Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Correspondence
Keith N. Leppard
Keith.Leppard{at}warwick.ac.uk
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ABSTRACT |
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Published ahead of print on 18 November 2002 as DOI 10·1099/vir.0·18820-0.
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Introduction |
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The idea that 55K and Orf6 were functionally related was first suggested by the observation of a physical complex between them by co-immunoprecipitation (Sarnow et al., 1984). More recently, an interaction between 55K and Orf3 has also been shown (Leppard & Everett, 1999
). Similarly, an association occurs between 55K and/or Orf6 and the cellular tumour suppressor protein p53, and these interactions lead to the inactivation and targeted degradation of p53, so blocking p53-dependent apoptosis (Sarnow et al., 1982a
; Kao et al., 1990
; Yew & Berk, 1992
; Dobner et al., 1996
). Orf3 may antagonise this effect on p53 through its interaction with 55K (König et al., 1999
).
The subcellular localization of these proteins has also been studied extensively. In infected cells, 55K is substantially localized to the nucleus. In the early phase of infection it associates with host cell nuclear structures termed ND10 or PODs (Doucas et al., 1996). Later, it becomes associated with the periphery of virus replication centres, dependent on the presence of Orf6 (Ornelles & Shenk, 1991
). When expressed by transfection in uninfected cells, 55K is unable to reach the nucleus without the co-expression of either Orf3 or Orf6 (Goodrum et al., 1996
; König et al., 1999
). In the absence of both these E4 proteins, it localizes to cytoplasmic bodies. In contrast, both E4 proteins, when expressed alone, enter the nucleus, Orf6 showing a diffuse localization (Goodrum et al., 1996
), while Orf3 localizes to, and causes the reorganization of, the same ND10 structures that are initial sites of 55K localization (Carvalho et al., 1995
; Doucas et al., 1996
). During infection, Orf3 shows essentially the same localization as during transfection, while Orf6 forms a reticular network in the nucleus that partially overlaps with the distribution of 55K in replication centres (Gonzalez & Flint, 2002
). Absence of either Orf3 or Orf6 during infection significantly alters the nuclear localization pattern of 55K (Leppard & Everett, 1999
). Finally, both Orf6 and 55K can shuttle independently between nucleus and cytoplasm (Dobbelstein et al., 1997
; Dosch et al., 2001
). Thus, one factor in the complex phenotypes observed when one or more of these proteins is absent from an infection may be the interdependence of these proteins for correct localization within the cell.
The association of both 55K and Orf3 with ND10 is likely to be functionally significant. ND10 are dynamic nuclear substructures that are defined by the presence of the promyelocytic leukaemia (PML) protein but which also contain a large number of other components, many of which are recruited into these structures only under specific physiological conditions (reviewed by Negorev & Maul, 2001). The function(s) of ND10 in uninfected cells remains unclear, with suggested roles in regulating gene expression, the cell cycle and apoptosis. Since a number of unrelated viruses target these structures in various ways (reviewed by Everett, 2001
; Regad & Chelbi-Alix, 2001
), it is probable that viruses either need to utilize cell functions that are provided by ND10 or their components and/or that they need to disrupt such a function that would otherwise be adverse to the infectious outcome. The fact that ND10 components are induced by the natural antiviral component interferon (Lavau et al., 1995
), coupled with the observation that overexpression of PML inhibits the growth of two interferon-sensitive viruses (Chelbi-Alix et al., 1998
), supports the latter idea.
The experiments reported here were designed to examine further the interplay between 55K, Orf3 and Orf6. In particular, they address the role of Orf3 and Orf6 in determining the localization of 55K to the insoluble nuclear substructure termed the nuclear matrix, of which ND10 form a part.
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Methods |
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Cell infection, transfection, fractionation and analysis.
Cells were plated at 50 % confluence for infection 24 h later with 10 p.f.u. per cell of virus. For plasmid transfection, cells were plated at 25 % confluence and transfected 24 h later using Lipofectin and Plus Reagent (Invitrogen), according to the manufacturer's protocols. To produce total extracts, cell monolayers were washed once in PBS and then lysed in sample buffer [200 µl per 106 cells of 2 % (w/v) SDS, 10 % (v/v) glycerol, 25 mM Tris/HCl, pH 6·8, and 0·1 M DTT)]. For nuclear matrix preparations, cells were fractionated as described (Leppard & Shenk, 1989; Leppard & Everett, 1999
) and the nuclear matrix pellet solubilized in 50 µl per 106 cells of sample buffer containing 0·1 M DTT. All samples were incubated at 100 °C for 510 min to denature the proteins and to degrade the DNA, prior to loading on a 10 % SDS-polyacrylamide gel (15 % for analysis of the smaller E4 proteins). After blotting, filters were blocked in PBST (0·05 % Tween 20 in PBS) containing 5 % w/v non-fat dried milk overnight at 4 °C. Antigens were detected by incubating the filter with primary antibody in PBST/milk for 1 h, washing in PBST for 3060 min with four buffer changes, then similarly incubating with an appropriate secondary antibody conjugated to HRP. Following washing, bound HRP was detected using Western Lightning Detection reagent (Perkin-Elmer), according to the manufacturer's instructions, with exposures taken on HyperECL film (Amersham). Films were scanned using an HP Scanjet 6100C/T and figures assembled in Microsoft Photodraw.
