JGV Papers in Press. Published October 30, 2014 as doi:10.1099/vir.0.071084-0
REVIEW
1 2 3
Viral and Cellular Sub-Nuclear Structures in
4
Human Cytomegalovirus Infected Cells
5 Blair L Strang
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Institute for Infection & Immunity, St George’s, University of London, London, UK
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Correspondence;
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E-mail:
[email protected], Telephone: +44 (0)208 266 6422
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Running Title: HCMV and Sub-Nuclear Structures
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No. of Words in Summary: 132
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No of Words in Text: 7965
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No. of References: 115
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1
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SUMMARY
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In human cytomegalovirus (HCMV) infected cells a dramatic remodeling of the
22
nuclear architecture is linked to the creation, utilization and manipulation of sub-
23
nuclear structures. This review outlines the involvement of several viral and
24
cellular sub-nuclear structures in areas of HCMV replication and virus-host
25
interaction that include viral transcription, viral DNA synthesis and the production
26
of DNA filled viral capsids. The structures discussed include those that promote
27
or impede HCMV replication (such as viral replication compartments and PML
28
nuclear bodies, respectively) and those whose role in the infected cell is unclear
29
(for example, nucleoli and nuclear speckles). Viral and cellular proteins
30
associated with sub-nuclear structures are also discussed. The data reviewed
31
here highlights advances in our understanding of HCMV biology and emphasizes
32
the complexity of HCMV replication and virus-host interaction in the nucleus.
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2
35
TEXT
36 37
The betaherpesvirus human cytomegalovirus (HCMV) is a widespread
38
opportunistic
pathogen
that
severely
39
immunodeficient populations (Mocarski et al., 2007). Like all herpesviruses,
40
HCMV undergoes either productive or latent infection. During productive infection
41
infectious virus is produced, while in latent infection the virus becomes largely
42
transcriptionally quiescent (Mocarski et al., 2007). Also, like other herpes viruses,
43
many events that are critical for productive HCMV replication take place within
44
the nucleus. These include essential steps in viral replication, including; the
45
transcriptional cascade of immediate early (IE) –early (E) –late (L) viral RNA
46
transcripts, synthesis of viral DNA and the production of DNA containing capsids
47
(Mocarski et al., 2007). Compared to the prototype human herpesvirus, the
48
alphaherpesvirus herpes simplex virus (HSV), certain features of productive
49
HCMV infection are not well characterized. However, it is clear that several sub-
50
nuclear structures are involved in HCMV genome replication and virus-host
51
interaction. Uncovering the roles of these structures has led to significant
52
advances in our understanding of HCMV biology. This review summarizes the
53
involvement of several viral and cellular sub-nuclear structures in productive
54
HCMV infection. The participation of each structure in HCMV replication and
55
virus-host interaction is described from entry of the viral genome into the nucleus
56
to the egress of viral capsids through the nuclear membrane. The data discussed
57
highlights recent advances in our understanding of sub-nuclear structure
3
affects
immunocompromised
and
58
function, introduces viral and cellular factors associated with sub-nuclear
59
structures and indicates what questions have yet to be answered.
60 61
Entry of the HCMV genome into the nucleus: the nuclear pore complex,
62
nuclear speckles, PML-NBs and pp150 bodies. HCMV genome entry into the
63
nucleus is poorly defined, but may be similar to movement of the HSV DNA
64
genome through the nuclear pore complex (NPC) (Kobiler et al., 2012) (Figure
65
1A). HCMV capsid and tegument proteins (potentially HCMV UL48 and UL77,
66
homologues of HSV UL36 and UL25, respectively) likely interact with proteins on
67
the cytoplasmic face of the NPC (potentially including, Nup214, Nup358 and
68
hCG1) (Table 1). This leads to opening, or “uncoating”, of the capsid. The
69
pressure of viral DNA on the capsid wall leads to release of a linear DNA form of
70
the HCMV genome. Viral DNA travels out of the capsid via a portal on a unique
71
vertex of the capsid comprised of UL104 (HSV UL6 homologue) (Kobiler et al.,
72
2012). The HCMV genome moves through the central channel of the NPC into
73
the nucleus (Kobiler et al., 2012).
74
The linear HCMV DNA genome circularizes upon entry into in the nucleus
75
via fusion of the genomic termini (McVoy & Adler, 1994) (Figure 1B). This could
76
be mediated by either homologous or non-homologous end joining DNA repair
77
mechanisms. The circular form of the genome is utilized as a template for viral
78
RNA transcription and DNA synthesis. Very early in infection viral genomes can
79
be found at the periphery of the nucleus where transcription from the viral
80
genome takes place (Ishov et al., 1997). At the periphery of the nucleus the
4
81
major viral immediate early transcriptional transactivator IE2 co-localizes with
82
cellular RNA polymerase II transcription factors TATA-binding protein (TBP) and
83
transcription factor IIB (TFIIB)(Ishov et al., 1997) (Figure 1B). It is possible, like
84
HSV RNA transcription, HCMV RNA transcription involves the nuclear lamina.
85
The nuclear lamina is a dense fibrillar protein network at the nuclear membrane
86
composed of lamin proteins and lamin-associated proteins (Figure 1A). In HSV
87
infected cells the lamina component lamin A/C is important for genome targeting
88
to the lamina and at the lamina hetero- or euchromatin on viral promoters
89
modulate HSV RNA transcription (Silva et al., 2008).
90
The majority of HCMV genomes entering the nucleus localize with
91
promyelocytic leukemia protein nuclear bodies (PML-NBs) (also referred to as
92
ND10 bodies) and SC35 containing structures within intrachromosomal space.
93
IE2 bridges PML-NBs and SC35 containing structures (Figure 1B). As
94
transcription progresses viral IE RNA transcripts co-localize with SC35-
95
contianing bodies, but not PML-NBs (Ishov et al., 1997). SC35 is a major protein
96
component of nuclear speckles which are dynamic nuclear structures composed
97
of RNA and protein and enriched in pre-mRNA splicing factors (Lamond &
98
Spector, 2003). A role for nuclear speckles in HCMV replication, or that of any
99
other human virus, is not often commented on. It is likely nuclear speckles are
100
involved in HCMV IE gene transcription, but it is unknown what transcriptional
101
events they might facilitate. In HSV infected cells it has been reported that
102
nuclear speckles are involved in export of viral RNA transcripts (Chang et al.,
103
2011). Thus, nuclear speckles may serve in the processing and export of HCMV
5
104
RNA early in infection. These RNAs likely include the viral RNAs transcribed from
105
the UL122-123 locus of the HCMV genome that are multiply spliced to produce
106
mRNA transcripts encoding the IE proteins in IE1 and IE2. The role of PML-NBs
107
in HCMV replication is one of the most studied and debated facets of HCMV
108
biology. The complex relationship between PML-NBs and HCMV cannot be fully
109
summarized here, but is discussed in detail elsewhere (Everett et al., 2013;
110
Tavalai & Stamminger, 2009, 2011). PML-NBs are dynamic structures that
111
comprise at least 70 constitutively or transiently present proteins, including PML
112
protein (which acts a scaffold for PML-NB formation) (Ishov et al., 1999). The
113
majority, if not all, PML-NB proteins are post-translationally modified with the
114
addition of small ubiquitin-related modifier (SUMO) moieties. PML-NBs contain
115
several proteins required for protein SUMOylation and the relationship protein
116
function and post-translational modification by SUMO is a rapidly developing
117
topic in HCMV biology and elsewhere (Everett et al., 2013).
