JGV Papers in Press. Published October 30, 2014 as doi:10.1099/vir.0.071084-0

REVIEW

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Viral and Cellular Sub-Nuclear Structures in

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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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SUMMARY

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In human cytomegalovirus (HCMV) infected cells a dramatic remodeling of the

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nuclear architecture is linked to the creation, utilization and manipulation of sub-

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nuclear structures. This review outlines the involvement of several viral and

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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

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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

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here highlights advances in our understanding of HCMV biology and emphasizes

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the complexity of HCMV replication and virus-host interaction in the nucleus.

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2

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TEXT

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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

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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

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alphaherpesvirus herpes simplex virus (HSV), certain features of productive

49

HCMV infection are not well characterized. However, it is clear that several sub-

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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

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HCMV infection. The participation of each structure in HCMV replication and

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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

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function, introduces viral and cellular factors associated with sub-nuclear

59

structures and indicates what questions have yet to be answered.

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Entry of the HCMV genome into the nucleus: the nuclear pore complex,

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nuclear speckles, PML-NBs and pp150 bodies. HCMV genome entry into the

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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

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vertex of the capsid comprised of UL104 (HSV UL6 homologue) (Kobiler et al.,

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2012). The HCMV genome moves through the central channel of the NPC into

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the nucleus (Kobiler et al., 2012).

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The linear HCMV DNA genome circularizes upon entry into in the nucleus

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via fusion of the genomic termini (McVoy & Adler, 1994) (Figure 1B). This could

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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

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genome takes place (Ishov et al., 1997). At the periphery of the nucleus the

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major viral immediate early transcriptional transactivator IE2 co-localizes with

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cellular RNA polymerase II transcription factors TATA-binding protein (TBP) and

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transcription factor IIB (TFIIB)(Ishov et al., 1997) (Figure 1B). It is possible, like

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HSV RNA transcription, HCMV RNA transcription involves the nuclear lamina.

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The nuclear lamina is a dense fibrillar protein network at the nuclear membrane

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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).

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The majority of HCMV genomes entering the nucleus localize with

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promyelocytic leukemia protein nuclear bodies (PML-NBs) (also referred to as

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ND10 bodies) and SC35 containing structures within intrachromosomal space.

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IE2 bridges PML-NBs and SC35 containing structures (Figure 1B). As

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transcription progresses viral IE RNA transcripts co-localize with SC35-

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contianing bodies, but not PML-NBs (Ishov et al., 1997). SC35 is a major protein

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component of nuclear speckles which are dynamic nuclear structures composed

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of RNA and protein and enriched in pre-mRNA splicing factors (Lamond &

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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

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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

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mRNA transcripts encoding the IE proteins in IE1 and IE2. The role of PML-NBs

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in HCMV replication is one of the most studied and debated facets of HCMV

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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;

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Tavalai & Stamminger, 2009, 2011). PML-NBs are dynamic structures that

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comprise at least 70 constitutively or transiently present proteins, including PML

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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).

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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

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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

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proteins involved in HCMV transcriptional repression and acts to facilitate

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assembly of PML-NBs containing transcriptionally repressive proteins at HCMV

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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

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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

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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

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(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

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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

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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

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proteasome independent loss of SUMOylated PML and Sp100 proteins (Ahn &

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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-

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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

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UL30, UL69, UL76, UL98 or TLR9 is sufficient to modestly reduce the number of

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PML-NBs in uninfected cells (Salsman et al., 2008). These proteins are

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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

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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

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reminiscent of PML cages trapping alphaherpesvirus varicella-zoster virus (VZV)

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capsids in infected cells via physical interaction between PML-NB proteins and

8

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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.

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Future consideration of PML-NB function in HCMV infected cells will

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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

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infection and analysis of PML-NB antagonism by the HSV protein ICP0 (Boutell &

190

Everett,

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papillomaviruses has been reported, but is less well understood. Prominent

192

findings include the ability of the adenovirus proteins E1B and E4orf3 to

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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

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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

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that pp150 nuclear bodies are sites of pp150-mediated transcriptional repression

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and are the destination of HCMV genomes not associated with PML-NBs

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(Bogdanow et al., 2013; Ishov et al., 1997) (Figure 1B).

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Therefore, upon engagement of the viral capsid with proteins on the

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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

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the viral genome and is antagonized by virally encoded proteins. Restriction of

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viral transcription by pp150 bodies is less well defined and is associated with

217

progression of the infected cell through the cell cycle.

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Replication of the viral genome: HCMV replication compartments,

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chromatin partitioning, protein quality control compartments and nuclear

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speckles. Once transcription of immediate early and early transcripts is

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underway viral proteins that replicate HCMV genomic DNA are produced. Viral

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DNA synthesis and several early and late transcriptional events occur within non-

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membrane bound compartments created by the virus composed of protein and

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nucleic acids. The formation of these “viral replication compartments” is poorly

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understood, but allows the recruitment and concentration of factors required for

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viral replication to specific areas of the infected cell. Analysis of alphaherpesvirus

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infection implies that a herpesvirus replication compartment can develop from a

229

single incoming viral genome (Kobiler et al., 2011). In HCMV infected cells

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replication compartments form in the vicinity of PML-NBs (Ahn et al., 1999). As

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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

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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.

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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

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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

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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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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

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells.

In human cytomegalovirus (HCMV)-infected cells, a dramatic remodelling of the nuclear architecture is linked to the creation, utilization and manipula...
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