JVI Accepted Manuscript Posted Online 10 June 2015 J. Virol. doi:10.1128/JVI.00722-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Human Papillomavirus Infectious Entry and Trafficking is a Rapid Process
2 3 4
Justyna Broniarczyk 1,2, Paola Massimi 1, Martina Bergant 3, Lawrence Banks 1
5 6 7 8 9 10 11
1. International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste, Italy 2. Department of Molecular Virology, Adam Mickiewicz University, Umultowska 89, 61614 Poznan, Poland 3. Centre for Biomedical Sciences and Engineering, University of Nova Gorica, Vipavska 13, 5000 Nova Gorica, Slovenia
12 13
Running Title: HPV trafficking
14 15 16 17
Corresponding author:
18
Lawrence Banks
19
International Centre for Genetic Engineering and Biotechnology,
20
Padriciano 99,
21
I-34149 Trieste, Italy
22
[email protected]
23
Word count for main abstract: 152
24
Word count main text: 2,201
25
1
26
Abstract
27 28
Previous studies have indicated that Human Papillomavirus (HPV) infectious entry is slow
29
requiring many hours after initial infection for the virus to gain entry into the nucleus.
30
However intracellular transport pathways are typically very rapid and in the context of a
31
natural HPV infection in a wounded epithelium, such slow intracellular transport would seem
32
at odds with a normal viral infection. Using synchronised cell populations we show that HPV
33
trafficking can however be a rapid process. In cells that are infected in the late S-early G2/M
34
phase of the cell cycle, HPV16 pseudovirion (PsV) reporter DNA gene expression is
35
detectable by 8hrs post-infection. Likewise reporter DNA can be visualised within the nucleus
36
in conjunction with PML nuclear bodies 1-2hrs post-infection in cells that are infected with
37
PsVs just prior to mitotic entry. This demonstrates that endosomal trafficking of HPV is
38
rapid, with mitosis being the main restriction on nuclear entry.
39 40
Importance
41
HPV infectious entry appears to be slow and requires mitosis to occur before the incoming
42
viral DNA can access the nucleus. In this study we show that HPV trafficking in the cell is
43
actually very rapid. This demonstrates that in the context of a normal virus infection, the cell
44
cycle state will have a major influence on the time it takes for an incoming virus to enter the
45
nucleus and initiate viral gene expression.
46 47 48 49 50 2
51
Introduction
52
Papillomaviruses (PV) are small, non-enveloped DNA viruses that infect epithelial cells.
53
Their icosahedral capsid is formed by two structural proteins: L1 and L2. PVs have oncogenic
54
potential and are associated with multiple human cancers including cervical cancer, other
55
anogenital cancers, and a significant number of head and neck tumours [1]. The HPV life
56
cycle is intimately linked to epithelial differentiation of keratinocytes, with the virus believed
57
to enter the undifferentiated proliferating basal compartment of the epithelium through
58
microtraumas in the skin. As the cells differentiate the viral genome is amplified and
59
ultimately new infectious virus particles are assembled in the nucleus of differentiated spinous
60
and granular keratinocytes [1].
61
PVs enter basal cells via endocytosis, but the precise mechanism appears diverse and is
62
dependent on the virus type [2, 3]. Virus capsids then disassemble in the late endosomes
63
and/or lysosomes in a pH-dependent manner. L2 interacts with components of the cellular
64
sorting machinery, such as sorting nexin 17, which is required for the lysosomal escape of the
65
L2-DNA complex [4]. L2 then targets the viral DNA to the perinuclear region of the cell
66
through a pathway that requires Dynein-mediated transport [5] and endocytic retromer
67
components [6], where the L2-DNA complex eventually accumulates in the trans-Golgi
68
network [7, 8]. HPV entry into the nucleus requires mitosis, during which the barrier between
69
nucleoplasm and cytosol is removed and the trans-Golgi network becomes dispersed, thereby
70
allowing the L2-DNA complex to gain access to the nucleus [9,10], where it is found in close
71
proximity with PML bodies in which viral genome expression is believed to initiate [11].
72
Whilst previous studies have shown that mitosis is an important element for completion of
73
infectious virus entry, expression of a HPV-16 Pseudovirion (PsV) reporter gene cannot be
74
detected earlier than 16h post-infection in asynchronously growing cells [3]. However,
75
classical endosomal cargo trafficking pathways are typically very quick [12], suggesting that 3
76
cells infected with HPVs when close to mitosis might allow nuclear entry and initiation of
77
viral gene expression at much earlier time points. To investigate this possibility we have
78
monitored HPV-16 PsV infection in cells following cell cycle synchronization. We now show
79
that intracellular transport of PsVs is remarkably quick, and depending upon the specific
80
phase of the cell cycle in which infection takes place, viral nuclear entry can occur within 1-
81
2h post infection and reporter gene expression can be detected as little as 8h post infection.
82
This demonstrates that the main restriction on nuclear entry of the L2-DNA complex and the
83
initiation of viral gene expression is completion of mitosis.
84 85
Materials and Methods
86
Cell lines
87
Spontaneously immortalized human keratinocyte cells (HaCaT) were maintained in
88
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum
89
(FBS) penicillin-streptomycin (100U/ml) and glutamine (300mg/ml).
90 91
PsV production
92
HPV-16 PsVs containing a luciferase reporter plasmid were generated in 293TT cells as
93
previously described [4, 13]. PsVs containing packaged 5-ethynyl-2′-deoxyuridine (EdU)-
94
labeled plasmid DNA were prepared by addition of 25 μM EdU to the 293TT cells at 24h
95
post-transfection and harvested after a further 24h.
