2 the establishment of replication complexes. 4 5 6 7 8 9...
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Serine phosphorylation of the hepatitis C virus NS5A protein controls 1
the establishment of replication complexes. 2
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Douglas Ross-Thriepland, Jamel Mankouri and Mark Harris * 4
School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury 5
Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK. 6
* Address for correspondence: School of Molecular and Cellular Biology, Faculty of 7
Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Tel +44 113 343 5632, 8
e-mail: [email protected] 9
Running title: Phenotype of NS5A serine phosphorylation 10
Word count: Abstract 225 (importance: 82), main text (including figure legends): 6951 11
8 Figures, 2 supplementary figures, 3 supplementary videos 12
13
JVI Accepts, published online ahead of print on 31 December 2014J. Virol. doi:10.1128/JVI.02995-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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ABSTRACT. The hepatitis C virus (HCV) non-structural 5A protein (NS5A) is highly 14
phosphorylated and involved in both virus genome replication and virion assembly. We, and 15
others, have identified serine 225 in NS5A as a phosphorylation site but the function of this 16
post-translational modification in the virus lifecycle remains obscure. Here we describe the 17
phenotype of mutations at serine 225, this residue was mutated to either alanine (S225A: 18
phosphoablatant) or aspartic acid (S225D: phosphomimetic) in the context of both the JFH-1 19
cell culture infectious virus and a corresponding subgenomic replicon. S225A exhibited a 10-20
fold reduction in genome replication whereas S225D replicated as wildtype. By confocal 21
microscopy we show that, in the case of S225A, the replication phenotype correlated with an 22
altered subcellular distribution of NS5A. This phenotype was shared by other mutations in the 23
low complexity sequence I (LCS I) – namely S229D, S232A and S235D, but not by mutations 24
with a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). 25
Together with other components of the genome replication complex (NS3, double-stranded 26
RNA and cellular lipids including phosphatidylinositol-4 phosphate), mutant NS5A was restricted 27
to a perinuclear region. This phenotype was not due to cell confluency or another 28
environmental factor, and could be partially transcomplemented by wildtype NS5A. We 29
propose that serine phosphorylation within LCS I may regulate the assembly of an active 30
genome replication complex. 31
IMPORTANCE: The mechanisms by which hepatitis C virus replicates its RNA genome remain 32
poorly characterised. We show here that phosphorylation of the viral non-structural protein 33
NS5A at serine residues is important for the efficient assembly of a complex that is able to 34
replicate the viral genome. This research implicates cellular protein kinases in control of virus 35
replication and highlights the need to further understand the interplay between the virus and the 36
host cell in order to develop potential avenues for future antiviral therapy. 37
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INTRODUCTION 38
The hepatitis C virus (HCV) currently infects an estimated 170 million individuals worldwide and 39
establishes a chronic infection in 85% of cases, which typically progress to liver cirrhosis and 40
hepatocellular carcinoma (23). The virus has a single stranded RNA genome that codes for a 41
~3000 amino acid polyprotein that is cleaved co- and post-translationally into 10 mature viral 42
proteins; Core, E1 and E2 envelope proteins, which make up the viral particle, followed by the 43
viroporin; p7, and the non-structural 2 (NS2) protein, which possesses autoprotease activity. 44
The remaining NS proteins; NS3, NS4A, NS4B, NS5A and NS5B are necessary and sufficient 45
for genome replication (15). With the advent of the full length clone of a genotype 2a isolate 46
(JFH-1), able to undergo the complete virus lifecycle in cell culture, significant progress has 47
been made in understanding how the different viral proteins contribute to the processes of 48
genome replication and virus assembly. In this regard, NS5A has been shown to play a critical 49
role in both of these processes, as well as being known to perturb numerous host pathways in 50
favour of virus persistence. 51
The 5’ untranslated region (UTR) of the viral genome contains an internal ribosome entry 52
sequence (IRES) that allows viral proteins to be translated in a cap-independent fashion 53
immediately after the viral genome is delivered into the cytoplasm. HCV proteins then rapidly 54
recruit and remodel ER-derived membranes to form a ‘membranous web’ (MW), a sub-cellular 55
structure comprised of single, double and multi-membrane vesicles (SMV, DMV and MMVs) 56
that are enriched in viral (eg NS3, NS5A and NS5B) and host cell (eg VAP-A) proteins (16, 19). 57
By analogy to other positive-sense RNA viruses, this enrichment of viral NS proteins, the 58
presence of double stranded (ds) RNA and the observation that isolated membrane fractions 59
retain replication activity, has led to the MW being proposed as the site of viral genome 60
replication (16). In Huh7 cells, the MW is extensively distributed throughout the cytoplasm, 61
correlating with the observed sub-cellular distribution of the NS5A and NS3 proteins as being 62
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discrete puncta, throughout the cytoplasm. The formation and maintenance of the MW has 63
been shown to be dependent on the activation of the phosphatidylinositol 4-kinase type III alpha 64
isoform (PI4KIIIα) by NS5A, and the subsequent elevated production of phosphatidylinositol-4-65
phosphate (PI4P) by this lipid kinase (1, 11, 18). Additionally, lipid droplets (LD) are a host 66
organelle responsible for the normal cellular storage of neutral lipids, and have been shown to 67
be central to the HCV lifecycle. While the site of virus assembly has not been elucidated, it is 68
known that both Core and NS5A coat the surface of LD, displacing the host factor adipocyte 69
differentiation-related protein (ADRP). In order for virus genomes that have been newly 70
synthesised in the MW to be packaged into virions, the Core-coated LD are thought to be 71
recruited to the MW, possibly in a NS5A directed manner, in order to allow the encapsidation of 72
viral RNA (4, 14). 73
