Results
Evolution of Pre-pVII in siadenoviruses and identification of
NLSs within Frog siadenovirus 1 (FrAdV1). Nuclear transport of AdV pVII
has been partially characterized for HAdV. Several studies reported that
HAdV5 pVII utilizes several different NLSs, enabling transportation to
the host cell nucleus via various import pathways, whereas very little
is known for other AdVs. While HAdV2 Pre-pVII localized to the
nucleolus, we have recently shown that the psittacine siadenovirus F
(PsSiAdV) Pre-pVII mainly localized in the nucleoplasm, when expressed
in the absence of any other viral proteins, implying important
differences in nuclear localization among diverse AdVs47. To gain insights into the evolutionary aspects of
pVII among siadenovirus members, we performed a phylogenetic analysis
using their primary amino acids sequences and identified FrAdV1 pVII as
the most ancient form within the genus Siadenovirus (Fig. 1A). In
order to better understand the nuclear transport of FrAdV1 Pre-pVII, we
analyzed its primary sequence bioinformatically with cNLSmapper, and by
visual inspection for putative nuclear localization signals (NLSs). Such
analysis revealed four putative NLSs (NLSa:43GYWRRKR SKK A53, NLSb:74RKK IPK TPVGVVGPLWQGTRKR 95;
NLSc:111GRK IKK AR AP120,
NLSd: 141PPRKRRR VA149), of
which NLSb was identified a putative bipartite NLS (Fig. 1B). FrAdV1 and
HAdV5 Pre-pVII share very little sequence identity (26.1%).
Accordingly, the identified NLSs have no clear counterparts in HAdV5
pVII (Fig. 1C).
Since the structure of Pre-pVII proteins is yet to be determined, we
employed AlphaFold2 to generate
models that may provide preliminary insights into their structure
(Supplementary Fig. S1). Predictions suggest HAdV5 Pre-pVII to be
disordered, with an unstructured N-terminus (comprised of a loose string
of five alpha helices, and two beta strands separated by flexible loops)
and a disordered C-terminus (Supplementary Fig. S1A-C). AlphaFold2 also
predicts FrAdV1 Pre-pVII to be an intrinsically disordered protein with
no structured regions (Supplementary Fig. S1D-E). Such disorder would
potentially enable exposure of the putative NLS regions, making them
accessible to the nuclear import receptors. Such a model would likely be
flexible and dynamic and could adopt multiple conformations and be
accessible to bind nuclear import receptors.
FrAdV1 Pre-pVII protein strongly accumulates in the nucleolus in
the absence of other viral proteins . To investigate the
subcellular localization of FrAdV1 Pre-pVII, we transiently expressed a
Pre-pVII GFP-fusion protein, and compared its subcellular localization
to similar fusions with HAdV2 Pre-pVII and PsSiAdV Pre-pVII, which are
known to accumulate into the nucleoli and nucleoplasm, respectively47. As further controls, GFP alone and GFP fused to
human cytomegalovirus (HCMV) DNA polymerase processivity factor UL44
were also expressed 48, while DsRed-fibrillarin was
used as a marker for nucleolar localization (Fig. 2A). As expected, GFP
alone localised with a diffuse pattern distributed between the nuclei
and cytoplasm, whereas UL44 strongly accumulated in the cell nucleus,
and both proteins were largely excluded from the nucleoli (Fig. 2B). As
recently shown, Pre-pVII from PsSiAdV exhibited a localization
reminiscent of UL44, while HAdV2 accumulated in the nucleoli and
colocalised with fibrillarin, although a considerable fraction of the
protein was detectable in the nucleoplasm (Fig. 2B). Intriguingly,
FrAdV1 Pre-pVII strongly accumulated in the nucleoli, with limited
detection in the nucleoplasm, displaying a different localization
pattern to its HAdV2 and PsSiAdV orthologues (Fig. 2B).
FrAdV1 Pre-pVII NLSs can bind to different cellular
transporters. Given their ability to interact with multiple IMPs, HAdVs
pVIIs are believed to be transported into the nucleus by multiple
pathways 10. We therefore assessed the binding of each
FrAdV-1 putative NLS to IMPα2 and IMPβ1 using a quantitative
fluorescence polarization (FP) assay, commonly used in the field23,25,34,35,47 (Figure 3 and Supplementary Table S3).
