Download Precursor of human adenovirus core polypeptide Mu targets the

Journal of General Virology (2004), 85, 185–196
DOI 10.1099/vir.0.19352-0
Precursor of human adenovirus core polypeptide
Mu targets the nucleolus and modulates the
expression of E2 proteins
T. W. R. Lee,1 F. J. Lawrence,2 V. Dauksaite,3 G. Akusjärvi,3 G. E. Blair1
and D. A. Matthews2
1
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Correspondence
David Matthews
2
Division of Virology, Department of Pathology and Microbiology, University Walk, Bristol
University, Bristol BS8 1TD, UK
[email protected]
3
Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, S-75123
Uppsala, Sweden
Received 13 May 2003
Accepted 25 September 2003
We have examined the subcellular localization properties of human adenovirus 2 (HAdV-2)
preMu and mature Mu (pX) proteins as fusions with enhanced green fluorescence protein
(EGFP). We determined that preMu is exclusively a nucleolar protein with a single nucleolar
accumulation signal within the Mu sequence. In addition, we noted that both preMu–EGFP and
Mu–EGFP are excluded from adenovirus DNA-binding protein (DBP)-rich replication centres in
adenovirus-infected cells. Surprisingly, we observed that cells in which preMu–EGFP (but not
Mu–EGFP) is transiently expressed prior to or shortly after infection with Ad2 did not express late
adenovirus genes. Further investigation suggested this might be due to a failure to express
pre-terminal protein (preTP) from the E2 region, despite expression of another E2 protein,
DBP. Deletion mutagenesis identified a highly conserved region in the C terminus of preMu
responsible for these observations. Thus our data suggest that preMu may play a role in
modulating accumulation of proteins from the E2 region.
INTRODUCTION
Human adenovirus particles consist of approximately 12
proteins enclosing a genome of linear double-stranded DNA
of approximately 36 kb. This viral DNA is covalently linked
to viral terminal protein and non-covalently bound to the
viral core proteins Mu, V and VII (reviewed by Shenk,
2001). Protein V, found only in mastadenoviruses (Davison
et al., 2003), is believed to form a link between the viral
DNA–core protein complex and the viral capsid (Matthews
& Russell, 1998b), while proteins VII and Mu are tightly
associated with the viral DNA (Chatterjee et al., 1985; Vayda
et al., 1983).
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 proteinrich 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
0001-9352 G 2004 SGM
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 viruscoded 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).
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T. W. R. Lee and others
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 crossreacts 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.
METHODS
Cells and viruses. HeLa cells, cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10 % foetal calf
serum, penicillin (100 IU ml21) and streptomycin (100 mg ml21),
were used to propagate human wild-type 2 adenovirus (HAdV-2).
Cells were infected and viruses purified as previously described
(Matthews & Russell, 1994). For transfection of plasmid constructs,
HeLa cells were grown on glass coverslips in six-well dishes. The
cells were transfected with 2 mg of each plasmid using Lipofectamine
2000 (Invitrogen).
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 promoterbased 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 49,6-diamidino-2-phenylindole (DAPI; Vector
Laboratories). Detection of EGFP-tagged proteins was performed
using a Zeiss Axiovert 135TV microscope with a Neofluor 406 oilimmersion 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,
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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 1006 UV oil-immersion lens.
Fluorouridine labelling of transfected cells. HeLa cells were
transfected with preMu–EGFP or Mu–EGFP constructs as described
and 18 h post-transfection the cells were incubated with 15 mM 59fluorouridine (59FU 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
preMu–EGFP and Mu–EGFP 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.
RESULTS
PreMu and Mu-EGFP fusion protein distribution
in uninfected HeLa cells
Fig. 1(A) shows the deletion mutants of preMu generated,
and localization of the EGFP fusion products in uninfected
cells. In each case the intracellular localization of these
products was consistent across >90 % of transfected cells.
The expression of full-length protein was confirmed by
Western blotting (see Fig. 6D) and nucleolar targeting was
confirmed by comparison with anti-B23 antibody and
phase-contrast microscopy (data not shown). Full-length
preMu–EGFP (1–79EGFP) demonstrated exclusive nucleolar targeting, as did 1–50EGFP. However, 32–79EGFP and
Mu (32–50EGFP) demonstrated nucleolar targeting with a
cytoplasmic background (representative images shown in
Fig. 1C).
