unable to express AcP35 stably in mammalian cells whereas, at least in our hands, it is exceedingly easy to generate cells lines overexpressing Bcl-2 (unpublished observations). It is possible that the basis of this difference is the interference of AcP35 with cellular transcription. Despite this, a number of P35-expressing cell lines have been described in the literature, such as in the above mentioned study about transformation. One could speculate that P35 interferes with gene expression in a way which is not compatible with normal cell division. For a cell to express P35 stably in spite of this, further mutations are perhaps necessary, and such mutations may affect other regulatory systems of the cell such as control of cell division. If this was the case, the interpretation could be that P35 does not actually promote malignant transformation but works to select for cells which already have further abnormalities.
The available evidence suggests that inhibition of apoptosis is a very important function of AcP35, at least in the tested model systems. However, as detailed above, there is evidence that P35 is involved in further biological processes both during infection of insect cells and when experimentally expressed in mammalian cells. We believe that the binding of P35 to hRPB11a could account for some of these additional functions.
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ACKNOWLEDGEMENTS |
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Received 4 June 2003;
accepted 13 August 2003.
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