NS5A is a highly phosphorylated protein, comprised of three domains and tethered to 74
membranes by an N-terminal amphipathic helix. Domain I is highly structured and sufficient for 75
NS5A to dimerise, while domains II and III are intrinsically disordered, but have recently been 76
shown to have elements of transient secondary structure throughout (6, 7, 10). These three 77
domains are linked by low complexity sequences (LCS) (26), a serine rich sequence links the 78
first two domains (LCS I) while a proline rich sequence links the last two domains (LCS II). 79
NS5A exists in two different phosphorylated forms that can be readily resolved by SDS-PAGE. 80
These forms, termed the basal and hyperphosphorylated species, are thought to represent 81
different functional pools of NS5A involved in different stages of the virus lifecycle, but as yet 82
there is little direct evidence to support this hypothesis. Until recently, the sites of NS5A 83
phosphorylation had not been unambiguously mapped, their locations having been inferred 84
from mutagenesis data. Recently however, we and others, have used mass spectrometry to 85
demonstrate that NS5A, purified from cells actively replicating the HCV subgenomic replicon 86
(SGR), is extensively phosphorylated within the LCS I, containing at a minimum seven 87
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phosphorylation sites (9, 13, 21). We also showed that NS5A contains phosphorylation sites in 88
domain I, domain II and LCS II. Previous work to characterise the role of LCS I phosphorylation 89
has focused almost entirely on a role in virus replication and/or assembly/release, and 90
numerous studies have shown several serines to be important for viral replication, but in a 91
genotype dependent manner. These studies have however, not explored the mechanism 92
through which the phosphorylation of LCS I might be acting. 93
We show here that the phosphorylation of serine 225 within LCS I is critical for the correct 94
localisation of viral proteins during infection. Ablation of serine 225 phosphorylation results in a 95
10-fold reduction in replicative fitness. We show using confocal microscopy that this phenotype 96
is concomitant with a dramatic relocalisation of both cellular and viral factors known to 97
participate in genome replication. This phenotype is shared with other serine mutations in LCS 98
I, further illustrating the complexity of phosphorylation in the region of NS5A. 99
100
MATERIALS AND METHODS 101
Plasmids. DNA constructs of and the full length pJFH-1 virus (27) and the luciferase sub-102
genomic replicon (SGR-luc-JFH-1) (25) were used throughout. Previously, the unique restriction 103
sites BamHI/AfeI flanking NS5A were introduced into both full length virus and SGR-luc-JFH-1 104
constructs, these constructs were denoted as mJFH-1 and mSGR-luc-JFH-1 respectively, and 105
shown to have no effect on virus genome replication or assembly and release (8). Mutagenesis 106
of NS5A was performed on a LITMUS28i (NEB) subclone containing an NsiI/HindIII fragment of 107
the mJFH-1 cDNA, by Quikchange site-directed mutagenesis (Stratagene), then cloned into 108
either mSGR-luc-JFH-1 or mJFH-1 via flanking BamHI/AfeI sites and confirmed by sequencing. 109
For the insertion of the SNAP/CLIP tag into NS5A, the gene was amplified from the pSNAPf 110
and pCLIPf vectors (NEB) using primers that inserted a flexible linker sequence (GSSGSS) 111
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either side of the insertion (primers sequences available upon request). This amplicon was 112
inserted into the aforementioned LITMUS28i sub-clone by BclI digestion and correct insertion 113
verified by sequencing. The NS5A(SNAP) or NS5A(CLIP) region was then inserted into the 114
mSGR-luc-JFH-1 by flanking RsrII/AfeI sites. For plasmid expression mutated NS5A coding 115
sequences were subcloned into the pCMV(NS3-5B) vector as described previously (20). All 116
mutations were verified by sequencing. 117
Cell culture. Huh7 cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM; Sigma) 118
supplemented with 10% foetal bovine serum (FBS), 100 IU penicillin/ml, 100 μg streptomycin/ml 119
and 1% non-essential amino acids in a humidified incubator at 37°C, 5% CO2. 120
Electroporation of replicon and virus RNA constructs. The preparation of in vitro 121
transcripts and electroporations for both mSGR-luc-JFH-1 and mJFH-1 were conducted as 122
described previously (20). In brief, 2 x 106 Huh7 cells in DEPC-PBS were electroporated with 5 123
μg of in vitro RNA transcripts using a square wave protocol, 260 V and 25 ms. Subsequently, 124
cells were resuspended in complete DMEM media and seeded at 1 x 104 cells/cm2 culture area, 125
into either 12- or 6-well dishes. 126
Virus titration. Supernatants were titrated on Huh7 cells in 96-well format as follows, clarified 127
virus supernatant were serially diluted 2-fold in complete DMEM media before addition of 100 μl 128
to cells seeded 8 hours prior at 8 x 103 cells/well, final volume 200 μl. The counting of infected 129
cells was automated by acquisition and analysis using an Incucyte ZOOM. Virus titre was 130
determined by averaging the positive cell count from 3 or more adjacent counted wells that 131
gave corresponding titres. 132
SDS-PAGE/Western blot. Cells were washed twice in PBS, lysed in 1 x Glasgow lysis buffer 133
(GLB - 1% (v/v) Triton X-100, 120 mM KCl, 30 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 10 134
mM PIPES-NaOH, pH 7.2 with protease and phosphatase inhibitors) and clarified at 2800 x g 135
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for 5 min at 4oC before determining and normalising protein concentration by BCA assay 136
(Pierce). Following separation by SDS-PAGE, proteins were transferred to a polyvinylidene 137
difluoride (PVDF) membrane and blocked in 50% (v/v) Odyssey blocking (OB) buffer (LI-COR) 138
in PBS. The membrane was incubated with primary antibodies overnight at 4oC, followed by 139
secondary antibodies for 2 h at RT, both prepared in 50% OB buffer. Primary antibodies used 140
were; anti-NS5A (sheep), 1:5,000 (12); anti-NS3 (sheep), 1:2,000 (in-house); and anti-141
glyceraldehyde-3-phosphate dehydrogenase (GAPDH, mouse), 1:20,000 (Sigma). Secondary 142
antibodies were anti-goat (800 nm) or anti-mouse (700 nm), used at 1:5,000 prior to imaging 143
fluorescence using a LI-COR Odyssey Sa infrared imaging system. Quantification of 144
fluorescently labelled Western blots were carried out using Image Studio v3.1 (LI-COR) using a 145
