1 School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
2 Division of Virology, Department of Pathology and Microbiology, University Walk, Bristol University, Bristol BS8 1TD, UK
3 Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, S-75123 Uppsala, Sweden
Correspondence
David Matthews
d.a.matthews{at}bristol.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Previous studies showed that protein V is associated with the nucleolus during the late phase of infection and, in the nucleus, V is excluded from viral DNA binding protein-rich centres where single-stranded DNA accumulates (Matthews & Russell, 1998b). Additional nuclear and nucleolar targeting sequences in protein V have been analysed, in which two highly basic nucleolar targeting regions were identified (Matthews, 2001
). In addition data were presented that protein V may disrupt nucleolar function by affecting the subcellular localization of the nucleolar antigens nucleolin and B23. Why adenovirus might disrupt nucleolar function is unknown, but recent data suggest that B23, for example, may have importance in initiating adenoviral DNA replication (Okuwaki et al., 2001
).
Nucleoli are known to be the sites of ribosome formation, where rRNA is synthesized, processed and incorporated into ribosomes (Pederson, 1998; Scheer & Hock, 1999
). In adenovirus infection the formation of 18S and 28S rRNA is reduced (Castiglia & Flint, 1983
) and late in infection the nucleoli are disrupted (Puvion-Dutilleul & Christensen, 1993
). Our interest in these phenomena led us to examine Mu protein because it is arginine-rich and contains sequences that are similar to a nucleolar targeting signal found in protein V (Matthews, 2001
).
Mu (also known as pX) is synthesized as a 79 aa precursor that undergoes both N- and C-terminal cleavage by a virus-coded protease to form mature Mu (19 aa). It has been shown that Mu protein precipitates DNA in vitro, suggesting that Mu helps condense DNA in the virus core by means of a charged-based interaction between the nine arginine residues in Mu and the phosphate DNA backbone (Anderson et al., 1989). Consistent with this, a recent study demonstrated that Mu protein enhanced transfection efficiency by up to 11-fold when used as an adjunct to liposome-mediated gene therapy (Murray et al., 2001
).
There is no published in situ data on the intracellular localization of Mu (or its precursor protein) during infection. Indeed, such a study is complicated by the observation that a monoclonal antibody against Mu cross-reacts with a similar region within protein VII (Lunt et al., 1988). Consequently, we decided to use tagged versions of Mu and preMu to examine the subcellular localization of the proteins in an infected cell, relative to viral DNA replication centres.
We found that preMu contains a nucleolar localization signal, the key element of which is located within Mu. We found no evidence that preMu or Mu affect the subcellular distribution of the nucleolar antigens nucleolin or B23. In addition, we could not demonstrate any gross defects in de novo rRNA synthesis. However, our data are consistent with preMu (but not Mu) being able to modulate accumulation of viral proteins derived from the E2 region.
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METHODS |
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Cloning of recombinant derivatives of preMu and ASF/SF2.
Regions of the open reading frame for preMu protein were amplified from HAdV-2 DNA using oligonucleotide primers and a PCR kit (PFX; Invitrogen). Synthetic oligonucleotides corresponding to the open reading frame of Mu were made and annealed to generate a DNA fragment for cloning. In this manner we also generated mutants of Mu with selected arginine codons replaced by alanine codons. The DNA fragments were cloned into a CMV promoter-based mammalian expression plasmid (pcJMA2egfp; a kind donation from J. Askham; Askham et al., 2000) to express the amino acid sequences produced as N-terminal fusions to enhanced green fluorescence protein (EGFP; Clontech). The open reading frame for the splicing factor ASF/SF2 was PCR-amplified from EGFP-ASF/SF2 (Sleeman et al., 1998
; kindly provided by A. I. Lamond) and cloned into pds-RedC1 (Clontech). In addition, synthetic oligonucleotides were designed to enable us to replace the open reading frame of EGFP with the amino acid sequences corresponding to the Myc or FLAG tags. The sequences of primers used in this manuscript are available on request.