96 97
Cell synchronization and infection
98
HaCaT cells were seeded in a 12 well plate at a density of 0,75 x 105 cells/well (aphidicolin
99
treatment) or 0,5 x 105 cells/well (thymidine treatment). After adherence HaCaT cells were
100
incubated 24h with 1µg/ml aphidicolin (Sigma) to induce arrest at the G1/S boundary. The 4
101
cells were then released by washing with PBS and fresh DMEM was added. Cells were
102
infected with HPV16 PsVs (1h, 4°C) at a concentration of 12ng/ml at different times after the
103
release as indicated in the text. Cells were harvested at different time points (0h, 4h, 8h, 12h,
104
16h) and infection was monitored by luminometric analysis of firefly luciferase activity using
105
a Luciferase Assay System kit (Promega).
106
For synchronization experiments using double thymidine block, the cells were incubated with
107
2mM thymidine (Sigma) for 16h. The cells were then washed with PBS, and fresh DMEM
108
was added for a further 9h, after which 2mM thymidine (Sigma) was again added for 16h.
109
The cells were then released by washing with PBS and infected 3h post-release (S phase) with
110
HPV16 PsVs. 1h after infection at 4°C the cells were washed with PBS, fresh medium was
111
added and cells were incubated for further 8h. After that time cells were collected and
112
infection was monitored by luminometric analysis as above. Experiments were done in
113
triplicate and each sample was measured three times. Throughout, equal amounts of protein
114
was used in the assays which was ascertained by prior measurement of total protein
115
concentration in the different cell extracts.
116 117
Flow cytometry
118
Cell cycle analysis was done with FACS analysis by measuring DNA content using
119
propidium iodide staining as described previously [14].
120 121
PsV trafficking assay
122
HaCaT cells seeded on coverslips were grown overnight and synchronised using aphidicolin
123
block as described above. At different times after release from G1/S (0h, 10h) cells were
124
prechilled to 4°C, infected with EdU-labeled PsVs, and incubated for 1h at 4°C with agitation
125
to allow viral attachment. Cells were then washed, DMEM was replaced and cells were 5
126
transferred to 37°C. At different time points after attachment (1h, 3h), cells were washed with
127
PBS and fixed in PBS + 3.7 % paraformaldehyde for 15 min at room temperature. EdU and
128
PML staining were done as previously described using Click-iT EdU Imagining Kit
129
(Molecular Probes) and anti-PML primary antibodies (Santa Cruz, 1:100) [4]. Cells were
130
counterstained with DAPI, washed in water and mounted on glass slides. Slides were
131
visualized using a Zeiss Axiovert 100 M microscope (Zeiss) attached to a LSM 510 confocal
132
unit or a Leica DMLB fluorescence microscope equipped with a Leica photo camera
133
(A01M871016).
134 135
Neutralization assay
136
Neutralization assays were performed by incubation of PsVs with neutralising antibody
137
H16.V5 at a final concentration of 1:1000 or pre-immune antibody for 1h at 4°C. Neutralized
138
PsVs were then added to HaCaT cells and infectivity was measured at different time points as
139
described above. Mock infections were also performed as additional negative controls
140 141
Results and Discussion
142
Previous studies have shown that mitosis is needed to complete HPV infection and that the
143
expression of a PsV reporter gene cannot be detected earlier then 16h post infection in
144
asynchronously growing cells [3, 10-11]. However, intracellular transport mechanisms are
145
relatively fast [12], suggesting that cells infected with HPVs when close to mitosis might
146
allow detection of viral gene expression and nuclear entry at much earlier time points post
147
infection.
148
To first identify the earliest time point at which PsV reporter gene expression can be detected
149
in asynchronously growing HaCaT cells, HPV-16 PsVs carrying luciferase were used to
150
infect HaCaTs and cell extracts were collected at different times post-infection. Luciferase 6
151
activity was measured and the results in Figure 1A show the first detection of luciferase
152
activity at 16h post infection, increasing to a maximum at 26h post infection (Figure 1A).
153
These results are very much in agreement with previous studies [3] and indicate that in
154
asynchronous cultures infectious HPV entry and initiation of gene expression is a relatively
155
slow process.
156
We then proceeded to investigate whether reporter gene activity could be detected at earlier
157
time points post-infection, if cells were exposed to virions close to mitosis. HaCaT cells were
158
synchronized in G1/S with aphidicolin and were then released from the G1/S block. After 7h
159
the cells were infected with HPV16 PsVs (time 0h) and cells were harvested at different times
160
post-infection (0h, 4h, 8h, 12h, 14h and 16h) and analysed for luciferase activity and for the
161
corresponding cell cycle profiles. The results demonstrate that when cells are infected with
162
PsVs when the cells are predominantly in S phase (Figure 1C panel (II)) the luciferase activity
163
can first be detected by 8h post-infection (Figure 1B), which is significantly quicker than in
164
asynchronously infected cells. As can be seen, no luciferase activity could be detected at 4h
165
post infection, a time when the majority of the cells were still passing through mitosis (Figure
166
1C panel (III)), whilst at the 8h time point a significant proportion of the cell population had
167
passed back into the G1 phase of the cell cycle, consistent with completion of mitosis (Figure
168
1C panel (IV)).
169
To verify that the luciferase signal observed at 8h post-infection was a result of infectious
170
virus entry, the assay was repeated but the PsVs were pre-incubated with the anti-L1 PsV
171
neutralizing antibody H16.V5 [15]. As can be seen from Figure 1D, prior incubation of the
172
PsVs with H16.V5 antibody abolished the luciferase activity at the 8h time point. In order to
173
confirm these results using a different method of cell synchronization, the neutralization assay
174
was repeated and infections performed on HaCaT cells that had been synchronized following
175
a double thymidine block. The PsVs were incubated with H16.V5 antibody and used to infect 7
176
HaCaT cells 3h post-release from the thymidine block. Luciferase activity was then measured
177
again at the 8h time point post-infection, and as can be seen in Figure 2A there was readily
178
detectable levels of reporter gene activity at this time point, and this was abolished by prior
179
incubation of the PsVs with the neutralizing antibody. Consistent with the results in Figure 1,
180
PsV infection at 3h post-release from the thymidine block occurred whilst the majority of the
181
cell population was in S phase (Figure 2B panel (II)) and no luciferase activity was detectable
182
at 4h post infection (data not shown) when most of the cells were in G2M (Figure 2B panel
183
(III)). However luciferase was detected at 8h post infection when the majority of the cell
184
population had re-entered G1 (Figure 2B panel (IV)). These results demonstrate that HPV
185
PsV infectious entry and subsequent reporter gene expression is much quicker when cells are
186
infected during the late S or the early G2/M phase of the cell cycle. This is perfectly
187
consistent with previous reports suggesting that infectious entry into the nucleus requires
188
mitosis [9-10], but it emphasizes that the intracellular trafficking of the virus is much faster
189
than previously assumed.