background subtraction method. 146
Immunofluorescence and confocal microscopy. Cells were washed once in PBS before 147
fixation for 20 mins in 4% (w/v) PFA, cells were subsequently permeabilised in 0.2% (v/v) 148
Triton-X-100, PBS before immunostaining with denoted antibody. Primary antibodies used 149
were; anti-NS5A (sheep), 1:1,000; anti-NS3 (sheep), 1:300 (in-house); anti-dsRNA (mouse), 150
1:200 (Scicons); anti-PI4P (mouse), 1:100 (Echelon). Various fluorescently conjugated 151
secondary antibodies were used at 1:1000 (Life Technology). Note, when conducting 152
immunostaining for dsRNA, all reagents were prepared as ‘RNAase free’ by DEPC treatment. 153
Lipid droplets were stained using the BODIPY(558/568)-C12 dye at 1:1000 (Life Technology) 154
added at the same time as the fluorescent secondary antibody. Confocal microscopy images 155
were acquired on a Carl Zeiss LSM 700 inverted microscope, post-acquisition analysis was 156
conducted using either Zeiss Zen 2012 or Fiji v1.49 software (22). 157
In cell labelling of SNAP/CLIP tag. Huh7 cells harbouring SNAP/CLIP replicons were fixed in 158
4% PFA and permeabilised in 0.2% Triton-X-100, PBS with the addition of 0.9 mM CaCl2 and 159
0.5 mM MgCl2 (PBS++). SNAP and CLIP substrates were dissolved to 1mM in DMSO, before 160
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preparing a 1μM labelling solution in PBS++ containing 10% (v/v) FBS, and addition to cells for 161
2 h at RT. For immunostaining of NS5A, antibody was added to SNAP/CLIP labelling solution, 162
followed by secondary antibody staining as detailed earlier. 163
Quantification of lipid droplets. For quantification of LD spatial arrangement, images were 164
acquired with the same acquisition parameters, but with variable gain to ensure correct 165
exposure. The spatial coordinates of LD were determined using the FindFoci function of the 166
GDSC plugin for Fiji, with the nuclear envelope being manually outlined (utilising the DAPI 167
staining as reference) and coordinates generated by Fiji. The distance from each lipid droplet to 168
the nuclear envelope was then determined using trigonometry. LD spatial distribution data was 169
generated for 12 randomly selected cells for each JFH-1 virus variant and data combined into a 170
box and whisker plot (Fig. 3D). 171
Statistics. Data sets analysed using a Students t-test assuming a two tailed, unequal variance 172
to determine statistical difference from wildtype (WT). n= 3 or greater throughout and * p<0.05, 173
** p<0.01, *** p<0.001. 174
175
RESULTS 176
Phosphorylation of serine 225 is important for genome replication, but not assembly, of 177
infectious virus. Previously we identified serine 225 as being one of a number of serines within 178
the LCS I of NS5A that is phosphorylated during the normal course of infection. To investigate 179
the role of serine 225 phosphorylation in the virus lifecycle we generated either a 180
phosphoablatant alanine substitution, or a phosphomimetic aspartic acid substitution at this 181
residue, in the context of both mSGR-luc-JFH-1 and mJFH-1 constructs, by site directed 182
mutagenesis as described previously (21). Huh7 cells were then electroporated with in vitro 183
transcripts of mSGR-luc-JFH-1 and genome replication followed over 72 h by luciferase activity. 184
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As seen previously, the phosphoablatant mutation S225A resulted in a 10-fold reduction in 185
genome replication efficiency, while the phosphomimetic mutation S225D had no discernible 186
effect (Fig. 1A). To investigate whether the phosphorylation of serine 225 had additional effects 187
on the release of infectious virus, in vitro transcripts of the mJFH-1 virus with the S225A/D 188
mutations were electroporated into Huh7 cells and released virus titre followed over 144 h by 189
focus forming assay. Figure 1B shows that the S225A mutant virus has around a 10-fold 190
reduction in infectious virus compared to WT, while the S225D mutant virus had titres 191
indistinguishable from wildtype. This reduction in released virus titre for the S225A mutant 192
correlated with the impairment to replication, in line with previous studies showing that genome 193
replication is the rate limiting step in the release of infectious virus (2). These data illustrate that 194
the phosphorylation of serine 225 is important in the replication of viral genomes, and not the 195
release of infectious virus, as previously concluded. 196
Cell lysates from infected cells were collected at 96 and 144 hpe, normalised for protein 197
concentration and analysed by SDS-PAGE/western blot. It was found that there was a reduction 198
in the level of cellular NS5A to a degree comparable with the reduction in virus replication (Fig. 199
1C), quantification of the western blot confirmed this (Fig. 1D). However, when the ratio of 200
hyper- to basal-phosphorylation was determined it was found that the S225A mutant, at both 201
early and late time points, exhibited a reduction in the extent of hyperphosphorylation (Fig. 1D). 202
This correlated with the involvement of the LCS I in hyperphosphorylation previously reported 203
by us and others (9, 13, 21). 204
It was noted when normalising protein concentration for SDS-PAGE that cells infected with the 205
S225A mutant virus were growing at a faster rate than wildtype and S225D mutant virus 206
infected cells (data not shown). To account for this, the released virus titre at 96 and 144 hpe 207
was normalised to cellular protein concentration, used here as a surrogate for cell number, and 208
it was observed that the S225A mutant still exhibited an approximate 10-fold reduction in virus 209
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release compared to wildtype (Fig. 1E). To ensure that cell confluency (known to negatively 210
affect HCV replication) was not affecting released virus titres, electroporated cells were also 211
passaged at 96 hpe, and at 144 hpe released virus titre determined and normalised to cellular 212
protein (Fig. 1E). As previously, the S225A mutant exhibited a 10-fold reduction in virus release. 213
Ablation of serine 225 phosphorylation alters the sub-cellular distribution of NS5A When 214
titrating virus by focus forming assay the sub-cellular distribution of NS5A(S225A) was 215
observed to be markedly different from that of the wildtype protein. Typically in wildtype JFH-1 216
infected cells NS5A was present throughout the cytoplasm in discrete puncta, however in cells 217