For immunofluorescence analysis, cells were grown on glass coverslips, fixed and antigens detected as described previously (Leppard & Everett, 1999). Nuclear matrix samples were centrifuged onto coverslips prior to fixation. Images were collected on a Leica SP2 confocal system using a 63x times; objective and assembled in Microsoft Photodraw without further manipulation.
Plasmid mutagenesis.
The K104R mutation in 55K was constructed by two-step PCR mutagenesis using as template a subclone containing the Ad5 E1b 55K coding region. Primers corresponded to positions 20212040, 23192339, 23392319 and 26662646 (all 5'
3', nucleotide positions from the Ad5 complete genome sequence) with the two central primers carrying sequence alterations specifying an A
G substitution at position 2329, converting codon 104 from AAG (lysine) to AGG (arginine). The second-round PCR product was cleaved at the KpnI (2048) and PstI (2502) sites in E1b and used to replace the equivalent fragment in the subclone. A KpnI (2048)HindIII (2804) fragment from this clone was then used to replace the equivalent segment of pwtXhoI-C (containing the Ad5 genomic DNA fragment 15788). The KpnIPstI region of this clone was sequenced to verify both the intended mutation and the absence of any other sequence alterations.
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Results |
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To examine further the effects of Orf3 and Orf6 expression on the localization of 55K, infections by an expanded panel of mutant viruses (Table 1) were studied using a Western blot assay. As shown in Fig. 1
(a), each of the viruses expressed 55K to a similar level when total cell extracts were analysed. However, nuclear matrix preparations produced in parallel showed marked differences between the viruses in the amounts of 55K found in this subcellular fraction (Fig. 1b
). As expected from previous immunofluorescence analysis, wt300 and dl355, which lack Orf6 expression, showed strong association of 55K with this fraction, whilst the E4 Orf1/2/3-deficient virus, dl1-3, showed minimal 55K in the matrix fraction (Leppard & Everett, 1999
). However, results from other viruses did not support the idea that Orf3 was required for efficient association of 55K with the matrix, since inOrf3, lacking only Orf3, and dl366*, lacking all of E4, showed high levels of 55K in the matrix fraction. Moreover, three other viruses showed minimal levels of 55K in the matrix, similar to dl1-3, only one of which, dl1-4, was expected to lack Orf3 expression.
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It was possible that the nuclear matrix-associated 55K in these various infections was not equivalently localized in each case. Therefore, nuclear matrix was prepared and analysed for 55K by immunofluorescence (Fig. 2). Among the infections with high levels of nuclear matrix 55K in Fig. 1(b), 55K was found in very similar distributions in both the presence (wt300, dl355 and dl366*+3) and absence (inOrf3 and dl366*) of Orf3. In contrast, infections with low amounts of matrix 55K showed only a weak diffuse staining (dl358, an Orf3-positive example, is shown). Thus the association of 55K with the nuclear matrix appears to be independent of Orf3 status.
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Matrix association of 55K is determined by Orf6 expression levels
Although the viruses tested, with the exception of in352, expressed the E4 proteins expected, they did not all do so to the same levels as wt300. Viruses in351, dl358 and dl355 overexpressed Orf2 (Fig. 1c), while inOrf3 and dl366*+4 overexpressed, and dl1-3 underexpressed, Orf4 (Fig. 1e
). Orf6/7 was grossly overexpressed by dl1-4 and to a lesser extent by dl1-3 and in352 (Fig. 1g
). Most notably, dl1-3, dl1-4, in352 and dl358 all overexpressed Orf6 (Fig. 1f
). These viruses were the same ones that showed reduced matrix association of 55K (Fig. 1b
).