190
These results indicate that PsV reporter DNA can gain access to the nucleus relatively quickly
191
post-infection, if infection occurs close to mitosis. To identify the minimum time required for
192
PsV DNA to gain access to the nucleus we used HPV16 PsVs containing reporter plasmid
193
DNA labelled with the thymidine analog, 5-ethynyl-2’deoxyuridine (EdU). HaCaT cells were
194
synchronized with aphidicolin as above, then released from the G1/S block and infected with
195
PsVs either immediately, when the cells were in G1 (Figure 1C panel (I) or after 10h when
196
the cells were still passing through G2M (Figure 1C panel (III)). The infected cells were fixed
197
at 1h post infection and EdU was detected in an azide–alkyne reaction. Cells were then
198
stained for PML, a major component of ND10 domains, where HPV genomes are typically
199
found in an established HPV infection [11]. As can be seen from Figure 3A EdU-labelled
200
DNA encapsidated by HPV-16 PsVs was detected only in the cytoplasm in cells infected 8
201
immediately after release from G1 and fixed 1h post-infection. In contrast, in cells that were
202
infected 10h post-release from G1 and fixed 1h post-infection, EdU staining was readily
203
detected within the nucleus and colocalision with PML could be observed (Figure 3B).
204
Similar results were also obtained when cells were analysed by confocal microscopy. As can
205
be seen from Figure 4A there is no co-localsiation of EdU with PML when cells are infected
206
immediately after release and fixed 1h post infection, whilst in cells infected 10h post –
207
release and analysed 1h post-infection there is clear co-localisation of PsV reporter DNA with
208
PML, as shown from the zeta-stack analysis (Figure 4B).
209
mitosis, PML bodies become fewer and larger [15], and in several instances, EdU labeled PsV
210
reporter DNA shows clear co-localisation with PML at such sites, using both epifluorescence
211
(Figure 3B) and confocal microscopy with zeta-stacks (Figure 5). Taken together these results
212
demonstrate that HPV PsV reporter DNA can gain access to the nucleus as soon as 1-2hrs
213
post infection if the infected cells are close to mitotic entry, and that association with PML
214
can also occur at this time.
215
Whilst previous studies have shown a requirement for mitosis for the L2-DNA complex to
216
gain entry into the nucleus, there has been little information on the speed with which HPV
217
virions and their packaged genomes are trafficked within the cell. We provide compelling
218
evidence that reporter gene expression can be detected as little as 8h post-infection if cells are
219
infected in late S phase. Likewise, PsV reporter DNA can be found associated with PML
220
nuclear bodies as little as 1-2h post-infection and, most strikingly, this can also be clearly
221
seen in cells which are undergoing mitosis and where nuclear envelope breakdown has
222
already occurred, suggesting that the association of the L2-DNA complex with PML nuclear
223
bodies first appears during mitosis. Current studies are focused on identifying the cellular
224
components that are required for recruiting the L2-DNA complex to these nuclear domains.
225
Acknowledgements
In cells actually undergoing
9
226
The authors are very grateful to Miranda Thomas for valuable comments on the manuscript.
227
Justyna Broniarczyk is a recipient of an Arturo Falaschi ICGEB Fellowship.
228 229
Statement of author contributions
230
Justyna Broniarczyk and Paola Massimi performed all the experiments and Martina Bergant
231
and Lawrence Banks helped with the study design and manuscript preparation.
232
10
233 234
References
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1. Doorbar J, Quint W, Banks L, Bravo IG, Stoler M, Broker TR, Stanley MA. 2012.
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The biology and life-cycle of human papillomaviruses. Vaccine Suppl 5: F55-70.
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2. Schelhaas M, Shah B, Holzer M, Blattmann P, Kühling L, Day PM, Schiller JT,
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Helenius A. 2012. Entry of human papillomavirus type 16 by actin-dependent, clathrin-
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and lipid raft-independent endocytosis. PLoS Pathog 8(4):e1002657.
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3. Spoden G, Kühling L, Cordes N, Frenzel B, Sapp M, Boller K, Florin L, Schelhaas
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requirements for entry. J Virol 87(13):7765-73.
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2013. Human papillomavirus types 16, 18, and 31 share similar endocytic
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5. Florin L, Becker KA, Lambert C, Nowak T, Sapp C, Strand D, Streeck RE, Sapp M.
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2006. Identification of a dynein interacting domain in the papillomavirus minor capsid
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protein L2. J Virol 80(13):6691-6.
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6. Popa A, Zhang W, Harrison MS, Goodner K, Kazakov T, Goodwin EC, Lipovsky A,
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Burd CG, DiMaio D. 2015. Direct binding of retromer to human papillomavirus type 16
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minor capsid protein L2 mediates endosome exit during viral infection. PLoS Pathog
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11(2):e1004699.