Fluorescent imaging.
Eighteen to twenty hours after transfection the cells were fixed using 4 % formaldehyde (v/v in PBS). Cells were washed in PBS prior to the coverslips being mounted with Vectashield containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Detection of EGFP-tagged proteins was performed using a Zeiss Axiovert 135TV microscope with a Neofluor 40x oil-immersion lens.
Immunofluorescence.
Formaldehyde-fixed cells on coverslips were permeabilized using Triton X-100 (1 %, v/v, in PBS) prior to blocking with dried skimmed milk (1 %, w/v, in PBS) for 1 h at room temperature. The following primary antibodies were used: anti-B23 (kindly provided by B. Valdez; Perlaky et al., 1997), anti-protein V (Matthews & Russell, 1998b
), anti-protein VI, (Matthews & Russell, 1994
), anti-DBP (Russell et al., 1989
), anti-hexon, anti-penton base (kind donations from W. C. Russell), and anti-terminal protein (kindly provided by R. T. Hay; Webster et al., 1997
). Appropriate secondary antibodies were linked to Texas Red or FITC (Vector Laboratories). Cells were mounted and viewed as described above; alternatively images were collected using a laser confocal microscope (Leica TCS SP) and a PlanApo 100x UV oil-immersion lens.
Fluorouridine labelling of transfected cells.
HeLa cells were transfected with preMuEGFP or MuEGFP constructs as described and 18 h post-transfection the cells were incubated with 15 mM 5'-fluorouridine (5'FU from Sigma) for 15 min. Cells were then fixed and permeabilized as outlined and FU incorporated into RNA was detected using monoclonal anti-BrdU antibody (Sigma) as described previously (Boisvert et al., 2000).
Western blotting.
HeLa cells were grown in six-well dishes without coverslips and transfected as described above. After 18 to 20 h, cells were harvested and prepared for SDS-PAGE. Western blotting was performed using rabbit antibodies against EGFP (Santa-Cruz), and secondary anti-rabbit immunoglobulin linked to HRP (Vector Laboratories). Detection was performed using ECL (Amersham-Pharmacia).
Infection/transfection studies.
In order to assess the location of preMuEGFP and MuEGFP during infection, cells were infected with HAdV-2 at an m.o.i. of 5 p.f.u. per HeLa cell. This inoculation was performed either 7 h before transfection with plasmid, simultaneous with transfection, 7 h after transfection or 24 h after transfection. Immunofluorescence patterns were observed at 20, 26 or 30 hours following infection.
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RESULTS |
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PreMuEGFP and MuEGFP did not abrogate rRNA synthesis or affect the localization of nucleolar antigens
An in situ rRNA synthesis assay (Boisvert et al., 2000) using fluorouridine labelling demonstrated that preMuEGFP had no effect on RNA synthesis in the nucleolus. In addition, as with protein V (Matthews, 2001
), blocking rRNA synthesis using actinomycin D did not affect the localization of preMuEGFP (data not shown). In contrast to protein V (Matthews, 2001
), preMuEGFP and MuEGFP did not affect the gross distribution of nucleolin or B-23. However, we could not find cells expressing high levels of preMuEGFP after 48 h. We were also unable to isolate stable cell lines expressing preMuEGFP using an inducible promoter system. Together these two observations indicate that expression of preMuEGFP is toxic to the cells.
PreMuEGFP and MuEGFP were excluded from the DBP-rich viral DNA replication centres during adenovirus infection
To determine if the localization of preMuEGFP or MuEGFP was affected by adenovirus infection, we studied the effects of transfecting these EGFP fusion protein expression plasmids into adenovirus-infected cells. As protein V had previously been shown to associate with the nucleolus and was excluded from the DBP-rich areas (Matthews & Russell, 1998a) we first assessed whether this also occurred with preMuEGFP and MuEGFP protein (Fig. 2A, B
). Immunofluorescent labelling of DBP enabled us to demonstrate that, as with protein V, both preMuEGFP and MuEGFP protein were excluded from DBP-rich areas. Other than this exclusion, the distribution of both preMuEGFP and MuEGFP was similar to that seen in uninfected cells.
