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Journal of General Virology (2015), 96, 239–253 Review DOI 10.1099/vir.0.071084-0 Viral and cellular subnuclear structures in human cytomegalovirus-infected cells Blair L. Strang Correspondence Institute for Infection & Immunity, St George’s, University of London, London, UK Blair L. Strang [email protected] Received 15 August 2014 Accepted 28 October 2014 In human cytomegalovirus (HCMV)-infected cells, a dramatic remodelling of the nuclear architecture is linked to the creation, utilization and manipulation of subnuclear structures. This review outlines the involvement of several viral and cellular subnuclear structures in areas of HCMV replication and virus–host interaction that include viral transcription, viral DNA synthesis and the production of DNA-filled viral capsids. The structures discussed include those that promote or impede HCMV replication (such as viral replication compartments and promyelocytic leukaemia nuclear bodies, respectively) and those whose role in the infected cell is unclear (for example, nucleoli and nuclear speckles). Viral and cellular proteins associated with subnuclear structures are also discussed. The data reviewed here highlight advances in our understanding of HCMV biology and emphasize the complexity of HCMV replication and virus–host interactions in the nucleus. Introduction The betaherpesvirus human cytomegalovirus (HCMV) is a widespread opportunistic pathogen that severely affects immunocompromised and immunodeficient populations (Mocarski et al., 2007). Like all herpesviruses, HCMV undergoes either productive or latent infection. During productive infection, infectious virus is produced, while in latent infection the virus becomes largely transcriptionally quiescent (Mocarski et al., 2007). Like other herpesviruses, many events that are critical for productive HCMV replication take place within the nucleus. These include essential steps in viral replication, such as: the transcriptional cascade of immediate-early (IE), early and late viral RNA transcripts, synthesis of viral DNA and the production of DNA-containing capsids (Mocarski et al., 2007). Compared with the prototype human herpesvirus, the alphaherpesvirus herpes simplex virus (HSV), certain features of productive HCMV infection are not well characterized. However, it is clear that several subnuclear structures are involved in HCMV genome replication and virus–host interactions. Uncovering the roles of these structures has led to significant advances in our understanding of HCMV biology. This review summarizes the involvement of several viral and cellular subnuclear structures in productive HCMV infection. The participation of each structure in HCMV replication and virus–host interactions is described from entry of the viral genome into the nucleus to the egress of viral capsids through the nuclear membrane. The data discussed highlight recent advances in our understanding of subnuclear structure function, introduce viral and cellular factors associated with subnuclear structures and indicate what questions have yet to be answered. 071084 G 2015 The Author Entry of the HCMV genome into the nucleus: the nuclear pore complex, nuclear speckles, promyelocytic leukaemia protein nuclear bodies and pp150 bodies HCMV genome entry into the nucleus is poorly defined but may be similar to movement of the HSV DNA genome through the nuclear pore complex (NPC) (Kobiler et al., 2012) (Fig. 1a). HCMV capsid and tegument proteins (potentially HCMV UL48 and UL77, homologues of HSV UL36 and UL25, respectively) probably interact with proteins on the cytoplasmic face of the NPC (potentially including Nup214, Nup358 and hCG1) (Table 1). This leads to opening of the capsid. The pressure of viral DNA on the capsid wall leads to release of a linear DNA form of the HCMV genome. Viral DNA travels out of the capsid via a portal on a unique vertex of the capsid comprised of UL104 (HSV UL6 homologue) (Kobiler et al., 2012). The HCMV genome moves through the central channel of the NPC into the nucleus (Kobiler et al., 2012) (Fig. 1a). The linear HCMV DNA genome circularizes upon entry into the nucleus via fusion of the genomic termini (McVoy & Adler, 1994) (Fig. 1b). This could be mediated by either homologous or non-homologous end-joining DNA repair mechanisms. The circular form of the genome is utilized as a template for viral RNA transcription and DNA synthesis. Very early in infection, viral genomes can be found at the periphery of the nucleus where transcription from the viral genome takes place (Ishov et al., 1997). At the periphery of the nucleus, the major viral IE transcriptional transactivator IE2 co-localizes with cellular RNA polymerase II transcription factors TATA-binding protein (TBP) and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 