118
PML-NBs are found throughout the nucleus. Upon entry of the viral
119
genome into the nucleus PML-NBs form on SUMO-modified PML proteins
120
juxtaposed to incoming viral genomes. PML-NBs act to repress viral transcription
121
via the co-operative function of the PML-NB proteins PML, Sp100 and hDaxx
122
(Glass & Everett, 2013). PML protein is one of the best characterized PML-NB
123
proteins involved in HCMV transcriptional repression and acts to facilitate
124
assembly of PML-NBs containing transcriptionally repressive proteins at HCMV
125
genomes (Tavalai & Stamminger, 2009, 2011). Sp100 promotes transcriptional
126
repression
through
its
interaction
with
6
the
transcriptional
repressor
127
heterochromatin protein (HP-1)(Seeler et al., 1998). SUMO modification of
128
Sp100 promotes interaction with HP1 (Seeler et al., 2001), but is not required for
129
localization to PML-NBs (Sternsdorf et al., 1999). hDaxx is recruited to PML-NB
130
via an interaction with SUMOylated PML (Everett et al., 2013) and acts as a
131
transcriptional co-repressor via its interaction with the chromatin remodeler ATRX
132
(Ishov et al., 2004; Xue et al., 2003). PML-NBs contain other transcriptionally
133
repressive cellular proteins (Tavalai & Stamminger, 2011) and it is possible other
134
PML-NB proteins may antagonize HCMV replication.
135
Crucially, HCMV encodes antagonists of PML-NB function (Table 1)
136
whose actions lead to removal of proteins from PML-NBs and the dispersal of
137
PML-NBs (Everett et al., 2013; Tavalai & Stamminger, 2009, 2011). In HCMV
138
infected cells, two major antagonists of PML-NB function are pp71 and IE1. pp71
139
degrades hDaxx by a proteasome-dependent, ubiquitin independent, mechanism
140
(Kalejta & Shenk, 2003) and displaces ATRX from PML-NBs (Lukashchuk et al.,
141
2008). The presence of IE1 promotes disruption of PML-NBs, induces the
142
proteasome independent loss of SUMOylated PML and Sp100 proteins (Ahn &
143
Hayward, 1997; Kang et al., 2006; Kim et al., 2011; Korioth et al., 1996; Lee et
144
al., 2004; Tavalai et al., 2011; Wilkinson et al., 1998; Xu et al., 2001) and
145
potentially induces the proteasome dependent degradation of un-SUMOylated
146
Sp100 late in infection (Tavalai et al., 2011). Importantly, IE1 does not act like its
147
well-studied counterpart expressed by HSV, ICP0. ICP0 possesses an E3
148
ubiquitin ligase activity directly involved in the proteasome dependent
149
degradation of PML protein (Boutell & Everett, 2013). IE1 has no known ubiquitin
7
150
ligase or SUMOylase activity (Kang et al., 2006), but is able to exert an effect
151
that leads to PML-NB disruption, loss of SUMOylated PML and an increase in the
152
abundance of unmodified PML. This would suggest that IE1 can recruit a cellular
153
protein to de-SUMOylate PML within PML-NBs or SUMO is removed from PML
154
by a cellular de-SUMOylase after PML-NBs have been dispersed in the presence
155
of IE1. The role of IE1 in Sp100 degradation is unclear. IE1 binding to Sp100
156
(Kim et al., 2011) may result in Sp100 degradation by an as yet unknown
157
mechanism. Alternatively, Sp100 degradation may be an indirect result of PML-
158
NB modification by IE1.
159
Other HCMV proteins may be involved in PML-NB destruction or
160
modification (Table 1). Expression of epitope tagged versions of US25, UL29
161
UL30, UL69, UL76, UL98 or TLR9 is sufficient to modestly reduce the number of
162
PML-NBs in uninfected cells (Salsman et al., 2008). These proteins are
163
expressed in different kinetic classes of viral transcription and have different
164
effects on HCMV replication, plus the function of several of these proteins is
165
unknown (Table 1). In transfection experiments epitope tagged HCMV proteins
166
US32, UL80a and UL35 co-localize with, but do not obviously degrade, PML-NBs
167
in uninfected cells (Salsman et al., 2008). Epitope tagged US32 and UL35 alter
168
the shape and size of PML-NBs in uninfected cells. The localization and function
169
of these proteins in relation to PML-NBs in HCMV infected cells has yet to be
170
elucidated. UL80a is a capsid protein. UL80a association with PML-NBs is
171
reminiscent of PML cages trapping alphaherpesvirus varicella-zoster virus (VZV)
172
capsids in infected cells via physical interaction between PML-NB proteins and
8
173
VZV capsids (Reichelt et al., 2011). This mechanism of cellular defense to
174
infection has not yet been observed in HCMV infected cells. An epitope tagged
175
version of the putative HCMV protein UL3 has been reported to alter PML-NBs in
176
uninfected cells (Salsman et al., 2008). However, it is now thought that the
177
HCMV genome does not contain an open reading frame encoding UL3 (Gatherer
178
et al., 2011). A final protein to consider in the alteration of PML-NBs in HCMV
179
infected cells is the HCMV viral kinase UL97. UL97 phosphorylates and
180
inactivates the PML-NB protein retinoblastoma (Rb) (Hume et al., 2008) and
181
expression of epitope tagged UL97 in uninfected simian cells (Prichard et al.,
182
2008), but not uninfected human cells (Kuny et al., 2010), notably reduces the
183
number of PML-NBs in the cell. Clarification of the roles, if any, of UL97 and Rb
184
proteins in PML-NB function in HCMV infected cells is required.
185
Future consideration of PML-NB function in HCMV infected cells will
186
benefit from further exploration of the interaction of PML-NBs with other viruses,
187
including other herpesviruses. Indeed, most detailed information concerning the
188
interaction of PML-NB with viral DNA genomes comes from the study of HSV
189
infection and analysis of PML-NB antagonism by the HSV protein ICP0 (Boutell &
190
Everett,
191
papillomaviruses has been reported, but is less well understood. Prominent
192
findings include the ability of the adenovirus proteins E1B and E4orf3 to
193
antagonize PML and hDaxx function (Schreiner et al., 2010; Ullman & Hearing,
194
2008) and the repression of papillomavirus RNA transcription by Sp100 (Stepp et
2013).
Interaction
of
PML-NB
9
interaction
with
adeno-
and
195
al., 2013). Future study of adeno- and papillomavirus replication may illuminate
196
important features of herpesvirus interaction with PML-NBs.
197
Sub-nuclear structures other than PML-NBs could also be involved in
198
repression of viral transcription. These include bodies which include the HCMV
199
tegument protein pp150 (UL32). pp150 is a substrate of cyclin A2-CDK (Table 1)
200
and is a sensor for cell cycle progression during HCMV infection (Table 1). By an
201
unknown mechanism, pp150 is involved in blocking viral transcription when
202
levels of cyclin A2-CDK are high (Bogdanow et al., 2013). It has been suggested
203
that pp150 nuclear bodies are sites of pp150-mediated transcriptional repression
204
and are the destination of HCMV genomes not associated with PML-NBs
205
(Bogdanow et al., 2013; Ishov et al., 1997) (Figure 1B).
206
Therefore, upon engagement of the viral capsid with proteins on the
207
cytoplasmic face of the nuclear pore complex, the HCMV genome is delivered
208
into the nucleus through the nuclear pore. Once in the nucleus, transcription from
209
the viral genome involves cellular transcription factors and may be facilitated by
210
interaction of the genome with the nuclear lamina at the periphery of the nucleus.
211
At the periphery of the nucleus the genome encounters nuclear speckles, which
212
likely facilitate the splicing and export to the cytoplasm of viral RNA transcripts,
213
and PML-NBs or pp150 bodies that restrict viral transcription. Restriction of viral
214
transcription by PML-NBs involves the recruitment of transcriptional repressors to
215
the viral genome and is antagonized by virally encoded proteins. Restriction of
216
viral transcription by pp150 bodies is less well defined and is associated with
217
progression of the infected cell through the cell cycle.