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Expression of preMuEGFP in HeLa cells inhibited preTP expression
We also examined preTP expression since, like DBP, it is also a product of the E2 transcription unit. In adenovirus-infected cells, those cells transfected with preMuEGFP and simultaneously infected with adenovirus failed to express preTP after 20 h (Fig. 4A), whereas we had previously shown that expression of DBP could be readily detected (Fig. 2A
). MuEGFP, protein VEGFP and EGFP expression did not affect the expression of preTP in these assays.
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Overexpression of REDASF/SF2 mislocalized a proportion of preMuEGFP in uninfected cells
This similarity of effect of preMuEGFP and REDASF/SF2 on late gene expression led us to examine whether overexpression of REDASF/SF2 would affect the intracellular localization of preMu-EGFP or vice versa. We therefore transfected uninfected cells with plasmids expressing both REDASF/SF2 and preMuEGFP. In >90 % of cells examined we observed mislocalization of a proportion of preMuEGFP into the nucleus. This nuclear preMuEGFP corresponded with REDASF/SF2 in extranucleolar sites (Fig. 5A). This effect on the distribution of preMuEGFP was only evident in cells overexpressing REDASF/SF2.
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Examination of preMuEGFP transfected cells (Fig. 5A) revealed that its subnucleolar distribution within the cell nucleolus was similar to that of protein VEGFP in uninfected cells (Matthews, 2001
). Thus, it appeared to be associated with those regions of the nucleolus where rRNA processing predominates and was excluded from the centres where inactive rDNA is stored.
REDASF/SF2 did not accumulate in the nucleolus in any of our experiments, and did not co-localize with protein VEGFP (data not shown). The deletion mutants 150EGFP, 3250EGFP, 3279EGFP, 169EGFP and 159EGFP also co-localized with REDASF/SF2, whereas 131EGFP and 5179EGFP did not co-localize with REDASF/SF2 (Fig. 6A).
Mutagenesis identified a region in the C-terminal one-third of preMu involved in blocking late gene expression
In order to explore which domains of preMu are responsible for the effect on late gene expression, the preMu deletion series described above was assessed (Fig. 6A). Only those proteins containing the sequence 3279EGFP affected late gene expression. Additional deletion mutants demonstrated that the sequence 169EGFP but not 159EGFP inhibited the expression of preTP and protein VI. In order to explore which regions of 3269EGFP were responsible for this effect, site-directed mutagenesis was performed in turn at sites 63, 67 and 69 within a 169EGFP construct (Fig. 6B
). These amino acids are highly conserved between adenovirus species (Fig. 6C
), and have large hydrophobic side-groups. In each case glycine (small hydrophilic) was used as a non-conservative substitution. Each of these mutants of 169EGFP continued to block the expression of late genes. The series of deletion and site-directed mutants described in this section all had the same subcellular localization as that of 3279EGFP, and were expressed at the expected size on Western blot (for mutants of key functional importance see Fig. 6D
).
Differentially tagged preMu fusion proteins also accumulate in the nucleolus in uninfected cells and blocked late gene expression in infected cells
The recombinant preMuEGFP protein is three times larger than preMu so we also C-terminally tagged our clone of preMu using Myc (12 aa), FLAG (8 aa) or dsRED (225 aa) instead of EGFP. We used these fusion proteins to confirm the nucleolar localization of preMu (Fig. 7A) and the effect on expression of late genes; in this example we have shown that cells in which preMuMyc is expressed exhibit greatly reduced levels of fibre protein (Fig. 7B
).