This correlation suggested that Orf6 overexpression might be the principal cause of loss of 55K matrix localization in infections by viruses such as dl1-3. However, since the four viruses overexpressing Orf6 also had severely reduced or absent Orf4 expression, it was possible that lack of Orf4 was a factor in preventing 55K association with the matrix. To test this, a co-infection between dl1-4 and dl338, an E4-wild-type, 55K non-expressing virus was analysed (Fig. 3). This infection provides overexpression of Orf6 against a background of expression of all the E4 proteins, including Orf4. As expected, dl338 alone showed no matrix-associated 55K, while dl1-4 showed a minimal level as compared to wild-type. Co-infection by dl338 and dl1-4 did not alter the minimal 55K matrix association phenotype of the latter virus. Thus, it is Orf6 overexpression, and not absence of Orf4, that is associated with reduced matrix association of 55K.
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A SUMO-modified form of 55K is more abundant in the absence of Orf6
The analysis of 55K from cells infected with the panel of E4 mutants showed the presence of a protein with an approximate molecular mass of 75 kDa (75K) that was immuno-reactive with the anti-55K mAb 2A6 (Fig. 1, *). This protein was present in the nuclear matrix fraction and was possibly enriched in this fraction relative to 55K. It was present in wild-type infections at low levels but at significantly increased levels in cells infected by dl355, dl366*, dl366*+3 and dl366*+4. These four viruses each lack expression of the Orf6 protein and so show strong association of 55K with the nuclear matrix. In contrast, those viruses showing negligible matrix-associated 55K lacked even the low level of this 75K species seen in wt300 infections. In a time-course analysis, the amount of this protein relative to 55K was maximal at 12 h p.i. and then declined significantly (Fig. 5
).
It has been reported recently that a minor proportion of 55K in infected cells is modified at lysine 104 by covalent attachment to SUMO-1, a ubiquitin-like protein (Endter et al., 2001). This modification has been described previously for both PML and another ND10-defining antigen, SP100 (
Sternsdorf et al., 1997; Müller et al., 1998
), as well as a diverse set of cellular and viral proteins, many of which can associate with ND10 (reviewed by Seeler & Dejean, 2001
). Given that the 75K form of 55K was of the size expected for SUMO-modified 55K, the possibility that it might be the SUMO-1-modified form described previously was tested. Codon 104 (lysine) within the E1b 55K reading frame in a clone of the Ad5 genome left-end (15788) was changed to arginine by site-directed mutagenesis (K104
R). This mutation has been shown to abolish SUMO-1 modification of 55K (Endter et al., 2001
). The ability of K104
R to produce the 75K form of 55K was then tested in transfection assays in comparison with the homologous wild-type plasmid (Fig. 7
). The wild-type plasmid gave a prominent band of approximately 75 kDa that co-migrated with the protein 55K* identified previously in dl355 samples. However, plasmid K104
R produced none of this 75K species, although 55K was expressed normally. To further confirm that the 75K species missing from K104
R transfections was the same protein as found in Orf6-deficient infections, the effect of Orf6 on expression of this 75K form of 55K from the plasmids was tested. Transfected cells were co-infected with viruses that either did (dl338) or did not (dl367) express E4 Orf6. Both viruses are 55K-deficient, so any 55K observed must derive from the plasmid. The intensity of the 75K band was significantly reduced by dl338 co-infection but was unaffected by the control virus, dl367. Thus, formation of 55K* depends on having a lysine residue at position 104, strongly suggesting that it is the SUMO-1-modified form of 55K described previously.
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Discussion |
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Orf6 overexpression displaced both subsets of matrix-associated 55K into a more soluble fraction. The fact that Orf3 was necessary for the observation of PML-associated 55K suggests that this association is mediated via the interaction of 55K with Orf3 (Leppard & Everett, 1999), which is known to associate with ND10 (Carvalho et al., 1995
). Alternatively, the presence of Orf3 may alter the kinetics of association of 55K with ND10 so as to permit the observation of the association as a significant fraction of total 55K. The ability of Orf6 to displace 55K from the matrix fraction depended on interaction between the two proteins. A recent study of the localization of 55K from mutant A143, which cannot bind to Orf6, supports this observation (Gonzalez & Flint, 2002
). Since Orf3 and Orf6 interactions with 55K define alternative localizations for the protein, it is possible that their interactions are mutually exclusive. However, this remains to be tested.
55K associated with replication centres is probably part of the soluble fraction of 55K rather than matrix-associated 55K, although this remains to be demonstrated directly. There are several lines of evidence for this idea. First, the conditions used to prepare nuclear matrix remove much of the viral DNA-binding protein, which is seen to accumulate predominantly in replication centres (Leppard & Everett, 1999). Second, viruses, such as dl1-3, that show little or no nuclear matrix 55K nevertheless replicate normally (Huang & Hearing, 1989
). Third, Orf6, which, as shown here, prevents matrix association of 55K, has been shown to be necessary for the movement of 55K into the periphery of replication centres (Ornelles & Shenk, 1991
). Thus, the role of nuclear matrix-associated 55K remains uncertain. The Orf3-independent association may simply represent the aberrant fate of the protein in the absence of the full constellation of interacting proteins. However, the Orf3-dependent association is more likely to be significant and may be connected with the reported transient blocking of 55K-mediated p53 inactivation by Orf3 (König et al., 1999
).