Mutations of Mu that affect nucleolar
localization
In order to assess which regions of Mu are important for
nucleolar targeting, a series of Mu mutants was generated
with arginine domains replaced with alanines (Fig. 1B).
This did not ablate nucleolar targeting; however, substitution of the arginines in positions 33 and 34 (mutant 1), 38
and 39 (mutant 2a) or 45 and 46 (mutant 3) resulted in
an apparent modest reduction in nucleolar targeting.
Substituting the two arginines at positions 40 and 41
(mutant 2b) appeared to distinctly reduce the nucleolar
targeting (Fig. 1C viii). The significance of these changes in
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Journal of General Virology 85
Properties of adenovirus preMu protein
Fig. 1. Identification of regions of Mu protein involved in nuclear and nucleolar targeting. (A) Schematic showing the regions
of preMu protein fused to EGFP. On the left, each protein is identified by the amino acids from the full-length preMu protein
sequence, expressed N-terminal to the EGFP sequences. On the right, the subcellular distribution of each fusion protein is
indicated as No. (nucleolar), Np. (nucleoplasmic) and/or Cyt. (cytoplasmic). (B) Mutants of Mu, formed by substitution of
selected arginine codons with alanine codons. Mu protein is shown, with arginines highlighted, followed by each mutant with
substitute alanines underlined. All mutants were formed as N-terminal fusions to EGFP. On the right the subcellular
localizations are described, as above: nucleolar (No.), nucleoplasmic (Np.) and cytoplasmic (Cyt). (C) Subcellular localization
of representative deletion mutants of preMu, and five Mu mutants. (i) Expression pattern of GFP alone; (ii) DAPI staining of cell
transfected with preMu–EGFP; (iii) preMu–EGFP expression pattern; (iv) DAPI staining of cell transfected with Mu–EGFP;
(v) Mu–EGFP expression pattern; (vi) Mu mutant 1; (vii) Mu mutant 2a; (viii) Mu mutant 2b; (ix) Mu mutant 3; (x) Mu mutant 4.
All images were scanned from slide film and labelled using Adobe Photoshop 4.0.
targeting, which although consistent across multiple experiments are difficult to quantify, is unclear. Substituting the
arginine in the sequence position 48 (mutant 4) made no
detectable difference to the nucleolar targeting characteristics of Mu. In addition, we noted that evenly distributing
the arginines within Mu (mutant 5) did not affect the
ability to localize EGFP in the nucleolus.
PreMu–EGFP and Mu–EGFP 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 preMu–EGFP
had no effect on RNA synthesis in the nucleolus. In addition, as with protein V (Matthews, 2001), blocking rRNA
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synthesis using actinomycin D did not affect the localization
of preMu–EGFP (data not shown). In contrast to protein V
(Matthews, 2001), preMu–EGFP and Mu–EGFP did not
affect the gross distribution of nucleolin or B-23. However,
we could not find cells expressing high levels of preMu–
EGFP after 48 h. We were also unable to isolate stable cell
lines expressing preMu–EGFP using an inducible promoter
system. Together these two observations indicate that
expression of preMu–EGFP is toxic to the cells.
PreMu–EGFP and Mu–EGFP were excluded
from the DBP-rich viral DNA replication centres
during adenovirus infection
To determine if the localization of preMu–EGFP or Mu–
EGFP was affected by adenovirus infection, we studied the
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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
preMu–EGFP and Mu–EGFP protein (Fig. 2A, B). Immunofluorescent labelling of DBP enabled us to demonstrate
that, as with protein V, both preMu–EGFP and Mu–EGFP
protein were excluded from DBP-rich areas. Other than
this exclusion, the distribution of both preMu–EGFP and
Mu–EGFP was similar to that seen in uninfected cells.
Expression of preMu–EGFP in HeLa cells
blocked late adenovirus gene expression
In order to further clarify the location of preMu–EGFP and
Mu–EGFP proteins in infected cells a series of fluorescent
antibody staining experiments was undertaken to establish
which adenoviral proteins co-localized with preMu– and
Mu–EGFP-tagged variants. We transfected cells with a range
of EGFP-tagged constructs and infected the cells at the
same time with adenovirus as described. We then assayed
the expression of protein VI at various times >20 h postinfection to ensure that late gene expression had begun.