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DISCUSSION |
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We suggest that, with the possible exception of the arginine at position 48, all the arginine motifs of Mu, particularly at positions 40 and 41, contribute to nucleolar accumulation. Nucleolar localization sequences (NoLS) similar to Mu have been described previously (Jarrous et al., 1999; Liu et al., 1997
). The nucleolar targeting of a 23 aa fragment of ribonuclease P subunit 38 is dependent on all nine lysine residues spaced through the sequence, though two adjacent lysines are most critical for entry to the nucleolus (Jarrous et al., 1999
). A single arginine present in the sequence had no role in nucleolar localization. Similarly, a comparison of homology between NoLS of 13 different proteins revealed that all are between 11 and 20 aa in length, contain between two and six arginine or lysine domains, with a gap between domains of between one and three non-basic amino acids (Liu et al., 1997
). Whilst these sequences are all broadly similar, there is no absolute consensus sequence. Van Eenennaam et al. (2001)
suggested that highly basic domains non-specifically target the nucleolus, and having entered the organelle by passive diffusion they are retained by binding to negatively charged molecules within the nucleolus. In common with these previously described NoLS, substitution of certain basic amino acids within the Mu NoLS reduces the intensity of the nucleolar targeting, but none of these amino acids are absolutely critical in this regard. Indeed, when the arginines are spaced evenly there is no apparent reduction in nucleolar accumulation. This may be due to either the existence of multiple redundant NoLS within the sequence or, more likely, targeting Mu to the nucleolus is primarily charge-based rather than dependent on a specific pattern of amino acids.
PreMu-EGFP and Mu-EGFP do not affect nucleolar antigens (B23 and nucleolin) nor do they affect rRNA synthesis and consequently their role in the nucleolus will require further investigation. Interestingly, neither protein VEGFP nor preMuEGFP are affected by inhibition of rRNA synthesis, something that markedly effects the distribution of rRNA processing proteins. Potentially they interact with proteins/structures within the nucleolus unaffected by treatment with actinomycin D. Examples of such nucleolar proteins are, RNA pol I, fibrillarin and p120 (Perlaky et al., 1997). Significantly, we have been able to lengthen the list of adenovirus proteins that may affect nucleolar structure and function.
We have shown that expression of preMu as a fusion protein in adenovirus-infected HeLa cells inhibits late gene expression. We have used specific antisera to a range of late viral proteins in order to establish that the defect is widespread. Normally, the progression to the late phase requires replication of viral DNA. Thus, the simplest conclusion is that there is a failure to enter the late phase, probably resulting from lack of DNA replication. Consistent with this, Western blotting revealed that preMuEGFP was not cleaved by the viral protease in our infection/transfection assays (data not shown). This is to be expected because the viral protease is a late gene and there is a failure to enter the late phase in preMuEGFP-transfected cells.
We had already established that viral DBP was being expressed in cells transfected with recombinant preMu proteins and we noted that there was precedence for this type of observation. A recombinant adenovirus that expressed ASF/SF2 under the control of an inducible promoter has been described (Molin & Akusjarvi, 2000). This report noted that when induced, ASF/SF2 blocked late gene expression but expression of DBP was evident. We have previously noted unpublished evidence that overexpression of ASF/SF2 inhibited preTP expression (Molin & Akusjarvi, 2000
). The lack of preTP expression would account for the lack of DNA replication and the loss of late protein expression. How ASF/SF2 might do this is not understood at present but it seems reasonable to postulate that there is a failure of the splicing pathway required to generate preTP mRNA.
Our data demonstrate that preMuEGFP has similar effects to ASF/SF2 in blocking adenovirus late protein expression. Consistent with a possible effect on DNA replication we noted that protein IX expression was reduced but not abolished. We therefore wanted to examine if our transfection/infection data could be repeated using the dsRED fusion of the splicing factor ASF/SF2 and we observed that both preMuEGFP and REDASF/SF2 block preTP expression but allowed DBP expression. Thus our data provided further evidence that both preMuEGFP and ASF/SF2 apparently modulate accumulation of E2 region proteins.