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7. Day PM, Thompson CD, Schowalter RM, Lowy DR, Schiller JT. 2013. Identification
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of a role for the trans-Golgi network in human papillomavirus 16 pseudovirus infection. J
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8. Zhang W, Kazakov T, Popa A, DiMaio D. 2014. Vesicular trafficking of incoming
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human papillomavirus 16 to the Golgi apparatus and endoplasmic reticulum requires γ-
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secretase activity. MBio 5(5):e01777-14. 11
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9. Aydin I, Weber S, Snijder B, Samperio Ventayol P, Kühbacher A, Becker M, Day
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PM, Schiller JT, Kann M, Pelkmans L, Helenius A, Schelhaas M. 2014. Large scale
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RNAi reveals the requirement of nuclear envelope breakdown for nuclear import of
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human papillomaviruses. PLoS Pathog 10(5):e1004162.
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10. Pyeon D, Pearce SM, Lank SM, Ahlquist P, Lambert PF. 2009. Establishment of
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human papillomavirus infection requires cell cycle progression. PLOS Pathog 5(2):
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e1000318.
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11. Day PM, Baker CC, Lowy DR, Schiller JT. 2004. Establishment of papillomavirus
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infection is enhanced by promyelocytic leukemia protein (PML) expression. Proc Natl
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Acad Sci U S A 101(39):14252-7.
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12. Huotari J, Helenius A. 2011. Endosome maturation. EMBO J 30(17):3481-500.
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13. Buck CB, Pastrana DV, Lowy DR, Schiller JT.
2005 Generation of HPV
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pseudovirions using transfection and their use in neutralization assays. Methods Mol Med
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119: 445–462.
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14. Banks L, Barnett SC, Crook T. 1990. HPV-16 E7 functions at the G1 to S phase transition in the cell cycle. Oncogene 5:833–7.
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15. Christensen ND, Dillner J, Eklund C, Carter JJ, Wipf GC, Reed CA, Cladel NM,
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Galloway DA. 1996. Surface conformational and linear epitopes on HPV-16 and HPV-
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18L1 virus-like particles as defined by monoclonal antibodies. Virology 223:174–184.
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16. Bernardi R, Pandolfi PP. 2007. Structure, dynamics and functions of promyelocytic
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leukaemia nuclear bodies. Nat Rev Mol Cell Biol 8(12):1006-16.
279 280
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Figure Legends
282 283
Figure 1. Comparative rate of HPV16 PsVs infectious entry in asynchronous and
284
synchronous HaCAT cells. Panel A. Asynchronously growing HaCaT cells were infected
285
with HPV-16 PsVs carrying a luciferase reporter plasmid and were harvested at different
286
time points post-infection (0h, 4h, 8h, 13h,16h, 19h, 22h, 26, 48h) and the luciferase activity
287
was measured. The results are expressed as the means of at least three independent
288
experiments and the standard deviations are shown. Panel B. Aphidicolin synchronised
289
HaCaT cells were infected with HPV16 PsVs carrying a luciferase reporter plasmid 7h post-
290
release from G1/S (time point 0h). Cells were harvested at different time points post-infection
291
(0h, 4h, 8h, 12h, 16h) and luciferase activity measured. The results are expressed as the means
292
of at least three independent experiments and the standard deviations are shown. Panel C. Cell
293
cycle analysis of the cells harvested in Panel B stained with propidium iodide and analyzed by
294
flow cytometry. Note that the cells were mostly arrested in G1 following aphidicolin
295
treatment (panel I), at the time of infection 7h post-release they were in S phase (panel II), and
296
had entered mitosis by 4h post-infection (panel III). A siginficant proportion of cells had re-
297
entered G1 at the 8h time point (panel IV) when luciferase activity was first detected. Panel
298
D. HPV16 PsV infections were performed as in Panel B except that PsVs were pre-incubated
299
with H16.V5 neutralising antibody or pre-immune (PI) antibody prior to infection and
300
luciferase activity measured 8h post-infection. The results are expressed as the means of at
301
least three independent experiments and the standard deviations are shown. Also shown are
302
mock infected cells.
303 304 305 13
306
Figure 2. HPV16 PsV reporter gene expression can be detected 8h post-infection.
307
Panel A. Double thymidine block synchronised HaCaT cells were infected with HPV16 PsVs
308
carrying a luciferase reporter plasmid 3h post-release from G1 arrest using PsVs that were
309
either pre-incubated with H16.V5 neutralising antibody or pre-immune (PI) antibody. After
310
8h the cells were harvested and luciferase activity measured. The results are expressed as the
311
means of at least three independent experiments and the standard deviations are shown and a
312
mock infection control was also included. Panel B. Cell cycle analysis of the HaCaT cells
313
used in Panel A. Note growth arrest in G1 following the double thymidine block (panel I),
314
entry into S phase 3h post-release (panel II) at the time of infection (time 0h), entry into G2M
315
4h post-infection (panel III) and re-entry into G1 8h post-infection (panel IV) at the time of
316
detection of luciferase activity in Panel A.
317 318
Figure 3. Detection of HPV16 PsV reporter DNA in proximity with PML 1h post-
319
infection. Aphidicolin synchronised HaCaT cells were infected with HPV16 PsVs carrying
320
EdU labelled luciferase reporter plasmid 1h (Panel A) or 10h (Panel B) post-release from G1/
321
S. After a further 1h at 37°C the cells were fixed and processed for the detection of EdU-
322
labelled DNA (red), PML (in green) and DAPI (blue). Note appearence of perinuclear
323
accumulation of EdU labelled DNA in Panel A but co-localisation with nuclear PML in Panel
324
B as indicated by the arrows and the expanded field.
325 326
Figure 4. Confocal image analysis showing HPV16 PsVs reporter DNA in conjunction
327
with PML. Aphidicolin synchronised HaCaT cells were infected with HPV16 PsVs carrying
328
EdU labelled luciferase reporter plasmid 1h (Panel A) or 10h (Panel B) post-release from G1/
329
S. After a further 1h at 37°C the cells were fixed and processed for the detection of EdU-
330
labelled DNA (red) and
PML (in green). Shown are the zeta-stacks demonstrating 14
331
cytoplasmic PsV EdU labelled DNA in Panel A and co-localisation with PML in two different
332
fields (upper and lower panels) in Panel B, with an expanded view of one of those fields.