To our surprise we observed that there was no detectable
adenovirus protein VI in any cells expressing preMu–EGFP
fusion, whilst un-transfected cells did express protein VI
(Fig. 3A). This effect was seen in cells with a wide range of
expression levels of preMu–EGFP (see Fig. 3A), suggesting
that preMu–EGFP inhibited the expression of protein VI,
a product of late gene transcription. The m.o.i. selected
ensured that >95 % of cells were infected and were
producing protein VI.
The expression of other proteins derived from adenovirus
late genes, such as hexon, fibre, penton base and protein V,
was also inhibited but expression of the intermediate gene
product protein IX was reduced, but not ablated (data not
shown). By comparison, Mu–EGFP fusion protein expression had no effect on the production of late proteins
(Fig. 3B). We also determined that a protein V–EGFP
fusion (Matthews, 2001) did not affect late gene expression
Fig. 2. Location of preMu–EGFP and Mu–EGFP in adenovirus-infected cells. Cells were infected with HAdV-2 and
simultaneously transfected with plasmid encoding preMu–EGFP (A) or Mu–EGFP (B). After 20 h the cells were fixed and
expression of DBP was detected using monoclonal antiserum. In these confocal images the cytoplasm is not visible. (A)
(i) Location of preMu–EGFP; (ii) immunofluorescent labelling of DBP; (iii) merge. (B) (i) Location of Mu–EGFP;
(ii) immunofluorescent labelling of DBP; (iii) merge. Scale, 10 mm.
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Properties of adenovirus preMu protein
Fig. 3. PreMu–EGFP but not Mu–EGFP blocked late gene expression in adenovirus-infected cells. Cells were infected with
HAdV-2 and simultaneously transfected with plasmids encoding preMu–EGFP (A) or Mu–EGFP (B). After 30 h the cells were
fixed and expression of protein preVI was detected using polyclonal antiserum. (A) (i) DAPI staining of cells; (ii) location of
three cells expressing notably different amounts of preMu-EGFP; (iii) immunofluorescent labelling of protein VI. (B) (i) DAPI
staining of cells; (ii) location of Mu-EGFP expressing cells; (iii) immunofluorescent labelling of protein VI. All images were
scanned from slide film and labelled using Adobe Photoshop 4.0.
(data not shown). Finally, we determined that expression of
preMu–EGFP 7 h before, after or during infection with
adenovirus precluded late gene expression for at least 30 h
post-infection.
Expression of preMu–EGFP 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 adenovirusinfected cells, those cells transfected with preMu–EGFP 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). Mu–EGFP, protein V–EGFP and EGFP expression did not affect the expression of preTP in these assays.
Molin & Akusjärvi (2000) demonstrated that overexpression of essential splicing factor ASF/SF2 during adenovirus
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infection resulted in a failure to express late genes (but
not DBP) and postulated that this was due to reduced
expression of preTP. We therefore used a RED–ASF/SF2
construct to determine if the results obtained using
preMu–EGFP could be repeated. Our data (Fig. 4B, C)
show that, like preMu–EGFP, RED–ASF/SF2 apparently
blocks the expression of preTP but not that of DBP. We
also noted that, like preMu–EGFP, RED–ASF/SF2 was
excluded from the DBP-rich replication centres.
Overexpression of RED–ASF/SF2 mislocalized a
proportion of preMu–EGFP in uninfected cells
This similarity of effect of preMu–EGFP and RED–ASF/
SF2 on late gene expression led us to examine whether
overexpression of RED–ASF/SF2 would affect the intracellular localization of preMu-EGFP or vice versa.
We therefore transfected uninfected cells with plasmids
expressing both RED–ASF/SF2 and preMu–EGFP. In
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Fig. 4. PreMu–EGFP and RED–ASF/SF2 reduced preTP expression in adenovirus-infected cells whereas RED–ASF/SF2
expression did not affect DBP expression in infected cells. Cells were infected with HAdV-2 and simultaneously transfected
with appropriate plasmids. After 20 h the cells were fixed and expression of protein was detected using appropriate antiserum.
(A) PreMu–EGFP and preTP expression. (i) DAPI staining of cells; (ii) distribution of cells expressing preMu–EGFP;
(iii) immunofluorescent labelling of preTP. (B) RED–ASF/SF2 and preTP expression. (i) DAPI staining of cells;
(ii) immunofluorescent labelling of preTP; (iii) distribution of cells expressing RED–ASF/SF2. (C) RED–ASF/SF2 and DBP
expression. (i) Immunofluorescent labelling of DBP; (ii) distribution of RED-ASF/SF2; (iii) merge. Scale, 10 mm.