infected with the S225A mutant virus NS5A localised into defined, condensed structures. To 218
investigate this further, Huh7 cells were electroporated with in vitro transcripts of mJFH-1 virus 219
(wildtype, S225A and S225D), fixed at 96 hpe and immunostained for NS5A as described. As 220
previously observed, wildtype NS5A was distributed into small, discrete puncta that were 221
abundant and relatively uniformly distributed throughout the cytoplasm (Fig. 2A). In contrast 222
NS5A(S225A) exhibited bright, condensed clusters that were less abundant and typically 223
localised around the nucleus (Fig. 2B). Importantly the phosphomimetic mutation S225D 224
exhibited a wildtype phenotype (Fig. 2C), strongly suggesting that phosphorylation of serine 225 225
is important for the normal sub-cellular localisation of NS5A. 226
The S225A mutation results in relocalisation of genome replication complexes. We then 227
asked whether the relocalisation of NS5A(S225A) resulted in any changes to the distribution of 228
other viral and cellular markers of replication complexes. Firstly, we looked at the distribution of 229
lipid droplets (LD), which serve as a neutral lipid reservoir for the host and have been shown 230
previously to be an essential host factor involved in HCV replication, with both NS5A and core 231
localising to their surface in infected cells (14). In the case of both wildtype and the two mutant 232
JFH-1 viruses, NS5A colocalised to the periphery of LD (Figs. 3A-C), and the distribution of the 233
LDs correlated with that of NS5A. Specifically, in cells infected with the S225A mutant virus, 234
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LDs clustered close to the nucleus, whereas in the context of either wildtype or S225D, the LDs 235
were more evenly distributed throughout the cytoplasm. In uninfected cells the distribution of 236
LDs more closely resembled the wildtype or S225D infected cells (Fig 3D). 237
To quantify this LD redistribution, we acquired images of 12 randomly selected cells for the 238
wildtype and each virus mutant, and quantified both the number of LDs, and their distance from 239
the nuclear membrane. With this quantitative approach we saw that in comparison to uninfected 240
cells, wildtype virus infected cells exhibited a broader distribution throughout the cytoplasm, 241
whereas in the context of the S225A mutation there was a significant shift of LD towards the 242
nucleus (Fig 3E). Interestingly however, the S225D mutation had an intermediate phenotype 243
that resembled uninfected cells, where there was a slight shift of LD towards the nucleus, 244
indicating that this mutation was not quite fully able to re-capitulate the effect of phosphorylation 245
at serine 225, at least with respect to LD distribution (Fig. 3E). With this data we were also able 246
to interrogate the total number of LD per cell. There was around a 2-fold increase in the number 247
of LD in cells infected with either wildtype or the S225D mutant viruses, compared to naïve cells 248
(Fig. 3E). This was not the case for the S225A mutant, where the number of LD per cell was 249
broadly comparable to uninfected cells (Fig. 3E), suggesting that NS5A(S225A) is unable to 250
increase the formation of LD upon infection. 251
The viral protease/helicase, NS3, is another key component of genome replication complexes, 252
therefore to further investigate the potential redistribution of these complexes by the S225A 253
mutation we co-stained infected cells with antibodies to both NS5A and NS3. As expected, NS3 254
extensively colocalised with NS5A in all cases, such that in the context of NS5A(S225A) it 255
exhibited a restricted perinuclear distribution, whereas for both wildtype and NS5A(S225D), 256
NS3 was extensively distributed throughout the cytoplasm (Fig. 4A). A similar phenotype was 257
observed for the distribution of double stranded (ds) RNA, a hallmark of viral genome replication 258
that can be detected using a specific antibody, J2 (24). As shown in Fig. 4B, in cells infected 259
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with mJFH-1 the dsRNA antibody detected small, bright puncta, typically of a uniform size and 260
distributed throughout the cytoplasm. In cells electroporated with a replication-defective mutant 261
(GND) however, there was little to no reactivity towards the dsRNA antibody, suggesting that 262
this staining was specific to cells undergoing virus genome replication (Fig 4B). Furthermore, 263
this dsRNA staining could be observed at very early time points post infection (24 h), further 264
supporting the notion that these puncta are active replication complexes, and not accumulation 265
of structured RNA in stress granules (data not shown). Whilst the distribution of dsRNA-positive 266
punctae was not as extensive as that of lipid droplets (Fig. 3) or NS3 (Fig. 4A), it again 267
correlated to the distribution of NS5A. Thus in cells infected with either wildtype or S225D 268
mutant viruses, dsRNA-positive punctae could be seen throughout the cytoplasm, whereas for 269
the S225A mutant virus they were less numerous and restricted to a perinuclear distribution. 270
The limited distribution of the dsRNA-positive punctae may be a consequence of the lack of 271
sensitivity of the antibody, or may indicate that there are only a limited number of active 272
replication complexes, much less than the total number of structures containing NS3 and NS5A. 273
Lastly, we investigated the distribution of a host cell factor, the phospholipid phosphatidylinositol 274
4-phosphate (PI4P). It has been well established that infection by HCV, and many other RNA 275
viruses, results in the increased production of PI4P lipids, and it is widely accepted that this is 276
one of the mechanisms through which HCV is able to induce the formation of the membranous 277
web (1, 11, 18). Using an antibody specific to PI4P we showed that in cells infected with 278
wildtype or S225D mutant viruses, PI4P levels were elevated in comparison to uninfected cells 279
(data not shown), and that there was a strong colocalisation between NS5A and PI4P, with 280
PI4P typically forming small irregular puncta (Fig. 4C). In contrast, cells infected with the S225A 281
mutant virus showed a dramatic reduction in the abundance of PI4P punctae and their 282
relocalisation to a perinuclear distribution along with NS5A (Fig. 4C). 283