333 334
Figure 5. Confocal image analysis showing HPV16 PsVs reporter DNA in conjunction
335
with PML in mitotic cells. Aphidicolin synchronised HaCaT cells were infected with HPV16
336
PsVs carrying EdU labelled luciferase reporter plasmid 10h post-release from G1/S. After a
337
further 3h at 37°C the cells were fixed and processed for the detection of EdU-labelled DNA
338
(red) and PML (in green). Shown are the zeta-stacks demonstrating PsV EdU labelled DNA
339
and co-localsiation with PML in two different fields (upper and lower panel) in cells that are
340
undergoing mitosis.
15
1
Human Papillomavirus Infectious Entry and Trafficking is a Rapid Process
2 3 4
Justyna Broniarczyk 1,2, Paola Massimi 1, Martina Bergant 3, Lawrence Banks 1
5 6 7 8 9 10 11
1. International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste, Italy 2. Department of Molecular Virology, Adam Mickiewicz University, Umultowska 89, 61614 Poznan, Poland 3. Centre for Biomedical Sciences and Engineering, University of Nova Gorica, Vipavska 13, 5000 Nova Gorica, Slovenia
12 13
Running Title: HPV trafficking
14 15 16 17
Corresponding author:
18
Lawrence Banks
19
International Centre for Genetic Engineering and Biotechnology,
20
Padriciano 99,
21
I-34149 Trieste, Italy
22
[email protected]
23
Word count for main abstract: 152
24
Word count main text: 2,201
25
1
26
Abstract
27 28
Previous studies have indicated that Human Papillomavirus (HPV) infectious entry is slow
29
requiring many hours after initial infection for the virus to gain entry into the nucleus.
30
However intracellular transport pathways are typically very rapid and in the context of a
31
natural HPV infection in a wounded epithelium, such slow intracellular transport would seem
32
at odds with a normal viral infection. Using synchronised cell populations we show that HPV
33
trafficking can however be a rapid process. In cells that are infected in the late S-early G2/M
34
phase of the cell cycle, HPV16 pseudovirion (PsV) reporter DNA gene expression is
35
detectable by 8hrs post-infection. Likewise reporter DNA can be visualised within the nucleus
36
in conjunction with PML nuclear bodies 1-2hrs post-infection in cells that are infected with
37
PsVs just prior to mitotic entry. This demonstrates that endosomal trafficking of HPV is
38
rapid, with mitosis being the main restriction on nuclear entry.
39 40
Importance
41
HPV infectious entry appears to be slow and requires mitosis to occur before the incoming
42
viral DNA can access the nucleus. In this study we show that HPV trafficking in the cell is
43
actually very rapid. This demonstrates that in the context of a normal virus infection, the cell
44
cycle state will have a major influence on the time it takes for an incoming virus to enter the
45
nucleus and initiate viral gene expression.
46 47 48 49 50 2
51
Introduction
52
Papillomaviruses (PV) are small, non-enveloped DNA viruses that infect epithelial cells.
53
Their icosahedral capsid is formed by two structural proteins: L1 and L2. PVs have oncogenic
54
potential and are associated with multiple human cancers including cervical cancer, other
55
anogenital cancers, and a significant number of head and neck tumours [1]. The HPV life
56
cycle is intimately linked to epithelial differentiation of keratinocytes, with the virus believed
57
to enter the undifferentiated proliferating basal compartment of the epithelium through
58
microtraumas in the skin. As the cells differentiate the viral genome is amplified and
59
ultimately new infectious virus particles are assembled in the nucleus of differentiated spinous
60
and granular keratinocytes [1].
61
PVs enter basal cells via endocytosis, but the precise mechanism appears diverse and is
62
dependent on the virus type [2, 3]. Virus capsids then disassemble in the late endosomes
63
and/or lysosomes in a pH-dependent manner. L2 interacts with components of the cellular
64
sorting machinery, such as sorting nexin 17, which is required for the lysosomal escape of the
65
L2-DNA complex [4]. L2 then targets the viral DNA to the perinuclear region of the cell
66
through a pathway that requires Dynein-mediated transport [5] and endocytic retromer
67
components [6], where the L2-DNA complex eventually accumulates in the trans-Golgi
68
network [7, 8]. HPV entry into the nucleus requires mitosis, during which the barrier between
69
nucleoplasm and cytosol is removed and the trans-Golgi network becomes dispersed, thereby
70
allowing the L2-DNA complex to gain access to the nucleus [9,10], where it is found in close
71
proximity with PML bodies in which viral genome expression is believed to initiate [11].
72
Whilst previous studies have shown that mitosis is an important element for completion of
73
infectious virus entry, expression of a HPV-16 Pseudovirion (PsV) reporter gene cannot be
74
detected earlier than 16h post-infection in asynchronously growing cells [3]. However,
75
classical endosomal cargo trafficking pathways are typically very quick [12], suggesting that 3
76
cells infected with HPVs when close to mitosis might allow nuclear entry and initiation of
77
viral gene expression at much earlier time points. To investigate this possibility we have
78
monitored HPV-16 PsV infection in cells following cell cycle synchronization. We now show
79
that intracellular transport of PsVs is remarkably quick, and depending upon the specific
80
phase of the cell cycle in which infection takes place, viral nuclear entry can occur within 1-
81
2h post infection and reporter gene expression can be detected as little as 8h post infection.
82
This demonstrates that the main restriction on nuclear entry of the L2-DNA complex and the
83
initiation of viral gene expression is completion of mitosis.
84 85
Materials and Methods
86
Cell lines
87
Spontaneously immortalized human keratinocyte cells (HaCaT) were maintained in
88
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum
89
(FBS) penicillin-streptomycin (100U/ml) and glutamine (300mg/ml).