10
218 219
Replication of the viral genome: HCMV replication compartments,
220
chromatin partitioning, protein quality control compartments and nuclear
221
speckles. Once transcription of immediate early and early transcripts is
222
underway viral proteins that replicate HCMV genomic DNA are produced. Viral
223
DNA synthesis and several early and late transcriptional events occur within non-
224
membrane bound compartments created by the virus composed of protein and
225
nucleic acids. The formation of these “viral replication compartments” is poorly
226
understood, but allows the recruitment and concentration of factors required for
227
viral replication to specific areas of the infected cell. Analysis of alphaherpesvirus
228
infection implies that a herpesvirus replication compartment can develop from a
229
single incoming viral genome (Kobiler et al., 2011). In HCMV infected cells
230
replication compartments form in the vicinity of PML-NBs (Ahn et al., 1999). As
231
compartments develop they contain viral transcriptional transactivators (including
232
IE2 and UL112-113 proteins), viral proteins involved in viral DNA synthesis
233
(including UL44 and UL57) and viral DNA (Ahn et al., 1999; Penfold & Mocarski,
234
1997; Strang et al., 2012b) (Table 1). Early in the formation of replication
235
compartments factors involved in the DNA damage response, including γH2AX
236
and ATM, localize to replication compartments (E et al., 2011) and may
237
recognize viral genomes within compartments. It is thought that viral
238
transcriptional transactivators drive E2F1 mediated activation of ATM signaling,
239
which activates DNA damage response factors such as γH2AX and p53 (E et al.,
240
2011). Activation of this pathway is likely required for modulation of cell cycle
11
241
related functions that promote viral replication. ATM signaling promotes the
242
development of replication compartments (E et al., 2011) but it is unclear if this is
243
an effect that directly relates to replication compartment development or an
244
indirect effect related to events that promote viral RNA transcription or viral DNA
245
synthesis.
246
During HCMV replication several compartments can form and then likely
247
coalesce to form a single compartment that occupies nearly all the nucleus, but
248
does not encroach into nucleoli (Penfold & Mocarski, 1997; Strang et al., 2012a;
249
Strang et al., 2012b) (Figures 2A and 2B). The mechanisms driving coalescence
250
of HCMV replication compartments have yet to be defined. Replication
251
compartment coalescence in HSV infected cells occurs via non-random
252
movement and is dependent upon nuclear actin, myosin and viral transcription
253
(Chang et al., 2011).
254
To provide areas in the nucleus for replication compartments to develop
255
the compaction and movement of chromatin is observed (O'Dowd et al., 2012;
256
Strang et al., 2012b). This process is termed “chromatin partitioning”. It is
257
unknown what drives this process, but UL98 and UL76 may be involved. UL98 is
258
an alkaline nuclease capable of digesting DNA (Sheaffer et al., 1997). UL76 is
259
member of the UL24 gene family, all of which contain a conserved putative PD-
260
(D/E)XK endonuclease motif (Knizewski et al., 2006). Although endonuclease
261
activity of UL76 has yet to be demonstrated, indirect evidence points to a role for
262
UL76 in the chromosome breakage required for chromatin partitioning. For
263
example, the presence of UL76 in uninfected cells is sufficient to stimulate
12
264
chromosomal aberrations (Siew et al., 2009) and in HCMV infected cells UL76 is
265
involved in stimulating a DNA damage response (Costa et al., 2013).
266
Several facets of HCMV replication compartment organization and
267
function late in infection have recently been described, each involving the
268
localization of HCMV protein UL44. UL44 is the putative processivity subunit of
269
the HCMV DNA polymerase (Ertl & Powell, 1992) and several different
270
phosphorylated forms of UL44 are present in the infected cell (Silva et al., 2011).
271
At least one form of UL44 accumulates at the periphery of replication
272
compartments throughout HCMV replication and UL44 is also found at
273
considerably lower levels within certain domains inside replication compartments,
274
plus in the nucleus outside of replication compartments and also in the cytoplasm
275
(Hamirally et al., 2009; Penfold & Mocarski, 1997; Strang et al., 2012b). Viral
276
DNA synthesis occurs at foci within the layer of UL44 the periphery of replication
277
compartments (Figure 2B) and newly synthesized viral DNA moves into the
278
interior of replication compartments (Strang et al., 2012b).
279
The viral ssDNA binding protein UL57 co-localizes with UL44 at the
280
periphery of replication compartments and is found throughout the interior of
281
compartments (Penfold & Mocarski, 1997; Strang et al., 2012b). UL57 at the
282
periphery of replication compartments is to be likely directly involved in viral DNA
283
synthesis. UL57 protein in the interior of replication compartments is associated
284
with viral DNA (Penfold & Mocarski, 1997) and is possibly involved in UL57
285
mediated DNA strand exchange events, including recombination and repair of
286
newly synthesized HCMV DNA.
13
287
Other than UL44 and UL57, other factors required for viral DNA synthesis
288
have been reported. These include the viral DNA polymerase subunit UL54,
289
replication factor UL84 and the helicase DNA complex (UL70, UL102 and UL105)
290
(Pari & Anders, 1993; Pari et al., 1993). However, the full repertoire of viral and
291
cellular proteins acting in concert during DNA synthesis has yet to be described.
292
These proteins likely include those viral and cellular proteins that associate with
293
UL44 and/or UL84 (Gao et al., 2008; Strang et al., 2010a; Strang et al., 2010b;
294
Strang et al., 2009), several proteins that have ill-defined roles in viral DNA
295
synthesis (for example, UL36, IRS1 and IE2 (Pari & Anders, 1993; Pari et al.,
296
1993; Sarisky & Hayward, 1996)), plus viral and cellular proteins involved in DNA
297
damages responses and viral DNA repair (E et al., 2011; O'Dowd et al., 2012;
298
Strang & Coen, 2010). How these and other proteins are organized within HCMV
299
replication compartments is unclear, but may involve UL44. UL44, a homodimer,
300
is a structural homologue of the cellular proliferating cell nuclear antigen (PCNA),
301
which exists as a timer (Appleton et al., 2004; Appleton et al., 2006; Loregian et
302
al., 2004a, b). PCNA binds multiple proteins of multiple functions at the DNA
303
replication fork (Moldovan et al., 2007). UL44 could play a similar role and bind
304
multiple proteins in replication compartments. Proteomic studies have indicated
305
that UL44 associates with viral and cellular proteins found in replication
306
compartments, including UL84 (Strang et al., 2010a). The binding of proteins to
307
UL44 is increasingly well characterized. Studies interrogating the binding of UL54
308
to UL44 reveal that, like PCNA, UL44 is able to bind proteins within “hydrophobic
309
pockets”; crevices in located in the amino termini of UL44 containing several
14
310
hydrophobic amino acids into which proteins binding UL44 insert their binding
311
domains (Appleton et al., 2004; Appleton et al., 2006; Loregian et al., 2004a, b).
312
Unexpectedly, UL44 can also associates with proteins outside of the hydrophobic
313
pockets as it has been demonstrated that the association of UL84 with UL44 is
314
not dependent upon binding in the hydrophobic pocket (Strang et al., 2009).
315
Rather, association of UL84 with UL44 takes place elsewhere in the amino
316
terminal domain of UL44 and is not dependent upon homodimerization of UL44
317
(Strang et al., 2009). It is unclear what drives selection of proteins for binding to
318
UL44, but there are likely determinants of protein binding in both UL44 and its
319
interacting proteins. Indeed, it has been demonstrated that the HCMV proteins
320
IRS1 and TRS1, who have identical amino termini that are required for
321
association with UL44, compete for UL44 binding (Strang et al., 2010b).