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From this confocal microscopy and immunofluorescence analysis we conclude that the 284
phosphoablatant mutation S225A in NS5A is sufficient to induce the relocalisation of numerous 285
factors associated with HCV genome replication. It is important to note that we did not observe 286
loss of colocalisation of NS5A with these factors, reflecting the fact that NS5A(S225A) retained 287
the ability to replicate and release infectious virus. 288
The relocalisation of NS5A(S225A) and viral/host factors is not a result of reduced HCV 289
replication. It was important to establish that this phenotype was specifically the result of the 290
loss of phosphorylation at serine 225, and not the result of the lower levels of replication (and so 291
viral proteins) that is observed with the S225A mutation. To achieve this we utilised two 292
previous characterised mutations within domain II of NS5A, P315A and L321A, that had been 293
previously shown to have a similar (approx. 10-fold) impairment to virus replication as S225A 294
(20). 295
As expected, both the NS5A(P315A) (Fig 5A), and NS5A(L321A) (data not shown), mutant 296
viruses exhibited an approximately 10-fold reduction in released virus which was comparable to 297
the S225A mutation, confirming that S225A, P315A and L321A exhibited a similar level of 298
impairment in virus replication. Both mutant viruses exhibited a similar reduction in levels of 299
NS5A expression as seen for S225A (Figs 5B and 5C and data not shown), and exhibited a 300
wildtype ratio of NS5A hyper- to basal-phosphorylation (Fig. 5C and data not shown), in 301
comparison to both S225 mutants, consistent with the previously proposed notion that S225 is a 302
site of phosphorylation in the hyperphosphorylated species of NS5A (21). Therefore, if the 303
phenotype of NS5A relocalisation was the result of reduced replication, it would be expected 304
that both P315A and L321A would show the same phenotype. 305
Huh7 cells infected with NS5A(P315A) (Fig 5D), or NS5A(L321A) (data not shown) mutant 306
viruses were analysed by confocal microscopy for the abundance and distribution of NS5A and 307
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LD. Reassuringly the distribution of NS5A for both these mutants was indistinguishable from 308
wildtype, with no evidence of the restriction to a perinuclear distribution. Quantitative analysis 309
revealed no significant difference between wildtype and the two mutants with regard to the 310
distance of LD from the nuclear membrane (Fig. 5E). Similar results were seen for the 311
distribution of NS3 and PI4P (Fig 5D), as well as for dsRNA (data not shown). It is noteworthy 312
that, although the distribution of PI4P lipids in NS5A(P315A) virus-infected cells was similar to 313
wildtype, there was an obvious reduction in their quantity, suggesting that this mutant might 314
have an independent effect on NS5A-mediated PI4K activation. This observation 315
notwithstanding, we conclude that the effect of the S225A mutation on the distribution of 316
replication complex components cannot be explained by a reduction in replicative fitness, rather 317
it is specifically the loss of phosphorylation at serine 225 that is responsible for this phenotype. 318
The S225A phenotype does not correlate with a loss of hyperphosphorylation. Although 319
our original observation was made during routine virus titration experiments we had not 320
screened an entire panel of serine mutants within LCS I. Given the complexity of the 321
phosphorylation patterns within this region, and the potential role of S146 in regulating 322
hyperphosphorylation (21), we therefore generated a complete panel of either phosphomimetic 323
or phosphoablatant mutants in all 8 serines in LCS I and S146, and examined their phenotypes 324
both by virus titration and immunofluorescence. Consistent with our previous analysis (21), and 325
that of Masaki et al (13), most mutations had no effect on virus titre, however S225A, S232A 326
and S235D all exhibited a significant reduction (approx. 10-fold) (Fig. 6A). S232A and S235D 327
also shared the restricted perinuclear distribution of NS5A and LD (Fig. 6B). As demonstrated 328
previously (13), S229D did not produce any infectious virus however it exhibited limited RNA 329
replication and protein expression (13, 21), and the distribution of both NS5A and LD was again 330
restricted to a perinuclear region. We conclude that phosphorylation of different serines within 331
LCS I can have opposing effects on the subcellular distribution of NS5A. 332
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Lastly, we examined the phenotype of a double mutant – S146D, S225D – we had previously 333
shown that this mutant replicated as well as wildtype but almost completely abolished 334
hyperphosphorylation (21). The distribution of NS5A in cells infected with this mutant was 335
comparable to wildtype (Supplementary Fig 1), confirming that the S225A phenotype of 336
restricted perinuclear distribution could not be explained by a defect in hyperphosphorylation. 337
As an aside, none of the mutants (including S225A) exhibited any significant differences in 338
sensitivity to DCV compared with wildtype. The EC50 values ranged from 10-57 pM (in our 339
hands a wildtype JFH-1 replicon had an EC50 of 57 pM), and although treatment with DCV 340
reduced the abundance of NS5A it did not alter the widespread cytoplasmic distribution (data 341
not shown). 342
The S225A phenotype is not the result of cell culture conditions. To enable us to 343
interrogate further the S225A phenotype, we utilised the SNAP and CLIP tag (NEB) technology. 344
This complementary tag system allows for specific conjugation of either benzylguanine (SNAP) 345
or benzylcytosine (CLIP) derivatives, typically fluorescently labelled, to the SNAP or CLIP tag 346
respectively. This approach has been recently described for NS5A (5), and we reasoned that it 347
would enable us to differentially label wildtype NS5A, S225A and S225D mutants within the 348
same population of cells. The advantage over traditional GFP- and RFP-tags is a combination 349
of the flexibility offered with the SNAP/CLIP system and the high sequence similarity between 350
the SNAP and CLIP tags. 351
The SNAP/CLIP tags were initially introduced into the wildtype mSGR-luc-JFH-1 replicon (8) at 352
position 431 in NS5A (domain III) as this site has previously been shown to tolerate large 353
insertions (Fig. 7A). We confirmed that the insertion of either tag had no discernible effect on 354