90 91
PsV production
92
HPV-16 PsVs containing a luciferase reporter plasmid were generated in 293TT cells as
93
previously described [4, 13]. PsVs containing packaged 5-ethynyl-2′-deoxyuridine (EdU)-
94
labeled plasmid DNA were prepared by addition of 25 μM EdU to the 293TT cells at 24h
95
post-transfection and harvested after a further 24h.
96 97
Cell synchronization and infection
98
HaCaT cells were seeded in a 12 well plate at a density of 0,75 x 105 cells/well (aphidicolin
99
treatment) or 0,5 x 105 cells/well (thymidine treatment). After adherence HaCaT cells were
100
incubated 24h with 1µg/ml aphidicolin (Sigma) to induce arrest at the G1/S boundary. The 4
101
cells were then released by washing with PBS and fresh DMEM was added. Cells were
102
infected with HPV16 PsVs (1h, 4°C) at a concentration of 12ng/ml at different times after the
103
release as indicated in the text. Cells were harvested at different time points (0h, 4h, 8h, 12h,
104
16h) and infection was monitored by luminometric analysis of firefly luciferase activity using
105
a Luciferase Assay System kit (Promega).
106
For synchronization experiments using double thymidine block, the cells were incubated with
107
2mM thymidine (Sigma) for 16h. The cells were then washed with PBS, and fresh DMEM
108
was added for a further 9h, after which 2mM thymidine (Sigma) was again added for 16h.
109
The cells were then released by washing with PBS and infected 3h post-release (S phase) with
110
HPV16 PsVs. 1h after infection at 4°C the cells were washed with PBS, fresh medium was
111
added and cells were incubated for further 8h. After that time cells were collected and
112
infection was monitored by luminometric analysis as above. Experiments were done in
113
triplicate and each sample was measured three times. Throughout, equal amounts of protein
114
was used in the assays which was ascertained by prior measurement of total protein
115
concentration in the different cell extracts.
116 117
Flow cytometry
118
Cell cycle analysis was done with FACS analysis by measuring DNA content using
119
propidium iodide staining as described previously [14].
120 121
PsV trafficking assay
122
HaCaT cells seeded on coverslips were grown overnight and synchronised using aphidicolin
123
block as described above. At different times after release from G1/S (0h, 10h) cells were
124
prechilled to 4°C, infected with EdU-labeled PsVs, and incubated for 1h at 4°C with agitation
125
to allow viral attachment. Cells were then washed, DMEM was replaced and cells were 5
126
transferred to 37°C. At different time points after attachment (1h, 3h), cells were washed with
127
PBS and fixed in PBS + 3.7 % paraformaldehyde for 15 min at room temperature. EdU and
128
PML staining were done as previously described using Click-iT EdU Imagining Kit
129
(Molecular Probes) and anti-PML primary antibodies (Santa Cruz, 1:100) [4]. Cells were
130
counterstained with DAPI, washed in water and mounted on glass slides. Slides were
131
visualized using a Zeiss Axiovert 100 M microscope (Zeiss) attached to a LSM 510 confocal
132
unit or a Leica DMLB fluorescence microscope equipped with a Leica photo camera
133
(A01M871016).
134 135
Neutralization assay
136
Neutralization assays were performed by incubation of PsVs with neutralising antibody
137
H16.V5 at a final concentration of 1:1000 or pre-immune antibody for 1h at 4°C. Neutralized
138
PsVs were then added to HaCaT cells and infectivity was measured at different time points as
139
described above. Mock infections were also performed as additional negative controls
140 141
Results and Discussion
142
Previous studies have shown that mitosis is needed to complete HPV infection and that the
143
expression of a PsV reporter gene cannot be detected earlier then 16h post infection in
144
asynchronously growing cells [3, 10-11]. However, intracellular transport mechanisms are
145
relatively fast [12], suggesting that cells infected with HPVs when close to mitosis might
146
allow detection of viral gene expression and nuclear entry at much earlier time points post
147
infection.
148
To first identify the earliest time point at which PsV reporter gene expression can be detected
149
in asynchronously growing HaCaT cells, HPV-16 PsVs carrying luciferase were used to
150
infect HaCaTs and cell extracts were collected at different times post-infection. Luciferase 6
151
activity was measured and the results in Figure 1A show the first detection of luciferase
152
activity at 16h post infection, increasing to a maximum at 26h post infection (Figure 1A).
153
These results are very much in agreement with previous studies [3] and indicate that in
154
asynchronous cultures infectious HPV entry and initiation of gene expression is a relatively
155
slow process.
156
We then proceeded to investigate whether reporter gene activity could be detected at earlier
157
time points post-infection, if cells were exposed to virions close to mitosis. HaCaT cells were
158
synchronized in G1/S with aphidicolin and were then released from the G1/S block. After 7h
159
the cells were infected with HPV16 PsVs (time 0h) and cells were harvested at different times
160
post-infection (0h, 4h, 8h, 12h, 14h and 16h) and analysed for luciferase activity and for the
161
corresponding cell cycle profiles. The results demonstrate that when cells are infected with
162
PsVs when the cells are predominantly in S phase (Figure 1C panel (II)) the luciferase activity
163
can first be detected by 8h post-infection (Figure 1B), which is significantly quicker than in
164
asynchronously infected cells. As can be seen, no luciferase activity could be detected at 4h
165
post infection, a time when the majority of the cells were still passing through mitosis (Figure
166
1C panel (III)), whilst at the 8h time point a significant proportion of the cell population had
167
passed back into the G1 phase of the cell cycle, consistent with completion of mitosis (Figure
168
1C panel (IV)).