322
UL44 is also indirectly involved in the recruitment of viral proteins to
323
replication compartments. The viral uracil DNA glycosylase UL114 is required for
324
efficient viral DNA synthesis and base excision repair (BER) (Prichard et al.,
325
1996). BER maintains genome integrity by removing uracil from HCMV genome,
326
which is the result of either misincorporation of uracil during viral DNA synthesis
327
or the spontaneous deamination of cytosine in the viral genome. Although UL114
328
localizes with UL44 within replication compartments (Prichard et al., 2005), it
329
does not bind UL44, as first thought (Ranneberg-Nilsen et al., 2008). Rather,
330
UL114 associates with the catalytic subunit of the viral DNA polymerase UL54
331
and UL114, UL54 and UL44 form a protein complex (Strang & Coen, 2010).
15
332
Thus, UL44 indirectly recruits UL114 to replication compartments via the
333
interaction of UL114 with UL54.
334
There are few reports of cellular proteins localizing to HCMV replication
335
compartments, although it is known that both p53 and replication protein A (RPA)
336
are recruited to the interior of replication compartments (Fortunato & Spector,
337
1998). Their roles within replication compartments are unknown but may involve
338
modulation of cellular responses to infection and promoting efficient viral DNA
339
synthesis.
340
Several
factors
influence
the
architecture
of
HCMV
replication
341
compartments. While the amino termini of UL44 is required for binding of UL44
342
associated proteins, the carboxyl terminus of UL44 is required for the
343
development of replication compartments and correct localization of UL44 in the
344
nucleus (Silva et al., 2010). Although the carboxyl termini of UL44 can be
345
extensively phosphorylated by both the viral kinase UL97 or the cellular kinase
346
cdk1, it is unlikely that these post-translational modifications affect replication
347
compartment function as mutational analysis of phosphorylated residues in the
348
UL44 carboxyl terminus has little effect on viral DNA synthesis and viral
349
replication (Silva et al., 2011). Efficient viral DNA synthesis is also required to
350
maintain UL44 localization and compartment structure (E et al., 2011; Penfold &
351
Mocarski, 1997; Strang et al., 2012a; Strang et al., 2012b). Unexpectedly, the
352
multifunctional cellular nucleolar protein nucleolin (Ginisty et al., 1999) is involved
353
in maintaining replication compartment architecture. UL44 and nucleolin
354
associate via the amino terminus of UL44 (Strang et al., 2010a) and in infected
16
355
cells nucleolin co-localizes with UL44, but not viral DNA, on the exterior of
356
replication compartments (Strang et al., 2010a; Strang et al., 2012a). The
357
absence of nucleolin in the infected cell causes mislocalization of UL44 at the
358
periphery of replication compartments and compromises efficient viral DNA
359
synthesis (Strang et al., 2012a). Nucleolin is also required for UL84 localization
360
with UL44 at the periphery of replication compartments, although in the absence
361
of nucleolin UL84 does not redistribute throughout the nucleus, but relocates to
362
the cytoplasm (Bender et al., 2014). Thus, nucleolin is required for both proper
363
nuclear and subnuclear localization of HCMV replication compartment proteins.
364
Furthermore, it is possible that in infected cells nucleolin, UL44, UL84 and either
365
IRS1 or TRS1 interact within a large-multiprotein complex (Strang et al., 2010a;
366
Strang et al., 2010b). Therefore, nucleolin may have an effect on the localization
367
of both IRS1 and TRS1 in the infected cell.
368
Although the organization of factors required for viral DNA synthesis in
369
replication compartments is increasingly well understood, perhaps the most
370
important unanswered questions involving the architecture of replication
371
compartments involve the localization of factors required for RNA transcription
372
from the viral genome. The location of transcriptionally active genomes within
373
replication compartments is unknown. There is little information on the
374
localization of viral and cellular proteins required for viral RNA transcription. In
375
infected
376
unphosphorylated (transcriptionally inactive) forms of RNA polymerase II
377
concentrate within nuclear structures thought to be replication compartments
cells
both
phosphorylated
17
(transcriptionally
active)
and
378
(Feichtinger et al., 2011). Within these structures unphosphorylated and
379
phosphorylated forms of RNA polymerase II localize with the viral protein UL69
380
(Feichtinger et al., 2011), which is known to be required for viral RNA processing
381
and export to the cytoplasm. The localization of RNA polymerase II in relation to
382
UL44 has yet to be described, however, viral proteins involved in transcription
383
and RNA metabolism, for example IE1, IE2 and UL84 (Bender et al., 2014; Silva
384
et al., 2010) are found throughout the nucleus and co-localize with UL44 in viral
385
replication compartments. Therefore, it is likely that viral transcription occurs
386
throughout replication compartments and, unlike viral DNA synthesis, is not
387
restricted to specific areas of replication compartments such as the layer of UL44
388
that accumulates at the periphery of replication compartments.
389
The
presence
of
sub-compartments
within
HCMV
replication
390
compartments has been reported. Components of the ubiquitin–proteasome
391
system (UPS) assemble into domains at the periphery of replication
392
compartments (Figure 2B), where they co-localize with UL57 (Tran et al., 2010).
393
The accumulation of UPS proteins at the periphery of HCMV replication
394
compartments is analogous to the formation of VICE (virus induced chaperone
395
enriched) domains at the periphery of HSV replication compartments. VICE
396
domains degrade misfolded proteins in a proteasome dependent fashion to
397
promote efficient virus replication (Livingston et al., 2009). Thus, compartments
398
containing UPS components at the periphery of HCMV replication compartments
399
may act as quality control areas for HCMV replication. It is unclear which HCMV
400
proteins other than UL57 localize to UPS compartments. However, HCMV UL76
18
401
co-localizes with the UPS component Rpn10/S5a in discrete areas of infected
402
cell nuclei termed aggressomes (Lin et al., 2013). Rpn10/S5a is found in UPS
403
compartments (Tran et al., 2010). Thus, some aggressomes may be UPS
404
compartments associated with replication compartments that contain both UL76
405
and Rpn10/S5a. It is unknown what role UL76 might have in UPS compartments.
406
Sub-compartments within the interior of replication compartments which do not
407
contain UL44 or viral DNA have been observed (Strang et al., 2012b). It is
408
possible these sub-compartments are the aforementioned UPS compartments.
409
The presence of SC35 containing structures on the exterior of HCMV
410
replication compartments has been reported (Rechter et al., 2009) (Figure 2B). It
411
is likely that, as in HSV infected cells (Chang et al., 2011), these are nuclear
412
speckles which remain associated with HCMV replication compartments
413
throughout virus replication. The directed movement of nuclear speckles with
414
HSV replication compartments, or vice versa, is associated with efficient viral
415
RNA transcription in HSV infected cells, in particular promoting the export of Late
416
HSV RNA transcripts to the cytoplasm (Chang et al., 2011). The function of
417
nuclear speckles associated with replication compartments in HCMV infected
418
cells has yet to be described, although it is likely they are involved in viral RNA
419
transcription.
420
In summary, HCMV replication compartments are necessary for the
421
concentration and organization of factors required for viral replication. The
422
organization of factors required for viral DNA synthesis in replication
423
compartments is increasingly well defined and involves UL44. Viral replication
19
424
compartments are dynamic structures whose architecture is influenced by UL44
425
localization, viral DNA synthesis and the cellular nucleolar protein nucleolin.
426
Nucleolin is not only required to maintain replication compartment architecture
427
but is also involved in the subcellular localization of at least one viral protein
428
found in replication compartments. The organization of factors required for RNA
429
transcription from the HCMV genome in replication compartments is not well
430
defined. However, it can be suggested that viral transcription takes place
431
throughout the interior of replication compartments, in contrast to viral DNA
432
synthesis, which takes place within a layer of UL44 at the periphery of replication
433
compartments. Within replication compartments there are sub-compartments,
434
some of which are associated with protein quality control. Nuclear speckles are
435
associated with viral replication compartments where they likely enable viral
436
transcription, possibly by facilitating the export of viral transcripts to the
437
cytoplasm.