Overexpression of Orf6 was observed for several viruses in this study resulting from mutations elsewhere in the E4 transcription unit. Such effects are not surprising, although their possible phenotypic significance has not been considered previously. Both dl1-3 and dl1-4 lack E4 reading frames and their associated splice acceptor sites (Huang & Hearing, 1989). In their absence, a given amount of transcription from the E4 promoter would be expected to result in proportionally more mRNA for the remaining reading frames. Moreover, the missing splice sites are differentially active at early times during infection, so their absence would be predicted to force the use of acceptor sites normally less active until later in infection (Dix & Leppard, 1993
). This can explain the increased expression of Orf6 and Orf6/7 by these viruses, particularly early in infection, but expression from the intact Orf4 reading frame in dl1-3 is reduced compared to wt300. However, the dl1-3 mutation also removes the splice acceptor normally used to produce Orf4 mRNAs. Thus, the reduced Orf4 expression from this virus likely represents the activity of an alternative or cryptic splice site.
Two other viruses, in352 and dl358, also showed elevated Orf6 expression. Both lack Orf4 expression due to frame-shifting mutations that would not be expected to affect cis-acting RNA processing signals (Halbert et al., 1985; this paper). Orf4 is known to negatively regulate the E4 promoter via its effect on E1a proteins (Bondesson et al., 1996
). However, relief of this inhibition in Orf4 mutants should apply equally to expression of all E4 proteins yet the elevation in Orf6 expression appeared to be specific. Alternatively, since the Orf4 reading frame overlaps the N terminus of Orf6 and Orf6/7, elevated Orf6 expression may be due to upregulation of translation initiation at the Orf6 AUG codon in mRNAs that would normally encode Orf4, consequent upon premature termination in Orf4. Finally, the absence of Orf4 may affect the pattern of splicing in E4 so as to increase Orf6 mRNA levels. Orf4 is known to affect splicing patterns during adenovirus infection via its effect on the phosphorylation state of SR proteins (Kanopka et al., 1996
).
The absence of Orf6 during infection led to the enhanced production of a minor 55K-immunoreactive species of around 75 kDa (55K*), which was shown to be modified on residue 104, the known site of SUMO-1 modification on 55K (Endter et al., 2001). Such modification is also characteristic of PML and is essential for the protein to form ND10 structures (Müller et al., 1998
). In addition, many other ND10 components are SUMO-1-modified, though typically this modification is not necessary for the protein to localize to ND10 (reviewed by Seeler & Dejean, 2001
). Given the propensity for SUMO-1-modified proteins to associate with ND10, it seemed likely that this biochemical subset of the protein might be equivalent to the ND10-associated subset seen by immunofluorescence. However, of the four viruses dl355, dl366*, dl366*+3 and dl366*+4 that showed increased levels of this modified 55K form, two (dl366* and dl366*+4) clearly did not show increased ND10-associated 55K as compared to wt300. Thus, the amount of SUMO-1-modified 55K was not directly related to the steady-state level of 55K associated with ND10. A common feature among these four viruses is that they lack Orf6 expression and so 55K association with the matrix fraction (ND10-associated or not) is maximal throughout infection. This suggests that SUMO modification of 55K is related to this association rather than to ND10 association specifically. As discussed, Orf3 may, through its affinity for both 55K and ND10, alter the kinetics of 55KND10 association so that a greater proportion of matrix-associated 55K is seen bound to these structures at steady-state. Perhaps these two components of matrix associated 55K are in dynamic equilibrium. Studies in live cells will be needed to address this question.
Data presented here show that the level of expression of Orf6 during infection is a dominant factor in determining whether or not E1b 55K associates with the nuclear matrix fraction of the cell, while Orf3 expression determines whether or not 55K is found localized with PML in reorganized ND10 within this fraction. Thus, high levels of Orf6 preclude the association of 55K with the matrix, which is a default localization for a significant fraction of 55K in the absence of all E4 proteins. This effect of Orf6 overexpression was dependent on the ability of the 55K protein to form a complex with Orf6. Thus, there are complex effects on 55K localization consequent upon expression of Orf3 and/or Orf6. Moreover, mutations in E4 outside of the Orf3 and Orf6 coding regions can affect expression of these proteins indirectly. These effects will contribute to the phenotypes observed for such mutants and need to be considered carefully in interpreting data from studies involving such viruses.
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ACKNOWLEDGEMENTS |
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Received 11 September 2002;
accepted 11 November 2002.