169
To verify that the luciferase signal observed at 8h post-infection was a result of infectious
170
virus entry, the assay was repeated but the PsVs were pre-incubated with the anti-L1 PsV
171
neutralizing antibody H16.V5 [15]. As can be seen from Figure 1D, prior incubation of the
172
PsVs with H16.V5 antibody abolished the luciferase activity at the 8h time point. In order to
173
confirm these results using a different method of cell synchronization, the neutralization assay
174
was repeated and infections performed on HaCaT cells that had been synchronized following
175
a double thymidine block. The PsVs were incubated with H16.V5 antibody and used to infect 7
176
HaCaT cells 3h post-release from the thymidine block. Luciferase activity was then measured
177
again at the 8h time point post-infection, and as can be seen in Figure 2A there was readily
178
detectable levels of reporter gene activity at this time point, and this was abolished by prior
179
incubation of the PsVs with the neutralizing antibody. Consistent with the results in Figure 1,
180
PsV infection at 3h post-release from the thymidine block occurred whilst the majority of the
181
cell population was in S phase (Figure 2B panel (II)) and no luciferase activity was detectable
182
at 4h post infection (data not shown) when most of the cells were in G2M (Figure 2B panel
183
(III)). However luciferase was detected at 8h post infection when the majority of the cell
184
population had re-entered G1 (Figure 2B panel (IV)). These results demonstrate that HPV
185
PsV infectious entry and subsequent reporter gene expression is much quicker when cells are
186
infected during the late S or the early G2/M phase of the cell cycle. This is perfectly
187
consistent with previous reports suggesting that infectious entry into the nucleus requires
188
mitosis [9-10], but it emphasizes that the intracellular trafficking of the virus is much faster
189
than previously assumed.
190
These results indicate that PsV reporter DNA can gain access to the nucleus relatively quickly
191
post-infection, if infection occurs close to mitosis. To identify the minimum time required for
192
PsV DNA to gain access to the nucleus we used HPV16 PsVs containing reporter plasmid
193
DNA labelled with the thymidine analog, 5-ethynyl-2’deoxyuridine (EdU). HaCaT cells were
194
synchronized with aphidicolin as above, then released from the G1/S block and infected with
195
PsVs either immediately, when the cells were in G1 (Figure 1C panel (I) or after 10h when
196
the cells were still passing through G2M (Figure 1C panel (III)). The infected cells were fixed
197
at 1h post infection and EdU was detected in an azide–alkyne reaction. Cells were then
198
stained for PML, a major component of ND10 domains, where HPV genomes are typically
199
found in an established HPV infection [11]. As can be seen from Figure 3A EdU-labelled
200
DNA encapsidated by HPV-16 PsVs was detected only in the cytoplasm in cells infected 8
201
immediately after release from G1 and fixed 1h post-infection. In contrast, in cells that were
202
infected 10h post-release from G1 and fixed 1h post-infection, EdU staining was readily
203
detected within the nucleus and colocalision with PML could be observed (Figure 3B).
204
Similar results were also obtained when cells were analysed by confocal microscopy. As can
205
be seen from Figure 4A there is no co-localsiation of EdU with PML when cells are infected
206
immediately after release and fixed 1h post infection, whilst in cells infected 10h post –
207
release and analysed 1h post-infection there is clear co-localisation of PsV reporter DNA with
208
PML, as shown from the zeta-stack analysis (Figure 4B).
209
mitosis, PML bodies become fewer and larger [15], and in several instances, EdU labeled PsV
210
reporter DNA shows clear co-localisation with PML at such sites, using both epifluorescence
211
(Figure 3B) and confocal microscopy with zeta-stacks (Figure 5). Taken together these results
212
demonstrate that HPV PsV reporter DNA can gain access to the nucleus as soon as 1-2hrs
213
post infection if the infected cells are close to mitotic entry, and that association with PML
214
can also occur at this time.
215
Whilst previous studies have shown a requirement for mitosis for the L2-DNA complex to
216
gain entry into the nucleus, there has been little information on the speed with which HPV
217
virions and their packaged genomes are trafficked within the cell. We provide compelling
218
evidence that reporter gene expression can be detected as little as 8h post-infection if cells are
219
infected in late S phase. Likewise, PsV reporter DNA can be found associated with PML
220
nuclear bodies as little as 1-2h post-infection and, most strikingly, this can also be clearly
221
seen in cells which are undergoing mitosis and where nuclear envelope breakdown has
222
already occurred, suggesting that the association of the L2-DNA complex with PML nuclear
223
bodies first appears during mitosis. Current studies are focused on identifying the cellular
224
components that are required for recruiting the L2-DNA complex to these nuclear domains.
225
Acknowledgements
In cells actually undergoing
9
226
The authors are very grateful to Miranda Thomas for valuable comments on the manuscript.
227
Justyna Broniarczyk is a recipient of an Arturo Falaschi ICGEB Fellowship.
228 229
Statement of author contributions
230
Justyna Broniarczyk and Paola Massimi performed all the experiments and Martina Bergant
231
and Lawrence Banks helped with the study design and manuscript preparation.
232
10
233 234
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1. Doorbar J, Quint W, Banks L, Bravo IG, Stoler M, Broker TR, Stanley MA. 2012.
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The biology and life-cycle of human papillomaviruses. Vaccine Suppl 5: F55-70.
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2. Schelhaas M, Shah B, Holzer M, Blattmann P, Kühling L, Day PM, Schiller JT,
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Helenius A. 2012. Entry of human papillomavirus type 16 by actin-dependent, clathrin-
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3. Spoden G, Kühling L, Cordes N, Frenzel B, Sapp M, Boller K, Florin L, Schelhaas
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requirements for entry. J Virol 87(13):7765-73.
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2013. Human papillomavirus types 16, 18, and 31 share similar endocytic
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5. Florin L, Becker KA, Lambert C, Nowak T, Sapp C, Strand D, Streeck RE, Sapp M.
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2006. Identification of a dynein interacting domain in the papillomavirus minor capsid
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protein L2. J Virol 80(13):6691-6.