438 439
Structures associated with HCMV replication: nucleoli, Cajal bodies and
440
perinucleolar compartments. To date, little attention has been paid to the role
441
of nucleoli and associated sub-nuclear structures such as Cajal bodies and
442
perinucleolar compartments in HCMV infected cells. It is increasing clear,
443
however, that these sub-nuclear structures may have important roles in HCMV
444
genome replication and virus-host interactions.
445
Nucleoli are dynamic non-membrane bound protein and RNA structures
446
that form on ribosomal DNA. A nucleolus has a tripartite structure in which the
20
447
fibrillar center is surrounded by a dense fibrillar component (containing the
448
majority of nucleolin found in nucleoli) that is surrounded by the granular
449
component. In HCMV infected cells nucleoli remain intact (Strang et al., 2012c)
450
and visualization of nucleolin in HCMV infected cells suggests changes in the
451
size of nucleoli upon infection (Strang et al., 2012a). Also, striking changes to
452
nucleoli composition and architecture occur as upon infection nucleolin levels in
453
nucleoli increase and nucleolin accumulates at the periphery of nucleoli (Strang
454
et al., 2012a) (Figure 2C). These changes in nucleolar composition and
455
architecture may relate to the recruitment
456
compartments, a nucleolar stress responses to infection or the production of
457
ribosomes in the HCMV infected cell. The recruitment of nucleolin to replication
458
compartments may be related to the interaction of nucleolin with viral proteins
459
found in replication compartments. It has been observed that the HCMV protein
460
UL84 co-localizes with nucleolin in nucleoli and at the periphery of replication
461
compartments (Bender et al., 2014). Therefore, it is possible that UL84 recruits
462
nucleolin to replication compartments from nucleoli, which results in changes to
463
nucleolar composition and architecture. Nucleoli can act as sensors of
464
environmental, genotoxic or osmotic stress and pathogen invasion (Boisvert et
465
al., 2007; Boulon et al., 2010; Hiscox, 2002; Hiscox et al., 2010). These stresses
466
result in a variety of outcomes including changes in nucleolar morphology and
467
nucleolar proteome, plus pleiotropic effects of p53 activation (Boulon et al.,
468
2010). The changes in nucleolar proteome and architecture observed in HCMV
469
infected cells could relate to a nucleolar stress response. The nucleolar
21
of
nucleolin to replication
470
localization of nucleolin in HCMV infected cells is akin to that seen in coronavirus
471
infected cells. It has been suggested that localization of nucleolin in coronavirus
472
infected cells is related to “nucleolar capping” (Dove et al., 2006). Capping is
473
associated with a nucleolar stress response caused by factors such as
474
transcriptional inhibition or DNA damage. These stresses cause condensation
475
and segregation of the fibriallar center and the granular component. This is
476
accompanied by the formation of “caps” of nucleoplasmic RNA binding proteins
477
upon the body of nucleoli (Boulon et al., 2010; Shav-Tal et al., 2005). It has been
478
reported that nucleolar capping is observed in HCMV infected cells (Gaddy et al.,
479
2010). However, it is unclear if the reported nucleolar caps are actually
480
associated with nucleoli. Furthermore, analysis of nucleoli in HCMV infected cells
481
by electron microscopy does not suggest the presence of nucleolar caps (Strang
482
et al., 2012c). It is unknown if HCMV infection stimulates p53 mediated nucleolar
483
stress responses, which include specific changes to the protein translational
484
profile of the cell (Boulon et al., 2010). Finally, the principal role of nucleoli in
485
uninfected cells is to produce ribosomes. Levels of certain ribosomal RNAs
486
(rRNAs) increase in infected cells (Tanaka et al., 1975). Therefore, ribosome
487
production during HCMV replication may not be compromised. However, the
488
increase in rRNA production in infected cells could be related to changes in
489
nucleolar composition and architecture.
490
An integral part of any of the possibilities discussed could be the action of
491
HCMV proteins in nucleoli. Several viral proteins can localize to nucleoli. It has
492
been reported that HCMV pp65 and UL84 proteins are a nucleolar antigens
22
493
(Arcangeletti et al., 2009; Bender et al., 2014). Also, transfection experiments
494
have indicated that several epitope tagged versions of HCMV proteins (TLR5,
495
TLR7, US33, UL29, UL31 and UL76) localize to the nucleolus in uninfected cells
496
(Salsman et al., 2008). These proteins are expressed in different kinetic classes
497
of viral RNA transcription and have different effects on HCMV replication, plus
498
the function of several of these proteins is unknown (Table 1). Also, it has been
499
reported that epitope tagged versions of the putative HCMV proteins TLR7 and
500
UL108 localize to nucleoli in uninfected cells (Salsman et al., 2008). However, it
501
is now thought that the HCMV genome does not possess open reading frames
502
encoding TLR7 or UL108 (Gatherer et al., 2011).
503
Cajal bodies and perinucleolar compartments are sub-nuclear structures
504
associated with nucleoli (Figure 2C) and both may be involved in viral RNA
505
production. Cajal bodies are non-membrane bound protein and RNA structures
506
involved in transcription and maturation of ribonuclear particles (Ogg & Lamond,
507
2002). A role for Cajal bodies in HCMV replication has yet to be described.
508
However, transfection of epitope tagged HCMV UL30 leads to disruption and/or
509
loss of Cajal bodies in uninfected cells (Table 1). Thus, it has been suggested
510
(Salsman et al., 2008) UL30 may allow HCMV to inhibit transcription and
511
ribonuclear particle maturation. Cajal bodies are thought to play a role in
512
adenovirus replication, wherein adenovirus infection results in fragmentation of
513
Cajal bodies. Also, Cajal bodies are required for efficient production of several
514
Late adenoviral RNA transcripts (James et al., 2010). Thus it is possible, as in
515
adenovirus infected cells, Cajal bodies are disrupted upon HCMV infection and
23
516
have a role in HCMV RNA transcription. However, Cajal bodies are generally
517
absent in slowly dividing or non-dividing cells (Ogg & Lamond, 2002) and may
518
not be present in the fibroblast, vascular and neuronal cell types within which
519
HCMV replicates during productive infection. It has been suggested that the
520
putative HCMV protein UL3 is also involved in modification of Cajal bodies
521
(Salsman et al., 2008). However, as mentioned above, it is now thought that an
522
open reading frame encoding UL3 is not present in the HCMV genome (Gatherer
523
et al., 2011).
524
Perinucleolar compartments are thought to be involved in RNA
525
polymerase II and III transcription. In HCMV infected cells at least one
526
perinucleolar compartment protein, polyryrimidine tract-binding protein (PTB), is
527
relocalized from the nucleus to the cytoplasm or degraded in the nucleus (Gaddy
528
et al., 2010). It has been suggested (Gaddy et al., 2010) that loss of PTB from
529
perinucleolar compartments is related to negative regulation of viral splicing by
530
PTB (Cosme et al., 2009).
531
In summary, upon HCMV infection of the cell nucleoli remain intact, but
532
changes to nucleolar composition and architecture are observed. These changes
533
may be related to a cellular nucleolar stress response, the recruitment of
534
nucleolin to HCMV replication compartments or the presence of viral proteins
535
within nucleoli. Subnuclear structures associated with nucleoli, including Cajal
536
bodies and perinucleolar compartments, may be involved in viral RNA production
537
in HCMV infected cells.
538
24
539
Production of DNA filled capsids: a viral structure putatively involved in
540
capsid assembly and genome packaging. Late viral transcription produces
541
proteins required to form viral capsids. The assembly of HCMV capsids and
542
packaging of genomes into them is increasingly well understood (Gibson, 2008;
543
McVoy et al., 2000; Scheffczik et al., 2002; Wang et al., 2012). However, it is
544
unclear where within viral replication compartments capsid assembly and
545
genome packaging take place. These questions have not been extensively
546
interrogated.