the replication kinetics of the mSGR-luc-JFH-1 replicon when compared to the untagged 355
wildtype replicon (Fig. 7B). As the SNAP and CLIP-tags share a very high sequence similarity, 356
yet retain different substrate specificity, it was important to establish that the insertion of the tag 357
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at an internal position did not introduce conformation changes that affected the substrate 358
specificity. As such, cells harbouring the mSGR-luc-JFH-1-NS5A(SNAP) replicon were fixed at 359
72 hpe, immunostained for NS5A and labelled with a CLIP-TMR fluorophore. The converse, 360
where NS5A(CLIP) was labelled with the SNAP-TMR fluorophore, was also carried out. Fig. 7D 361
shows that both the SNAP and CLIP tags retain both catalytic activity and specificity towards 362
the appropriate SNAP/CLIP substrate, with SNAP-TMR only labelling the NS5A(SNAP) and 363
CLIP-TMR only labelling the NS5A(CLIP). The corresponding immunostaining for NS5A also 364
showed a very high degree of correlation, confirming that there was no sub-population of either 365
NS5A(SNAP) or NS5A(CLIP) that was not labelled with the SNAP/CLIP substrates. 366
We proceeded to use the SNAP/CLIP system to establish whether there was a hitherto 367
unknown environmental factor that was inducing the S225A phenotype. To do this we 368
generated mSGR-luc-JFH-1-NS5A(S225A-SNAP) and mSGR-luc-JFH-1-NS5A(S225D-CLIP) 369
replicons. As shown in Fig. 7B, these two mutants retained their replication phenotypes (10-fold 370
impairment for S225A and wildtype for S225D) when expressed as SNAP/CLIP fusions 371
Analysis of cell lysates by western blot showed comparable abundance and hyper/basal 372
phosphorylation ratios of NS5A to that observed previously for the untagged mutants (Fig. 7C). 373
As seen in the context of virus infected cells (Figs. 3 and 4), cells harbouring the NS5A(SNAP-374
S225A) replicon exhibited both a reduced abundance, and restricted perinuclear distribution, of 375
NS5A (Fig. 7E)), in comparison to either wildtype (SNAP tagged) or NS5A(S225D-CLIP) 376
replicon harbouring cells. 377
To confirm that the S225A phenotype was not due to an environmental factor such as 378
differences in cell confluency, we electroporated either mSGR-luc-JFH-1-NS5A(CLIP) or 379
mSGR-luc-JFH-1-NS5A(S225A-SNAP) replicon RNAs into Huh7 cells and co-seeded the 380
electroporated cells into single cultures. By confocal microscopy we observed that within the 381
same population of cells, in the same field of view, those harbouring the wildtype NS5A(CLIP) 382
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replicon had a normal distribution of NS5A, while those harbouring the NS5A(S225A-SNAP) 383
replicon continued to show the restricted NS5A localisation (Fig. 8A). 384
To further investigate the NS5A(S225A) phenotype we utilised the SNAP/CLIP tag technology 385
to ask if NS5A(S225A) could be transcomplemented by wildtype NS5A. In our experience the 386
simultaneous introduction of two replicons into a single cell is a rare event, for reasons that are 387
not clear, particularly given the ratio of RNA molecules to cells in a typical electroporation 388
experiment. However, after co-electroporation of mSGR-luc-JFH-1-NS5A(CLIP) and mSGR-389
luc-JFH-1-NS5A(S225A-SNAP) replicon RNAs into Huh7 cells we did observe some cells that 390
were stained with both SNAP-505 and CLIP-TMR fluorophores, consistent with the presence of 391
both actively replicating replicons in a single cell. In these cells we observed two distinct 392
phenotypes; either the presence of wildtype NS5A(CLIP) was sufficient to restore the 393
localisation of NS5A(S225A-SNAP) to that of wildtype, resulting in a broad distribution of both 394
and high level of colocalisation (Fig. 8B); or the distribution of NS5A(S225A-SNAP) remained in 395
bright, condensed and amorphous structures, with wildtype NS5A(CLIP) being distributed 396
normally throughout the same cell (Fig. 8C). 397
398
DISCUSSION 399
We present here our investigation of the phenotype of mutations at residue serine 225 in NS5A 400
of JFH-1. We propose that the phosphorylation of serine 225 is important for genome 401
replication; mutation of this residue to alanine (S225A) resulted in a relocalisation of NS5A and 402
other components of the HCV genome replication complex (NS3, dsRNA and PI4P lipids) to a 403
perinuclear distribution, in comparison to either wildtype or a phosphomimetic (aspartic acid) 404
substitution (S225D). This altered sub-cellular localisation was accompanied by a 10-fold 405
reduction in genome replication, again in comparison to wildtype or S225D. This phenotype, 406
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together with the reduction in genome replication, was shared with three other mutants in LCSI 407
(S229D, S232A and S235D) but importantly was not seen in the context of other NS5A mutants 408
with a similar replication defect (P315A and L321A). These data are consistent with serine 409
phosphorylation in LCSI either positively (S225 and S232), or negatively (S229 and S235), 410
regulating the formation and distribution of replication complexes. This conclusion also fits well 411
with the concept of a phosphorylation-mediated switch of NS5A functions between replication 412
and virus assembly, although the mechanics of such a switch remain elusive. Intriguingly, S232 413
corresponds to the residue which was the target for a dramatic culture adaptation (20,000-fold) 414
in the context of genotype 1b replicons (S2204I in polyprotein numbering). Despite the 415
conservation of the sequence of LCS I between HCV genotypes these observations suggest 416
that this does not manifest in a concomitant conservation of function as both we (Fig 6A), and 417
others (13) do not see any major effect of mutations at this residue on JFH-1 virus replication. In 418
the future it will be important to compare the sites of phosphorylation and phenotypes in a range 419
of HCV genotypes to address these questions. 420
How might the altered distribution of the mutant NS5A be explained? A clue may come from 421
the experiments examining the distribution of PI4P lipids (Fig 4C). PI4P lipids are produced by 422
PI4K, and previous work from other laboratories (1, 11, 18) has demonstrated that the PI4KIIIα 423
isoform is required for HCV genome replication. Indeed PI4KIIIα has been shown to bind to 424
NS5A, the binding site mapping to a short region at the C-terminus of domain I (residues 202-425