Interestingly, NLSa interacted with IMPβ1 with high affinity, NLSd
interacted with high affinity with IMPα2, while NLSb and NLSc bound with
low affinity to both IMPα2 and IMPβ1 (Figure 3 and Supplementary Table
S3). These results suggest that FrAdV1 Pre-pVII can interact with
different IMPs via distinct NLSs, consistent with its ability to be
transported into the nucleus via several nuclear import pathways.
FrAdV1 Pre-pVII NLSs can confer IMPα/β dependent and independent
nuclear import to heterologous proteins. Since the FrAdV1 Pre-pVII
strongly accumulated in the nucleoli of transfected cells and contains
four putative NLSs (Fig. 1BC) which can mediate binding to several IMPs
(Fig. 3), we analysed their ability to alter the subcellular
localization of a reporter protein. To this end, we analysed the
subcellular localization of fusion proteins between GFP and FrAdV1
Pre-VII putative NLSs when transiently expressed in mammalian cells
(Fig. 4A), followed by quantitative analysis on nuclear and nucleolar
accumulation levels (Fig. 4BC). We also expressed GFP alone and GFP
fused to Simian Virus 40 (SV40) large tumour antigen (LTA) NLS, as
negative and positive controls for nuclear accumulation respectively. As
expected, GFP alone equally distributed between nucleus and cytoplasm
(Fig. 4B) with a Fn/c of 1.2 (Fig. 4C); GFP-SV40-NLS strongly
accumulated in the cell nucleus (Fig. 4B), with a Fn/c of 6.9 (Fig. 4C);
and both proteins were partially excluded from the nucleoli with a
Fno/Fn < 1 (Fig. 4D). Importantly, fusion to each FrAdV1
Pre-pVII-NLS affected GFP subcellular localization, although to
different extents. The highest levels of nuclear accumulation were
observed for GFP-FrAdV1 pVII-NLSd, with a Fn/c of 7.2 (Fig. 4C), and
similarly to GFP alone, it was mainly excluded from nucleoli (Fno/n
< 1, Fig. 4D). On the other hand, while nuclear accumulation
mediated by either NLSa or NLSc was considerably lower as compared to
NLSd, both sequences could significantly increase GFP targeting to the
nucleolus (Fig. 4D). Finally, NLSb slightly increased GFP nuclear
targeting (Fn/c of. c. 2, see Fig. 4C), but not its nucleolar
accumulation (Fno/n < 1, see Fig. 4D). Our results suggest
that FrAdV1 Pre-pVII contains four sequences which could contribute to
its nuclear localization (NLSa-d), and two (NLSa and NLSc) which could
contribute to its nucleolar targeting. Since we and others have
extensively shown that the peptide inhibitor Bimax2 can abolish IMPα/β
dependent nuclear import 21,25,49, we decided to test
its effect on the activity of FrAdV1 Pre-pVII NLSs. We therefore tested
the effect of co-expression with mCherry-Bimax2 on the nuclear
accumulation of the FrAdV1 Pre-pVII NLSs GFP fusion proteins
(Supplementary Fig. S2). As expected, expression with Bimax2 completely
abolished nuclear targeting of GFP-SV40 NLS (Supplementary Fig. S2),
consistent with its ability to bind exclusively to IMPα/β via IMPα.
Importantly, Bimax2 significantly reduced nuclear accumulation mediated
by NLSd but not by NLSa, NLSb and NLSc (Supplementary Fig. S2B). These
results are consistent with FrAdV1 Pre-pVII NLSd being a cNLS
functionally interacting with IMPα.