547
There is little data regarding the localization of HCMV capsid proteins
548
within the infected cell nucleus. However, it has been reported that a “sponge-
549
like” web of UL80 proteins forms throughout the infected cell nucleus, dependent
550
upon UL80 self-interaction (Loveland et al., 2007; Nguyen et al., 2008). UL80
551
proteins include the capsid assembly protein precursor (UL80.5) and the
552
maturational protease precursor (UL80a), which interact with the major HCMV
553
capsid protein UL86 (Wood et al., 1997). It is possible the web of UL80 proteins
554
could act as a scaffold for capsid assembly and genome packaging within viral
555
replication compartments.
556
Packaging of viral DNA into nascent viral capsids likely takes place within
557
replication compartments. The viral protein responsible for DNA binding during
558
the packaging process, UL56, is found within replication compartments. UL56
559
localization to replication compartments is dependent on viral DNA synthesis and
560
UL56 may interact with UL44 within replication compartments (Giesen et al.,
561
2000). Furthermore, it has been speculated that packaging of viral DNA into
25
562
capsids is responsible for the movement of newly synthesized viral DNA from the
563
periphery of replication compartments to the interior of replication compartments
564
(Strang et al., 2012b). Therefore, further study is required to understand the relationship between
565 566
the
production of
DNA
containing
viral
capsids
and
viral
replication
567
compartments. It can be proposed, however, that viral capsid proteins play a role
568
in organizing capsid production by creating a sub-nuclear structure on which
569
capsid assembly and DNA packaging might take place.
570 571
Exit of capsids from the nucleus: the nuclear lamina. To escape the nucleus
572
DNA containing capsids must first travel from within viral replication
573
compartments to the nuclear periphery. It is unknown what facilitates this process
574
in HCMV infected cells. In HSV infected cells directed capsid movement in the
575
nucleus is dependent upon myosin and actin (Forest et al., 2005). It is possible
576
HCMV also utilizes these cellular cargo transporters to facilitate capsid
577
movement. Recently, it has been reported that mutation of HCMV UL84 leads to
578
a defect in virus replication associated with a defect in nuclear egress of capsids
579
and the clustering of capsids around nucleoli (Strang et al., 2012c). This would
580
suggest that UL84 is, directly or indirectly, involved in the efficient movement of
581
HCMV capsids within the nucleus. It has yet to be determined what relevance the
582
mislocalization of capsids to nucleoli has on movement of capsids in the nucleus.
583
At the periphery of the nucleus capsids must traverse the physical barrier
584
of the nuclear lamina and pass though the nuclear membrane. There have been
26
585
several recent advances in our understanding of these processes. HCMV
586
proteins mediate events at the nuclear lamina required for nuclear egress. The
587
viral nuclear egress complex (UL50 and UL53) recruits the viral kinase UL97 to
588
the nuclear lamina in HCMV infected cells (Sharma et al., 2014) (Table 1).
589
Phosphorylation of the lamina component lamin A/C by UL97 promotes lamina
590
disruption, akin to that observed during cell division, resulting in thinning and the
591
appearance of gaps in the lamina (Hamirally et al., 2009). This should facilitate
592
capsid movement through the lamina to the nuclear membrane. The requirement
593
for other viral (including c-ORF29 and UL45) and cellular (including emerin, p32
594
and protein kinase C) proteins implicated in nuclear egress in this model remains
595
unclear (Milbradt et al., 2014; Miller et al., 2010). It is unknown what mechanism
596
selects only DNA containing capsids for nuclear egress in HCMV infected cells.
597
In HSV infected cells this process is governed by the interaction of the nuclear
598
egress complex and capsid proteins (Yang & Baines, 2011). A similar
599
mechanism may operate in HCMV infected cells.
600
Once through the lamina, capsids must traverse the nuclear membrane in
601
a process now thought to be similar to the passage of large ribonucleoprotein
602
particles from the nucleus to the cytoplasm (Jokhi et al., 2013; Speese et al.,
603
2012). Briefly, capsids must bud through the inner leaflet of the nuclear
604
membrane into the intramembrane space. A fusion event with the outer leaflet of
605
the membrane grants capsids access the cytoplasm. Capsids then engage the
606
cytoplasmic membrane envelopment processes, via a cytoplasmic viral assembly
607
compartment (Alwine, 2012), which leads to virion exit from the cell. It is thought
27
608
that the development of the cytoplasmic assembly compartment next to the
609
nucleus deforms the nuclear membrane. This creates a concave surface on the
610
nucleus, giving it a “kidney bean” shape (Azzeh et al., 2006). To gain direct
611
access to the cytoplasmic assembly compartment capsids likely exit the nucleus
612
through the concave face of the nuclear membrane.
613
In summary, the process that facilitates the movement of DNA containing
614
capsids to the nuclear periphery is ill-defined, but likely involves cellular cargo
615
transporters. Once at the nuclear membrane a virally encoded nuclear egress
616
complex possibly selects DNA containing capsids for nuclear egress. This
617
nuclear egress complex then facilitates capsid movement through the nuclear
618
lamina by promoting modification of lamina components that results in thinning
619
and the appearance of gaps in the lamina. The capsid moves through the inner
620
and outer leaflets of the nuclear membrane via the intramenbrane space. Once in
621
the cytoplasm capsids participate in a complex membrane envelopment process
622
via a viral cytoplasmic assembly compartment that leads to virion exit from the
623
cell. There may be a relationship between the construction of the viral
624
cytoplasmic assembly compartment, deformation of the nucleus and exit of
625
capsids through the nuclear membrane proximal to assembly compartments.
626 627
Further questions and future perspectives. As discussed here there have
628
been notable advances in our understanding of HCMV replication and virus-host
629
interaction by examining sub-nuclear structures. However, a full examination of
630
the questions that remain is limited by several factors. These include our inability
28
631
to fully dissect viral gene expression or protein function and explore the
632
composition of viral and cellular sub-nuclear structures. To address these issues
633
technological development is required. Of particular importance to interrogating
634
the role of virally encoded factors will be the continued development of robust
635
systems to produce recombinant HCMV viruses, including viruses with lethal
636
mutations in their genomes. The study of viral genomes containing lethal
637
mutations can currently be achieved by studying cells transfected with bacmids
638
containing mutated HCMV genomes, for example Silva et al., (2010), although
639
this methodology has its limitations. Examination of viral cellular sub-nuclear
640
structure composition requires the development of well characterized antibodies
641
recognizing unmodified and post-translationally modified forms of viral and
642
cellular proteins. Recombinant viruses and antibodies can then be coupled with
643
increasingly sophisticated molecular and cellular techniques. These include live
644
cell imaging, super resolution microscopy, correlative light electron microscopic
645
analysis (Sharma et al., 2014) chemical biology techniques (for example “click
646
chemistry” (Strang et al., 2012b)) and proteomic analysis of sub-nuclear
647
structures (Andersen et al., 2002; Hiscox et al., 2010).
648
A number of observations discussed above rely on the study of
649
transfection experiments using epitope tagged versions of viral proteins in
650
uninfected cells (Kuny et al., 2010; Prichard et al., 2008; Salsman et al., 2008). It
651
is unclear what effect over expression during transfection or epitope tagging
652
might have on protein function. Thus, data based on transfection experiments
653
should be cautiously interpreted. Furthermore, certain aspects of this review rely
29
654
on the study of HSV. However, several features of HSV replication differ from
655
HCMV replication, likely due to evolutionary divergence between herpesviruses.