210) (17), and disruption of this interaction had a profound effect on replication (100-fold 426
reduction), blocked the stimulation of PI4P formation, and led to a collapse of the membranous 427
web. In this regard, the restricted distribution and clustering of NS5A seen in the context of the 428
S225A mutation is reminiscent of the NS5A localisation in cells silenced for expression of 429
PI4KIIIα (18). We propose therefore that the replication defect of S225A may in part be 430
explained by the inability of this NS5A mutant protein to effectively recruit and activate PI4KIIIα. 431
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This simplistic explanation is complicated by the results of experiments in which we expressed 432
NS5A in the context of an NS3-5B polyprotein from a CMV promoter driven plasmid vector 433
(data not shown). In this situation the non-structural proteins can form a replication complex but 434
in the absence of a cognate RNA template no genome replication can occur. Intriguingly, under 435
these conditions the S225A phenotype is not apparent: the distribution and abundance of both 436
NS5A and PI4P in transfected cells is very similar for both wildtype and S225A. Thus we would 437
further propose that the function of S225 phosphorylation in regulating interactions between 438
NS5A and PI4KIIIα (and/or other cellular components) is only manifest in the context of a 439
replication complex that is actively replicating the viral genome. What is difficult to reconcile is 440
the observation that inhibiting the interaction between NS5A and PI4KIIIα increased 441
hyperphosphorylation of NS5A (17), concomitant with a reduction in virus genome replication. 442
In contrast, our data show a loss of hyperphosphorylation of S225A (Fig. 1D) and indeed our 443
previous data (21) failed to observe a correlation between hyperphosphorylation and replication. 444
This discrepancy serves to highlight the complexity of NS5A phosphorylation, particularly 445
regarding the multiple phosphorylation events within LCS I. There is a need to fully 446
characterise both the kinases involved and the consequences of phosphorylation to clarify the 447
function of these post-translational modifications in the virus lifecycle. 448
An alternative explanation for the restricted distribution of the S225A mutant NS5A is that it was 449
impaired in its ability to traffic around the cytoplasm. In an elegant set of experiments using 450
both tetracysteine and GFP-tagged NS5A containing viruses, it was shown recently that NS5A 451
was present in two classes of motile structures – the majority were relatively static but some 452
exhibited rapid, long-range and sporadic movement throughout the cytoplasm (5). The rapid 453
movement was shown to involve the microtubule network and was dynein-dependent. It is thus 454
formally possible that NS5A(S225A) is impaired in interactions with microtubules or dynein, 455
preventing its movement around the cytoplasm and leading to its accumulation in the 456
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perinuclear region. However, we do not believe this is the case because live cell imaging of 457
either wildtype or S225A/D mutant viruses in which NS5A was Emerald-GFP (emGFP)-tagged 458
did not reveal any gross differences in the motility of NS5A positive structures (Supplementary 459
video). In addition, a defect in dynein dependent trafficking might intuitively be expected to 460
result in a distribution around the periphery of the cell, as dynein moves towards the minus end 461
of microtubules which is usually at the cell centre. 462
The use of the SNAP/CLIP tagging system allowed us to differentially label NS5A(S225A-463
SNAP) and wildtype NS5A(CLIP) within one population of cells, thereby visualising in the same 464
field of view, cells that were either harbouring S225A and wildtype replicons. Importantly this 465
analysis demonstrated that the distribution of NS5A(S225A) was still markedly different from 466
wildtype (Fig. 7E), illustrating that the altered localisation of NS5A was not due to an 467
environmental factor. However, the SNAP/CLIP system also revealed that the presence of 468
wildtype NS5A was able to complement the S225A defect in only a proportion of cells . This 469
suggests that additional, as yet undefined, stochastic factors such as stage of cell cycle or 470
availability of cellular factors, may contribute to the observed phenotype. 471
In conclusion, this study highlights an important role of phosphorylation in regulating the 472
function of NS5A. Clearly there is much to do to define the mechanisms underpinning this 473
effect, in particular we need to understand the molecular interactions that are modulated by 474
phosphorylation of NS5A at serine 225 and other serines in LCS I. Whilst a number of 475
candidates such as PI4KIIIα are already apparent, there are many other NS5A interacting 476
proteins that should be considered and a proteomics approach to compare the interactome of 477
both wildtype and mutant forms of NS5A is an appropriate way forward. Such experiments are 478
currently underway in our laboratory. 479
480
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ACKNOWLEDGEMENTS. 481
We thank John McLauchlan (Centre for Virus Research, University of Glasgow) for the SGR-482
JFH-1-luc construct, Takaji Wakita (National Institute for Infectious Diseases, Tokyo) for pJFH-483
1, Thomas Pietschmann (Hannover) for the NS3 antibody, and Joseph Shaw (Leeds) for help 484
with the daclatasvir EC50 analysis. Finally, we thank Volker Lohmann and Dave Rowlands for 485
insightful comments on this work. This work was funded by a Wellcome Trust Senior 486
Investigator Award to MH (096670MA). JM is a Royal Society University Research Fellow 487
(grant number UF100419), the confocal microscope used for live cell imaging of HCV infected 488
cells was funded by a Royal Society equipment grant (grant number RG110306). 489
490
FIGURE LEGENDS. 491
Figure 1. Characterisation of the phosphorylation site serine 225. (A) Huh7 cells were 492
electroporated with in vitro transcripts of the indicated mSGR-luc-JFH-1 genomes, seeded into 493
96-well format and incubated for 4, 24, 48 and 72 h post-electroporation (hpe). Luciferase 494
activity was determined at 24, 48 and 72 hpe, and normalised to the 4 hpe value. (B) Huh7 cells 495
were electroporated with in vitro transcripts of the indicated mJFH-1 virus genomes, seeded into 496