>90 % of cells examined we observed mislocalization
of a proportion of preMu–EGFP into the nucleus. This
nuclear preMu–EGFP corresponded with RED–ASF/SF2 in
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extranucleolar sites (Fig. 5A). This effect on the distribution
of preMu–EGFP was only evident in cells overexpressing
RED–ASF/SF2.
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Properties of adenovirus preMu protein
Fig. 5. Uninfected cells expressing preMu–EGFP and RED–ASF/SF2 indicated a proportion of preMu–EGFP was present in
the same regions of the nucleus as RED–ASF/SF2. In both cases, uninfected cells were transfected with appropriate
plasmids and examined 20 h later. (A) Cells transfected with preMu–EGFP and RED–ASF/SF2 and examined 20 h later.
(i) Location of preMu–EGFP in the nucleus and nucleolus. (ii) RED–ASF/SF2. (iii) Merge. (B) Cells transfected with preMu–
EGFP only and counter-stained 20 h later with monoclonal antiserum to detect the splicing factor sc35. (i) PreMu–EGFP in
the nucleus and nucleolus (ii) Anti-sc35. (iii) Merge. Note that in this set of images the preMu-EGFP image has been
deliberately overexposed compared to (A) (i) in order that the location of very low levels on preMu–EGFP in the nucleus could
be detected. In all cases the white bar represents 10 mm.
In contrast, Fig. 5(B) shows that we consistently observed
only a very low level diffuse staining of preMu–EGFP in
the nucleus of cells not transfected with RED–ASF/SF2. For
clarity, we deliberately raised the sensitivity of the confocal
microscope for Fig. 5(B) so that this low-level background
nuclear staining of preMu–EGFP could be clearly seen.
Note there was no correspondence with structures revealed
by anti-sc35 serum which detects another essential splicing
factor, sc35.
Examination of preMu–EGFP transfected cells (Fig. 5A)
revealed that its subnucleolar distribution within the cell
nucleolus was similar to that of protein V–EGFP 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.
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RED–ASF/SF2 did not accumulate in the nucleolus in
any of our experiments, and did not co-localize with protein V–EGFP (data not shown). The deletion mutants
1–50EGFP, 32–50EGFP, 32–79EGFP, 1–69EGFP and
1–59EGFP also co-localized with RED–ASF/SF2, whereas
1–31EGFP and 51–79EGFP did not co-localize with RED–
ASF/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 32–79EGFP affected late
gene expression. Additional deletion mutants demonstrated
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Fig. 6. Identification of regions of preMu protein involved in inhibiting late gene expression in adenovirus-infected cells, and
regions required for co-localization with ASF/SF2. (A) Schematic representation of deletion mutants of preMu–EGFP fusion
protein as in Fig. 1, with indication of the effects of these mutants, and further deletion mutants 1–59EGFP and 1–69EGFP,
on both the expression of adenovirus late gene products and co-localization of mutants with Red–SF2 in adenovirus-infected
cells. (B) Amino acid sequence of a fragment of preMu (51–79), showing the cleavage sites for the further deletion mutants
1–59EGFP and 1–69EGFP, as well as the point mutants formed by replacing the isoleucine at position 63, proline at position
67 and isoleucine at position 69 with glycine in turn. (C) Comparison of amino acid sequence of fragment of preMu protein
after the Mu region, showing sequences from representatives of the four official genera of adenoviruses: Mastadenovirus
[human adenovirus serotypes 2 (WMADH2), 12 (A45393) and 40 (Q64858) and mouse adenovirus 1 (O10443)];
Atadenovirus [duck adenovirus 1 (NP044708)]; Aviadenovirus [fowl adenovirus 1 (CELO) (CAB69562)]; and Siadenovirus
[frog adenovirus 1 (NP 062441)]. Conserved amino acids are in bold type, amino acids selected for mutation are denoted with
an asterisk and amino acids required for blocking late gene expression are underlined. (D) Western blot of EGFP fusion
constructs of key functional importance, to demonstrate that these mutants were expressed at the appropriate size. PreMu–
EGFP and 1–69EGFP ablated the expression of late genes in HAdV-2-infected cells, whilst Mu–EGFP, EGFP alone and
1–59EGFP did not.
that the sequence 1–69EGFP but not 1–59EGFP inhibited
the expression of preTP and protein VI. In order to explore
which regions of 32–69EGFP were responsible for this
effect, site-directed mutagenesis was performed in turn at
sites 63, 67 and 69 within a 1–69EGFP construct (Fig. 6B).