656
These include antagonism of the PML response (Everett et al., 2013), nucleolar
657
organization (Lymberopoulos & Pearson, 2007; Strang et al., 2012a), the
658
localization of nucleolin (Lymberopoulos & Pearson, 2007; Strang et al., 2012a),
659
and replication compartment organization (Liptak et al., 1996; Strang et al.,
660
2012b). Thus, there are areas where examination of HSV, or other herpes
661
viruses, may not benefit HCMV research. In sum, these points underscore the
662
need to generate reagents and methodologies to examine events that occur in
663
HCMV infected cells.
664
The use of laboratory viral strains, cell types studied and virus latency
665
must be considered in future experiments. Most of the data outlined in this review
666
is derived from the study of HCMV laboratory strains. The genomic complexity of
667
HCMV is still being addressed. Primary strains of HCMV contain several more
668
open reading frames than laboratory strains of HCMV (Dolan et al., 2004). Also,
669
recent high resolution transcriptome mapping (Gatherer et al., 2011) and
670
ribosome profiling (Stern-Ginossar et al., 2012) have revealed the presence of
671
many previously unrecognized viral transcripts and non-canonical open reading
672
frames. Further investigation is required to discover how recently identified
673
HCMV factors might influence viral or cellular sub-nuclear structures. These
674
factors include newly identified transcripts from the US33 and TRL9 loci
675
(Gatherer et al., 2011) which may localize to the nucleolus of HCMV infected
676
cells (Salsman et al., 2008) (Table 1). Also, the HCMV transcriptome contains a
30
677
number of long (Gatherer et al., 2011) and short (Grey et al., 2005) non-coding
678
RNAs. As yet, there is no data indicating HCMV non-coding RNAs are involved in
679
the function of viral or cellular sub-nuclear structures.
680
The HCMV transcriptome varies between cell types (Towler et al., 2012),
681
suggesting cell-type specific
requirements for
viral
factors.
Transcripts
682
differentially expressed between cell lines tested include those encoding UL44
683
(involved in viral replication compartment architecture), UL98 (suggested to be
684
involved in chromatin partitioning), TRL5 (thought to be localized to the
685
nucleolus), UL80 proteins (thought to be involved in capsid assembly within
686
replication compartments), plus UL97 and UL50 (involved in capsid egress
687
through the nuclear lamina) (Towler et al., 2012) (Table 1). Thus, the function of
688
viral and cellular sub-nuclear structures could vary between cell types.
689
Virus latency is a hallmark of herpesvirus infection. However, our
690
understanding the establishment, maintenance and reactivation from latency of
691
HCMV is incomplete. Recent in vitro models of virus infection in hematopoietic
692
cells (for example, CD14+ and CD34+ cells) have illuminated important aspects
693
of HCMV latency. The loss of hDaxx in CD34+ cells is required for transcription
694
of IE viral genes upon infection by laboratory, but not primary, strains of HCMV
695
(Saffert et al., 2010). This would argue that PML-NBs repress HCMV
696
transcription from laboratory HCMV strains and primary HCMV strains posses as
697
yet unidentified functions that control viral transcription during latency. Co-
698
infection of CD34+ cells with both laboratory and primary strains of HCMV blocks
699
the ability of a histone deacetylases to activate IE gene transcription from the
31
700
laboratory strain of HCMV (Saffert et al., 2010). Thus, transcriptional repression
701
of primary HCMV strains may relate to chromatin modification by histone
702
deacetylases. Also, it is unclear if hDaxx has a major role in repression of HCMV
703
gene transcription during latency. In the embryonic carcinoma cell line NT2D1,
704
which can recapitulate the differentiation-dependent regulation of IE gene
705
expression observed during latency, repression of IE gene expression has been
706
reported in the presence (Saffert & Kalejta, 2007) and absence (Groves &
707
Sinclair, 2007) of hDaxx in undifferentiated NT2D1 cells. This may be due to
708
differences in virus strains used in each study. Further experiments are required
709
to ascertain the relationship between virus strain, cell type, cell differentiation and
710
gene expression during HCMV latency.
711
There is little other data regarding the involvement of other sub-nuclear
712
structures in HCMV latency. Furthermore, the latent HCMV transcriptome in
713
either CD14+ or CD34+ cells (Rossetto et al., 2013), does not produce high
714
levels of transcripts encoding HCMV proteins thought to be localize to the cellular
715
sub structures during productive HCMV replication (Table 1). Thus, if cellular
716
sub-nuclear structures are involved in HCMV latency, different repertoires of viral
717
factors interact with sub-nuclear structures during productive and latent HCMV
718
infections.
719
32
720
ACKNOWLEDGMENTS
721 722
Thanks to Andrew Davison, Wade Gibson, David Pasdeloup and Frazer Rixon
723
for insightful discussions, plus Chris Boutell and Mayuri Sharma for comments on
724
the manuscript. I would also like to thank the anonymous reviewers for their
725
constructive comments, which have greatly improved this manuscript. I sincerely
726
apologize to colleagues whose important work was not discussed or cited due to
727
space limitations. This work was supported by St George’s, University of London.
728 729
33
730
FIGURE LEGENDS
731 732
Figure 1 Entry of the HCMV genome through the nuclear pore complex and
733
association with viral and cellular factors at the periphery of the nucleus.
734
(A) The viral capsid (shown in yellow) docks at the nuclear pore complex. The
735
linear form of the viral DNA genome exits the capsid through the portal on a
736
unique vertex of the capsid (shown in gray). (B) The linear form of the genome
737
circularizes and associates with the viral transcriptional transactivator IE2 (shown
738
in pink), PML-NBs containing PML (shown in purple) and SC35 containing bodies
739
thought to be nuclear speckles (shown in yellow) or, potentially, pp150 containing
740
bodies (shown in orange). The cellular transcription factors TFIIB (shown in blue)
741
and TBP (shown in green) also associate with the viral genome.
742 743
Figure 2 Viral replication compartments, nuclear speckles, nucleoli and
744
nucleoli associated sub-nuclear structures. (A) Viral replication compartments
745
form and coalesce to form a single viral replication compartment that dominates
746
nearly the entire nuclear space. Throughout these processes the viral protein
747
UL44 (shown in red) accumulates at the periphery of replication compartments.
748
(B) Viral DNA replication occurs at foci (shown in yellow) distributed throughout
749
the layer of UL44 at the periphery of replication compartments. UPS
750
compartments (shown in blue) are found within replication compartments. SC35
751
containing structures thought to be nuclear speckles (shown in yellow) are found
752
on the exterior of replication compartments. (C) Viral replication compartments
34
753
do not encroach into nucleoli. In HCMV infected cells nucleolin (shown in green)
754
accumulates at the periphery of nucleoli. Cajal bodies (shown in pink) and
755
perinucleolar compartments (shown in orange) may be involved in HCMV
756
replication and virus-host interaction.