6-well format and cell supernatant harvested at denoted time point, released virus titre was 497
subsequently determined by focus forming assay. (C) At 96 and 144 hpe cells from (B) were 498
lysed and analysed by SDS-PAGE/western blot. (D) Total NS5A levels and the ratio of hyper- to 499
basal-phosphorylation were quantified from fluorescent western blots. (E) In order to account for 500
the differences in cell growth rate the released virus titre was normalised to total protein, cells 501
were also passaged 1:4 at 96 hpe before harvesting released virus supernatants at 144 hpe. 502
Figure 2. Sub-cellular distribution of wildtype and serine 225 mutant NS5A in JFH-1 503
infected cells. Huh7 cells were electroporated with in vitro transcripts of either wildtype mJFH-504
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1 (A), NS5A(S225A) (B) or NS5A(S225D) (C) mutants, cells were seeded onto coverslips and 505
incubated for 96 h prior to fixation, immunostaining for NS5A (sheep, 1:1000) and imaging by 506
confocal microscopy. 507
Figure 3. Analysis of lipid droplet redistribution in infected cells. Huh7 cells were 508
electroporated with in vitro transcripts of either wildtype mJFH-1 (A), NS5A(S225A) (B) or 509
NS5A(S225D) (C) mutants, cells were seeded onto coverslips and incubated for 48, 72 and 96 510
hpe, prior to fixation, immunostaining for NS5A and LDs and imaging by confocal microscopy. 511
Uninfected control cells were also processed at 96 h post-seeding. Spatial data for LD was 512
determined from 12 cells for each virus using the GDSC plug-in for Fiji, this data was used to 513
determine the number of LD per cell as well as the distance of each LD from the nuclear 514
envelope (E), box-and-whisker plots show the 10-90% distribution. ***, P< 0.05, significant 515
difference from wildtype. 516
Figure 4. Colocalisation of NS5A phospho-mutants with host and viral factors. Huh7 cells 517
were electroporated with in vitro transcripts of either wildtype mJFH-1, NS5A(S225A) or 518
NS5A(S225D) mutants, and seeded onto coverslips. At 96 hpe cells were fixed and 519
immunostained for NS5A and either NS3 (A), dsRNA (B) or the phospholipid, PI4P (C). An 520
NS5B GND (non-replicating) control is included for the dsRNA staining. Colocalisation scatter 521
plots are of a 200 x 200 μm area containing >10 cells, and show the correlation of absolute 522
values. The correlation value, r, is shown for each image. 523
Figure 5. An NS5A domain II mutation that impairs replication does not have the same 524
redistribution phenotype as NS5A(S225A). Huh7 cells were electroporated with in vitro 525
transcripts of either wildtype mJFH-1, NS5A(S225A), NS5A(S225D) or NS5A(P315A) mutants . 526
(A) Released virus titre was determined at 96 hpe by focus forming assay. Virus titre was 527
normalised to cellular protein in order to account for differences in cell growth rate. (B) At 96 528
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hpe cells were lysed and analysed by SDS-PAGE/Western blot for NS5A and GAPDH. (C) 529
Total NS5A levels and the ratio of hyper- to basal-phosphorylation were quantified from 530
fluorescent western blots. (D) At 96 hpe, electroporated cells were fixed and immunostained for 531
NS5A and either LD, PI4P or NS3 as described in Figure 4. (E) Spatial data for LD was 532
determined from 12 cells for each virus using the GDSC plugin for Fiji, box-and-whisker plots 533
show the 10-90% distribution. ***, P< 0.05, significant difference from wildtype. 534
Figure 6. Other serine mutations in LCSI share the condensed redistribution phenotype 535
with S225A. (A) Huh7 cells were electroporated with in vitro transcripts of either wildtype 536
mJFH-1 or the indicated mutants . Released virus titre was determined at 96 hpe by focus 537
forming assay. (B) At 96 hpe, electroporated cells were fixed and immunostained for NS5A and 538
LD as described in Figure 3. Only the three mutants that shared the phenotype are shown here 539
with wildtype for comparison. See Supplementary Figure 1 for complete data set. (C) Summary 540
table of mutant phenotypes (D) Amino acid sequence of LCSI. * indicates serine residues with a 541
redistribution phenotype. 542
Figure 7. Generation and validation of mSGR-luc-JFH-1 replicons containing either 543
NS5A-SNAP or NS5A-CLIP fusions. (A) Schematic of the mSGR-luc-JFH-1 replicon showing 544
the region of NS5A where the SNAP/CLIP tag was inserted, along with the flanking, flexible 545
linker sequences. JFH-1 NS5A amino acid numbering. (B) Huh7 cells were electroporated with 546
the indicated mSGR-luc-JFH-1 replicon RNAs, and replication followed over 72 h by 547
determining luciferase activity, shown normalised to the 4 hpe. (C) Cells were lysed at 72 hpe 548
and analysed by SDS-PAGE/Western blot for NS5A and GAPDH. A non-specific band (ns) is 549
seen at equal levels in all lysates (D). Huh7 cells electroporated with the NS5A-SNAP (left 550
panel) or NS5A-CLIP (right panel) replicon RNAs were also seeded onto coverslips, and at 72 551
hpe were fixed and immunostained for NS5A, as well as labelled with either SNAP-TMR or 552
CLIP-TMR before imaging by confocal microscopy. (E) Huh7 cells electroporated with the 553
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indicated mSGR-luc-JFH-1 replicon RNAs were seeded onto coverslips, fixed at 96 hpe and 554
immunostained for NS5A. 555
Figure 8. The redistribution of NS5A(S225A) is not the result of an environmental factor 556
and can be partially transcomplemented by wildtype NS5A. (A) Huh7 cells electroporated 557
with either mSGR-luc-JFH-1-NS5A(CLIP) or mSGR-luc-JFH-1-NS5A(S225A-SNAP) RNAs 558
were co-seeded onto the same coverslip, and at 96 hpe cells were fixed and labelled with either 559
SNAP-505 (green) or CLIP-TMR (red) before imaging by confocal microscopy. (B, C) In vitro 560
transcripts of mSGR-luc-JFH-1-NS5A(CLIP) and mSGR-luc-JFH-1-NS5A(S225A-SNAP) were 561
pooled prior to coelectroporating into Huh7 cells and seeding onto coverslips. At 96 hpe cells 562
were fixed and labelled with SNAP-505 and CLIP-TMR prior to imaging by confocal microscopy. 563
(B) Example of a population of Huh7 cells showing colocalisation between wildtype NS5A(CLIP) 564
and NS5A(S225A-SNAP). (C) Example of an Huh7 cell showing a difference in cellular 565
localisation between NS5A(CLIP) and NS5A(S225A-SNAP). Colocalisation scatter plots are of 566
individual cells, and show the correlation of absolute values. 567
568
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