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6. Popa A, Zhang W, Harrison MS, Goodner K, Kazakov T, Goodwin EC, Lipovsky A,
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Burd CG, DiMaio D. 2015. Direct binding of retromer to human papillomavirus type 16
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minor capsid protein L2 mediates endosome exit during viral infection. PLoS Pathog
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7. Day PM, Thompson CD, Schowalter RM, Lowy DR, Schiller JT. 2013. Identification
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of a role for the trans-Golgi network in human papillomavirus 16 pseudovirus infection. J
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8. Zhang W, Kazakov T, Popa A, DiMaio D. 2014. Vesicular trafficking of incoming
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human papillomavirus 16 to the Golgi apparatus and endoplasmic reticulum requires γ-
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PM, Schiller JT, Kann M, Pelkmans L, Helenius A, Schelhaas M. 2014. Large scale
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10. Pyeon D, Pearce SM, Lank SM, Ahlquist P, Lambert PF. 2009. Establishment of
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11. Day PM, Baker CC, Lowy DR, Schiller JT. 2004. Establishment of papillomavirus
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infection is enhanced by promyelocytic leukemia protein (PML) expression. Proc Natl
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Acad Sci U S A 101(39):14252-7.
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12. Huotari J, Helenius A. 2011. Endosome maturation. EMBO J 30(17):3481-500.
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13. Buck CB, Pastrana DV, Lowy DR, Schiller JT.
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279 280
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Figure Legends
282 283
Figure 1. Comparative rate of HPV16 PsVs infectious entry in asynchronous and
284
synchronous HaCAT cells. Panel A. Asynchronously growing HaCaT cells were infected
285
with HPV-16 PsVs carrying a luciferase reporter plasmid and were harvested at different
286
time points post-infection (0h, 4h, 8h, 13h,16h, 19h, 22h, 26, 48h) and the luciferase activity
287
was measured. The results are expressed as the means of at least three independent
288
experiments and the standard deviations are shown. Panel B. Aphidicolin synchronised
289
HaCaT cells were infected with HPV16 PsVs carrying a luciferase reporter plasmid 7h post-
290
release from G1/S (time point 0h). Cells were harvested at different time points post-infection
291
(0h, 4h, 8h, 12h, 16h) and luciferase activity measured. The results are expressed as the means
292
of at least three independent experiments and the standard deviations are shown. Panel C. Cell
293
cycle analysis of the cells harvested in Panel B stained with propidium iodide and analyzed by
294
flow cytometry. Note that the cells were mostly arrested in G1 following aphidicolin
295
treatment (panel I), at the time of infection 7h post-release they were in S phase (panel II), and
296
had entered mitosis by 4h post-infection (panel III). A siginficant proportion of cells had re-
297
entered G1 at the 8h time point (panel IV) when luciferase activity was first detected. Panel
298
D. HPV16 PsV infections were performed as in Panel B except that PsVs were pre-incubated
299
with H16.V5 neutralising antibody or pre-immune (PI) antibody prior to infection and
300
luciferase activity measured 8h post-infection. The results are expressed as the means of at
301
least three independent experiments and the standard deviations are shown. Also shown are
302
mock infected cells.
303 304 305 13
306
Figure 2. HPV16 PsV reporter gene expression can be detected 8h post-infection.
307
Panel A. Double thymidine block synchronised HaCaT cells were infected with HPV16 PsVs
308
carrying a luciferase reporter plasmid 3h post-release from G1 arrest using PsVs that were
309
either pre-incubated with H16.V5 neutralising antibody or pre-immune (PI) antibody. After
310
8h the cells were harvested and luciferase activity measured. The results are expressed as the
311
means of at least three independent experiments and the standard deviations are shown and a
312
mock infection control was also included. Panel B. Cell cycle analysis of the HaCaT cells
313
used in Panel A. Note growth arrest in G1 following the double thymidine block (panel I),
314
entry into S phase 3h post-release (panel II) at the time of infection (time 0h), entry into G2M
315
4h post-infection (panel III) and re-entry into G1 8h post-infection (panel IV) at the time of
316
detection of luciferase activity in Panel A.
317 318
Figure 3. Detection of HPV16 PsV reporter DNA in proximity with PML 1h post-
319
infection. Aphidicolin synchronised HaCaT cells were infected with HPV16 PsVs carrying
320
EdU labelled luciferase reporter plasmid 1h (Panel A) or 10h (Panel B) post-release from G1/
321
S. After a further 1h at 37°C the cells were fixed and processed for the detection of EdU-
322
labelled DNA (red), PML (in green) and DAPI (blue). Note appearence of perinuclear
323
accumulation of EdU labelled DNA in Panel A but co-localisation with nuclear PML in Panel
324
B as indicated by the arrows and the expanded field.
325 326
Figure 4. Confocal image analysis showing HPV16 PsVs reporter DNA in conjunction
327
with PML. Aphidicolin synchronised HaCaT cells were infected with HPV16 PsVs carrying
328
EdU labelled luciferase reporter plasmid 1h (Panel A) or 10h (Panel B) post-release from G1/
329
S. After a further 1h at 37°C the cells were fixed and processed for the detection of EdU-
330
labelled DNA (red) and
PML (in green). Shown are the zeta-stacks demonstrating 14
331
cytoplasmic PsV EdU labelled DNA in Panel A and co-localisation with PML in two different
332
fields (upper and lower panels) in Panel B, with an expanded view of one of those fields.
333 334
Figure 5. Confocal image analysis showing HPV16 PsVs reporter DNA in conjunction
335
with PML in mitotic cells. Aphidicolin synchronised HaCaT cells were infected with HPV16
336
PsVs carrying EdU labelled luciferase reporter plasmid 10h post-release from G1/S. After a
337
further 3h at 37°C the cells were fixed and processed for the detection of EdU-labelled DNA
338
(red) and PML (in green). Shown are the zeta-stacks demonstrating PsV EdU labelled DNA
339
and co-localsiation with PML in two different fields (upper and lower panel) in cells that are
340
undergoing mitosis.
15