These amino acids are highly conserved between adenovirus species (Fig. 6C), and have large hydrophobic sidegroups. In each case glycine (small hydrophilic) was used as
a non-conservative substitution. Each of these mutants of
1–69EGFP 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
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that of 32–79EGFP, 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 preMu–EGFP 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)
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Properties of adenovirus preMu protein
Fig. 7. Differential tagging of preMu does not affect nucleolar localization or inhibition of late gene expression. (A) Cells were
transfected with plasmid encoding preMu–Myc and allowed to express for 20 h prior to fixation and staining. (i) Location of
preMu–Myc in uninfected cells as detected using an anti-Myc antiserum; (ii) Phase-contrast photograph of the same cells.
(B) Cells were infected with HAdV-2 and simultaneously transfected with plasmids encoding preMu–Myc. After 30 h the cells
were fixed and expression of protein preVI was detected using polyclonal antiserum. (i) Location of preMu–Myc-expressing
cells; (ii) Immunofluorescent labelling of fibre; (iii) DAPI staining of cells.
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 preMu–Myc is expressed exhibit greatly
reduced levels of fibre protein (Fig. 7B).
DISCUSSION
Our data reveal that, as with protein V (Matthews & Russell,
1998b), preMu protein demonstrates nuclear and nucleolar
targeting. Furthermore, the single nucleolar accumulation
motif within the preMu protein corresponds to Mu. In this
regard we can apparently separate nuclear targeting from
nucleolar accumulation as has been described for protein V
(Matthews, 2001). For example, clone 1–50EGFP is nuclear
and nucleolar whereas 32–50 is also cytoplasmic, suggesting
that this clone is not actively targeted to/retained within
the nucleus. Since there is no nuclear localization motif
within 1–31EGFP we suggest that the motifs required to
target or retain 1–50EGFP within the nucleus span the
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N-terminal cleavage site for Mu. This could imply that a
bipartite nuclear localization signal spans this cleavage site.
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
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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 V–EGFP
nor preMu–EGFP 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 preMu–EGFP
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 preMu–EGFP-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.
194
Our data demonstrate that preMu–EGFP 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 preMu–EGFP and RED–ASF/SF2 block preTP expression but allowed DBP expression. Thus our data provided
further evidence that both preMu–EGFP 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 RED–ASF/
SF2 clone was also excluded from the DBP-rich centres.
The exclusion of proteins preMu–EGFP and Mu–EGFP
from the adenoviral DBP-rich areas is compatible with a
model in which preMu influences mRNA biogenesis.
However, we also note that preMu–EGFP 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
RED–ASF/SF2 together with preMu–EGFP in uninfected
cells leads to a proportion of preMu–EGFP accumulating
in the nucleus with RED–ASF/SF2. Clearly, this phenomenon requires overexpression of Red–ASF/SF2 but it
indicates that preMu–EGFP and Red–ASF/SF2 might colocalize under certain conditions in uninfected cells. Intriguingly, only clones with the arginine-rich Mu sequence
present acted in this manner. However, this property of
preMu–EGFP is not sufficient to block late gene expression
in infected cells. For example, Mu–EGFP is affected by
RED–ASF/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 preMu–
EGFP and RED–ASF/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.
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Journal of General Virology 85
Properties of adenovirus preMu protein
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, preMu–Myc, preMu–FLAG and preMu–dsRED
proteins all showed identical characteristics to preMu–
EGFP. 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.
ACKNOWLEDGEMENTS
We would like to thank R. T. Hay, W. C. Russell and B. Valdez for
antisera. We also thank the Molecular Medicine Unit Confocal User
Group for invaluable assistance in using the microscope. The research
is supported by the Medical Research Council (D. A. M. and
T. W. R. L.), the Wellcome Trust (F. J. L.) and the Swedish Cancer
Society (G. A. and V. D.). D. A. M. and T. W. R. L. are Fellows of the
Medical Research Council.
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