757 758
35
759
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Strang, B. L. & Coen, D. M. (2010). Interaction of the human cytomegalovirus uracil DNA glycosylase UL114 with the viral DNA polymerase catalytic subunit UL54. J Gen Virol 91, 2029-2033. Strang, B. L., Boulant, S. & Coen, D. M. (2010a). Nucleolin associates with the human cytomegalovirus DNA polymerase accessory subunit UL44 and is necessary for efficient viral replication. J Virol 84, 1771-1784. Strang, B. L., Geballe, A. P. & Coen, D. M. (2010b). Association of human cytomegalovirus proteins IRS1 and TRS1 with the viral DNA polymerase accessory subunit UL44. J Gen Virol 91, 2167-2175. Strang, B. L., Boulant, S., Kirchhausen, T. & Coen, D. M. (2012a). Host cell nucleolin is required to maintain the architecture of human cytomegalovirus replication compartments. mBio 3, 301-311. Strang, B. L., Sinigalia, E., Silva, L. A., Coen, D. M. & Loregian, A. (2009). Analysis of the association of the human cytomegalovirus DNA polymerase subunit UL44 with the viral DNA replication factor UL84. J Virol 83, 7581-7589. Strang, B. L., Boulant, S., Chang, L., Knipe, D. M., Kirchhausen, T. & Coen, D. M. (2012b). Human cytomegalovirus UL44 concentrates at the periphery of replication compartments, the site of viral DNA synthesis. J Virol 86, 2098-2095. Strang, B. L., Bender, B. J., Sharma, M., Pesola, J. M., Sanders, R. L., Spector, D. H. & Coen, D. M. (2012c). A mutation deleting sequences encoding the amino terminus of human cytomegalovirus UL84 impairs interaction with UL44 and capsid localization. J Virol 86, 11066-11077. Tanaka, S., Furukawa, T. & Plotkin, S. A. (1975). Human cytomegalovirus stimulates host cell RNA synthesis. J Virol 15, 297-304. Tavalai, N. & Stamminger, T. (2009). Interplay between Herpesvirus Infection and Host Defense by PML Nuclear Bodies. Viruses 1, 1240-1264. Tavalai, N. & Stamminger, T. (2011). Intrinsic cellular defense mechanisms targeting human cytomegalovirus. Virus research 157, 128-133. Tavalai, N., Adler, M., Scherer, M., Riedl, Y. & Stamminger, T. (2011). Evidence for a dual antiviral role of the major nuclear domain 10 component Sp100 during the immediate-early and late phases of the human cytomegalovirus replication cycle. J Virol 85, 9447-9458. Towler, J. C., Ebrahimi, B., Lane, B., Davison, A. J. & Dargan, D. J. (2012). Human cytomegalovirus transcriptome activity differs during replication in human fibroblast, epithelial and astrocyte cell lines. J Gen Virol 93, 1046-1058. Tran, K., Mahr, J. A. & Spector, D. H. (2010). Proteasome subunits relocalize during human cytomegalovirus infection, and proteasome activity is necessary for efficient viral gene transcription. J Virol 84, 3079-3093. Ullman, A. J. & Hearing, P. (2008). Cellular proteins PML and Daxx mediate an innate antiviral defense antagonized by the adenovirus E4 ORF3 protein. J Virol 82, 7325-7335. Wang, J. B., Zhu, Y., McVoy, M. A. & Parris, D. S. (2012). Changes in subcellular localization reveal interactions between human cytomegalovirus terminase subunits. Virol J 9, 315.
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1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142
Wilkinson, G. W., Kelly, C., Sinclair, J. H. & Rickards, C. (1998). Disruption of PML-associated nuclear bodies mediated by the human cytomegalovirus major immediate early gene product. J Gen Virol 79 ( Pt 5), 1233-1245. Wood, L. J., Baxter, M. K., Plafker, S. M. & Gibson, W. (1997). Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains. J Virol 71, 179-190. Xu, Y., Ahn, J. H., Cheng, M., apRhys, C. M., Chiou, C. J., Zong, J., Matunis, M. J. & Hayward, G. S. (2001). Proteasome-independent disruption of PML oncogenic domains (PODs), but not covalent modification by SUMO-1, is required for human cytomegalovirus immediate-early protein IE1 to inhibit PMLmediated transcriptional repression. J Virol 75, 10683-10695. Xue, Y., Gibbons, R., Yan, Z., Yang, D., McDowell, T. L., Sechi, S., Qin, J., Zhou, S., Higgs, D. & other authors (2003). The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies. Proc Natl Acad Sci U S A 100, 10635-10640. Yang, K. & Baines, J. D. (2011). Selection of HSV capsids for envelopment involves interaction between capsid surface components pUL31, pUL17, and pUL25. Proc Natl Acad Sci U S A 108, 14276-14281. Yu, D., Silva, M. C. & Shenk, T. (2003). Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proc Natl Acad Sci U S A 100, 12396-12401.
1143 1144
44
1145 1146 1147
Table 1 HCMV proteins associated with sub-nuclear structures.
HCMV associated proteins UL48
Kinetic Class of HCMV protein a transcript L
HCMV protein function Capsid transport
Requirement for HCMV c replication E
UL77
E
Portal capping protein
E
TLR9 UL29 UL30 UL35 UL69 UL82 (pp71)
L L Unknown E E-L L
Unknown A A NE A A
UL76 UL80a UL97 UL98 UL122-123 (IE1)
Unknown L L E-L IE, L
UL122-123 (IE2)
IE, L
Unknown Temperance factor Unknown Unknown Multiple regulatory functions Transcriptional transactivator Unknown Capsid protein Viral kinase Alkaline nuclease Transcriptional transactivator Transcriptional transactivator
US25 US32
E-L L
Unknown Unknown
Nuclear Speckles
UL122-123 (IE2)
IE, L
pp150 bodies
UL32 (pp150)
Viral Replication Compartments
Nuclear Structure Nuclear pore
PML-NB
b
Known or proposed cellular partner(s) Nup214, Nup358 hCG1 Nup214, Nup358 hCG1
References Kobiler et al. (2012)
Kobiler et al. (2012)
NE NE
Unknown Unknown Unknown Unknown SUMO hDaxx, ATRX Unknown Unknown Rb Unknown PML Sp100 Sp100 SC35 PML SUMO Unknown
Salsman et al. (2008) Salsman et al. (2008) Salsman et al. (2008) Salsman et al. (2008) Salsman et al. (2008) Kaletja & Shenk (2003) Lukashchuk et al. (2008) Salsman et al. (2008) Salsman et al. (2008) Prichard et al. (2008) Salsman et al. (2008) Tavalai et al. (2011) Tavalai et al. (2011) Tavalai et al. (2011) Ishov et al. (1997) Ishov et al. (1997) Salsman et al. (2008) Salsman et al. (2008)
Transcriptional transactivator
E
SC35
Ishov et al. (1997)
L
Cell cycle regulator
E
A2-CDK
Bogdanow et al. (2013)
UL122-123 (IE2)
IE, L
Transcriptional transactivator
E
Unknown
UL112-113
E, E-L
A
Unknown
UL44
E-L
Transcriptional transactivator DNA polymerase subunit
Ahn et al. (1999) Penfold & Mocarski (1997) Ahn et al. (1999)
E
Nucleolin
UL57
E
ssDNA binding protein
E
Unknown
UL84
E-L
DNA replication factor
E
hnRNP K
Nucleoli
TLR5 TRL9 UL29 UL31 UL76 UL83 (pp65) UL84
E L L L Unknown L E
Unknown Unknown Temperance factor Unknown Unknown Interferon antagonist Replication factor
Unknown Unknown A NE A NE E
Unknown Unknown Unknown Unknown Unknown Unknown Unknown
Salsman et al. (2008) Salsman et al. (2008) Salsman et al. (2008) Salsman et al. (2008) Salsman et al. (2008) Arcangeletti et al. (2009) Bender et al. (2014)
Cajal Bodies
UL30
Unknown
Unknown
A
Unknown
Salsman et al. (2008)
Nuclear
UL50
Unknown
Nuclear Egress Complex
E
Unknown
Sharma et al. (2014)
45
A E A E E E
Ahn et al. (1999) Penfold & Mocarski (1997) Strang et al. (2012)b Penfold & Mocarski (1997) Strang et al. (2012)b Kagele et al. (2012) Gao et al. (2008)
Lamina
UL53 UL97
L E-L
Nuclear Egress Complex Viral kinase
E A
Unknown Lamin A/C
Sharma et al. (2014) Sharma et al. (2014)
1148 1149
a
1150
Early (E), Late (L), Early to Late (E-L).
1151
b
1152
Virology (Mocarski et al., 2007).
1153
c
1154
augments replication (A).
From Chambers and co-workers (Chambers et al., 1999); Immediate Early (IE),
Protein functions described in references indicated in the Table and/or Fields
From Yu and co-workers (Yu et al., 2003); essential (E), non-essential (NE),
1155
46