High-resolution crystal structure uncovers the classical binding
interface of IMPα FrAdV1 Pre-pVII NLSd. Based on our observation that
the FrAdV1 Pre-pVII NLSd bound to with high affinity to IMPα2, we
undertook X-ray crystallography of this complex to gain a deeper
understanding of the interactions between FrAdV1 Pre-pVII and host
cellular transporters. The protein crystallization process was carried
out using the hanging-drop vapor diffusion method, resulting in the
formation of large rod-shaped crystals within a three-day incubation
period (Fig. 5). These crystals diffracted to 2.2 Å at the Australian
Synchrotron on the MX2 beamline, and the data were indexed in P212121,
with unit cell parameters of a = 77.29, b = 89.29, and c = 95.80. The
structure was solved using Phaser 41 through molecular
replacement with the model derived from PDB entry 3UKX. The resolved
structure identified one chain of FrAdV1 Pre-pVII NLSd
(PPRKRRR VA-149) bound to IMPα2ΔIBB (residues 72-498) with a
well-characterized lysine at the P2 site. Lys144 is the predominant
binding determinant of the major binding site forming hydrogen bonds
with Gly150, Thr155, and Asp192 at the P2 binding pocket of IMPα2, with
a salt bridge formed between Asp192 of IMPα2 and Lys144 of the NLSd
peptide. Arg145 of NLSd interact with the P3 binding site of IMPα2 at
Asn188 and Asn288, via hydrogen bonding whereas Arg146 and Arg147 bound
at P4 and P5 positions (Fig. 5) 39,40. The full data
collection and refinement statistics is given in Supplementary Table S4.
Furthermore, when an excessive
amount of peptide is employed, NLSd was found at IMP⍺ minor binding site
(Fig. 5B). Our results are therefore consistent with NLSd being a cNLS
functionally interacting with IMPα major binding site.
FrAdV1 Pre-pVII can accumulate in the nucleolus in a Ran
independent process. Since nuclear import of HAdV2 Pre-pVII and mature
pVII has been shown to rely on IMPα/β, and IMPβ2, respectively10, we analysed the effect of inhibitors of the IMPα/β
and IMPβ2 nuclear import pathways on the subcellular localization of
FrAdV1 Pre-pVII. Inhibitors tested include mcherry-Bimax230, which impairs IMPα/β dependent nuclear import,
M9M-RFP 29, which inhibits IMPβ2 dependent nuclear
import, and DsRed-RanQ69L 31, affecting Ran dependent
nuclear import (Fig. 6A). GFP-UL44, which is imported into the nucleus
by IMPα/β, was also expressed as a control 48. As
previously, in the absence of nuclear import inhibitors, both GFP-UL44
and FrAdV1 Pre-pVII strongly localised to the cell nucleus (Fig. 6B),
with a Fn/c > 20 (Fig. 6C). Co-expression with either
mCherry-Bimax2 (Fn/c 0.3) or DsRed-RanQ69L (Fn/c 1.1) strongly impaired
nuclear accumulation of GFP-UL44, while no significant reduction was
observed in the presence of RFP-M9M (Fig. 6C). Surprisingly, GFP-FrAdV1
Pre-pVII nuclear import was not significantly impaired by any inhibitor
tested. The lack of response to either Bimax2 or M9M can be interpreted
as the consequence of the ability of Pre-pVII to interact with multiple
IMPs, while nuclear localization in the presence of RanQ69L could be the
consequence of its ability to passively enter the nucleus due to its low
molecular weight, followed by interaction with nuclear components.
Molecular dissection of the role of FrAdV1 individual in nuclear
and nucleolar targeting. To verify these hypotheses, we
dissected the contribution of each NLS identified here to FrAdV1 Pre-VII
nuclear and nucleolar targeting. Therefore, we quantitatively analysed
the subcellular localization of transiently expressed GFP fusion
proteins with FrAdV1 Pre-pVII and several substitution derivatives
thereof, whereby NLS basic residues were replaced either by A or T (Fig.
7A), using DsRed-Fibrillarin as a nucleolar marker. Importantly,
individual inactivation of single NLSs did not significantly reduce
nuclear accumulation (Fig. 7B and C), with barely no signal being
detectable in the cytosol of cells expressing GFP-FrAdV1 Pre-pVII wt
(Fn/c of 17.7), mNLSa (Fn/c of 14.7), mNLSb (Fn/c of 14.8), mNLSc (Fn/c
of 17.9) and mNLSd (Fn/c of 15.69). Simultaneous inactivation of either
NLSa and NLSc (mNLAac, Fn/c of 11), NLSa and NLSd, (mNLSad, Fn/c of 12)
or NLSc and NLSd (mNLScd, Fn/c of 15) only mildly reduced nuclear
localization (Fig. 7C). These results confirm that, when any of FrAdV1
Pre-VII NLS is individually inactivated, the others can functionally
compensate for its absence. However, simultaneous inactivation of NLSa,
NLSc and NLSd was sufficient to significantly reduce nuclear
accumulation (mNLSacd, Fn/c of 7.7). This data suggests that NLSb is
functional in the context of full-length protein, but not sufficient to
mediate optimal nuclear targeting. Accordingly, further inactivation of
NLSb completely abolished nuclear accumulation (mNLSabcd, Fn/c 1.5).