Previous studies have established that transcription and splicing are separate from DNA replication and DBP-rich regions of the infected nucleus (Bridge et al., 1993, 1995
; Pombo et al., 1994
). As expected therefore, our REDASF/SF2 clone was also excluded from the DBP-rich centres. The exclusion of proteins preMuEGFP and MuEGFP from the adenoviral DBP-rich areas is compatible with a model in which preMu influences mRNA biogenesis. However, we also note that preMuEGFP in infected cells does have a significant nuclear component not normally seen in uninfected cells. This might reflect preMu being involved in disruption of nucleolar function as well as in viral RNA biogenesis.
In support of this idea we observed that co-expression of REDASF/SF2 together with preMuEGFP in uninfected cells leads to a proportion of preMuEGFP accumulating in the nucleus with REDASF/SF2. Clearly, this phenomenon requires overexpression of RedASF/SF2 but it indicates that preMuEGFP and RedASF/SF2 might co-localize under certain conditions in uninfected cells. Intriguingly, only clones with the arginine-rich Mu sequence present acted in this manner. However, this property of preMuEGFP is not sufficient to block late gene expression in infected cells. For example, MuEGFP is affected by REDASF/SF2 in uninfected cells but is not associated with ablation of late gene expression in infected cells. Moreover, as they are both excluded from DBP-rich centres in infected cells we postulate that preMu and ASF/SF2 might be present in the same complex during infection. Thus, the nature of the relationship between preMuEGFP and REDASF/SF2 is unclear but it is notable that ASF/SF2 can be isolated from purified nucleoli (Andersen et al., 2002).
Deletion mutagenesis enabled us to determine that the C-terminal portion of preMu was required to affect the accumulation of preTP. Examination of this region in a wide range of adenoviruses revealed that this region is highly conserved amongst all the major lineages of adenovirus. We attempted to define individual amino acids that might be key to preMu's ability to block preTP but were unsuccessful. In the current absence of structural data on preMu, this might imply that an extended region of preMu is involved in blocking preTP expression; such ideas are difficult to test.
The data we had accumulated pointed to a role for preMu in mRNA biogenesis, and the similarities with ASF/SF2 point to the simple conclusion that preMu may also be capable of modulating splice-site selection. However, splicing of the E2 region is not well understood at present and so the potential for particular effects on the accumulation of preTP mRNA cannot be excluded at this stage (e.g. specific effects on preTP mRNA transport). Indeed, it has not yet been shown that ASF/SF2 modulates the splicing of E2 mRNA, so claims that both these proteins modulate the splicing of E2 must be treated with caution at this time. That late viral proteins might affect splice selection has some precedence in the adenovirus system. Recent evidence suggests that unscheduled expression of pre-IIIa protein (derived from L1) caused alterations to the accumulation of late mRNA species (Molin et al., 2002). Clearly, it is possible that several members of the late family of proteins influence post-transcriptional events during the late phase of infection.
We were conscious of the possibility that the EGFP tag may have a significant influence on preMu function. However, preMuMyc, preMuFLAG and preMudsRED proteins all showed identical characteristics to preMuEGFP. Therefore, we consider that the use of a number of alternate tags provides some measure of assurance that the effects we observe result from the properties of preMu and not from the fusion protein as a whole.
PreMu is the first adenovirus late protein that has been shown to have an effect on accumulation of early proteins in infected cells. Prior to this study, the role of preMu, other than as a precursor to Mu, was unknown. We note that the effects are likely to be mediated through the highly conserved C-terminal one-third of preMu. This suggests that modulation of E2 protein expression by preMu may be conserved amongst all the adenovirus genera. In addition, our data regarding the nucleolar targeting ability of Mu protein further enhances our understanding of the relationship of adenovirus with the nucleolus and suggests that Mu may also play a role in the disruption of the nucleolus. Further investigation will focus on the mechanism of preMu's effect on E2 mRNA, and the activity of preMu in the nucleolus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 May 2003;
accepted 25 September 2003.
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