Therefore, FrAdV1 contains multiple functional NLSs responsible of
active nuclear import, which are apparently functionally redundant.
On the other hand, almost any substitution introduced in FrAdV1 Pre-pVII
NLSs significantly affected nucleolar accumulation, although to a
different extent (Fig. 7B). Quantitative analysis revealed that
GFP-FrAdV1 Pre-pVII nucleolar accumulation (Fno/n of c. 20) was
minimally affected by substitutions within NLSa (mNLSa, Fno/n of 13.5)
or NLSd (mNLSd, Fno/n of 10.8), while it was clearly affected by
substitution of either NLSb (mNLSb, Fno/n of 3.7) and especially NLSc
(mNLSc, Fno/n of 1.2). Overall, all derivatives containing substitutions
within NLSc failed to accumulate in the nucleoli, confirming that NLSc
(111GRK IKK AR AP120)
has a major role in nucleolar accumulation, while NLSb
(RKK IPK TPVGVVGPLWQGTRKR -95) also contributes
to nucleolar targeting.
FAdV1 Pre-pVII is transported into the nucleus by IMPα/β and
IMPβ, specifically recognising individual NLSs. To more precisely
characterize the contribution of each NLS identified in this study to
the functional interaction with specific IMPs, we analyzed the effect of
overexpressing inhibitors of specific nuclear import pathways on the
subcellular localization of the FrAdV Pre-pVII substitution derivatives.
To this end, mammalian cells were transfected to express GFP-fusion
proteins (Fig. 8A) in the presence and absence of mcherry-Bimax2,
M9M-RFP or DsRed-RanQ69L, and microscopic images were captured by CLSM
(Fig. 8B), followed by quantitative analysis of the levels of nuclear
(Fn/c; Fig. 8C) and nucleolar (Fno/n; Fig 8D) accumulation. Despite the
nuclear accumulation of FrAdV1 Pre-pVII not being affected by any
inhibitor tested, nuclear import of all substitution derivatives lacking
NLSc (mNLSc, mNLSac, mNLScd and mNLSabc) was significantly inhibited by
RanQ69L (Fig. 8B and C, see Supplementary Figs S3-S5). This suggests
that Pre-pVII is capable of passively diffusing into the nucleus, likely
due to its small size, and accumulating in the nucleolus in the absence
of active transport by binding to nucleolar components dependent on
NLSc, which functions as a NoLS. In the absence of a NoLS, nuclear
accumulation is dependent on Ran-dependent active nuclear import, and
therefore inhibited by RanQ69L. Intriguingly, M9M did not inhibit
nuclear import of any FrAdV1 derivative (Fig. 8BC, Supplementary Figs S6
and S7), despite the reduced accumulation of a GFP-GST-FUS fusion
protein, which has been shown to be functionally depend on transportin
(also called IMPβ2; Supplementary Fig. S8). These findings suggest that
transportin does not play a major role in FrAdV1 Pre-pVII nuclear
import. Finally, nuclear accumulation of most FrAdV1 Pre-pVII
derivatives was not affected by Bimax2 co-expression, unless both NLSa
and NLSc were simultaneously inactivated, such as in the case of FrAdV1
Pre-pVII mNLSac, and Pre-pVII mNLSacd (Fig. 8B and C, Supplementary Figs
S9 and S10). Taken together, our data suggest that each signal
identified in this study plays a specific and independent role in
mediating FrAdV1 Pre-pVII subcellular trafficking. NLSa confers IMPβ1
dependent nuclear import, NLSb is an atypical sequence which contributes
both to IMPα/β nuclear import and nucleolar targeting, and NLSc is the
main NoLS, mediating nucleolar localization by interacting with
intranuclear components, while NLSd is a cNLS conferring IMPα/β nuclear
import (Fig. 9).