Printed in Great Britain 239 B. L. Strang Capsid (a) Portal Nuclear membrane Nuclear pore complex Nuclear lamina (b) PML IE2 SC35 ? TFIIB pp150 TBP Fig. 1. Entry of the HCMV genome through the nuclear pore complex and association with viral and cellular factors at the periphery of the nucleus. (a) The viral capsid (shown in yellow) docks at the nuclear pore complex. The linear form of the viral DNA genome exits the capsid through the portal on a unique vertex of the capsid (shown in grey). (b) The linear form of the genome circularizes and associates with the viral transcriptional transactivator IE2 (shown in pink), promyelocytic leukaemia protein (PML)containing nuclear bodies (shown in purple) and SC35-containing bodies thought to be nuclear speckles (shown in yellow) or, potentially, pp150-containing bodies (shown in orange). The cellular transcription factors transcription factor IIB (TFIIB) (shown in blue) and TATA-binding protein (TBP) (shown in green) also associate with the viral genome. transcription factor IIB (TFIIB) (Ishov et al., 1997) (Fig. 1b). It is possible that, like HSV RNA transcription, HCMV RNA transcription involves the nuclear lamina. The nuclear lamina is a dense fibrillar protein network at the nuclear membrane composed of lamin proteins and lamin-associated proteins (Fig. 1a). In HSV-infected cells, the lamina component lamin A/C is important for genome targeting to the lamina, and at the lamina hetero- or euchromatin on viral promoters modulates HSV RNA transcription (Silva et al., 2008). The majority of HCMV genomes entering the nucleus localize with promyelocytic leukaemia protein nuclear bodies (PML-NBs, also referred to as ND10 bodies) and SC35-containing structures within intrachromosomal space. IE2 bridges PML-NBs and SC35-containing structures (Ishov et al., 1997) (Fig. 1b). As transcription progresses, viral IE RNA transcripts co-localize with SC35containing bodies but not PML-NBs (Ishov et al., 1997). SC35 is a major protein component of nuclear speckles, which are dynamic nuclear structures composed of RNA and protein and enriched in pre-mRNA splicing factors (Lamond & Spector, 2003). A role for nuclear speckles in 240 HCMV replication, or that of any other human virus, is not often commented on. It is likely that nuclear speckles are involved in HCMV IE gene transcription, but it is unknown what transcriptional events they might facilitate. In HSV-infected cells, it has been reported that nuclear speckles are involved in export of viral RNA transcripts (Chang et al., 2011). Thus, nuclear speckles may serve in the processing and export of HCMV RNA transcripts early in infection. These RNAs probably include the viral RNAs transcribed from the UL122–123 locus of the HCMV genome that are multiply spliced to produce mRNA transcripts encoding the IE proteins IE1 and IE2. The role of PML-NBs in HCMV replication is one of the most studied and debated facets of HCMV biology. The complex relationship between PML-NBs and HCMV cannot be fully summarized here but is discussed in detail elsewhere (Everett et al., 2013; Tavalai & Stamminger, 2009, 2011). PML-NBs are dynamic structures that comprise at least 70 constitutively or transiently present proteins, including PML protein (which acts a scaffold for PML-NB formation) (Ishov et al., 1999). The majority of, if not all, PML-NB proteins are post-translationally modified with the addition of small ubiquitin-related modifier (SUMO) moieties. PML-NBs contain several proteins required for protein SUMOylation and the relationship between protein function and post-translational modification by SUMO is a rapidly developing topic in HCMV biology and elsewhere (Everett et al., 2013). PML-NBs are found throughout the nucleus. Upon entry of the viral genome into the nucleus, PML-NBs form on SUMO-modified PML proteins juxtaposed to incoming viral genomes. PML-NBs act to repress viral transcription via the co-operative function of the PML-NB proteins PML, Sp100 and hDaxx (Glass & Everett, 2013). PML protein is one of the best characterized PML-NB proteins involved in HCMV transcriptional repression and acts to facilitate assembly of PML-NBs containing transcriptionally repressive proteins at HCMV genomes (Tavalai & Stamminger, 2009, 2011). Sp100 promotes transcriptional repression through its interaction with the transcriptional repressor heterochromatin protein (HP1) (Seeler et al., 1998). SUMO modification of Sp100 promotes interaction with HP1 (Seeler et al., 2001) but is not required for localization to PML-NBs (Sternsdorf et al., 1999). hDaxx is recruited to PML-NBs via an interaction with SUMOylated PML (Everett et al., 2013) and acts as a transcriptional co-repressor via its interaction with the chromatin remodeller ATRX (Ishov et al., 2004; Xue et al., 2003). PML-NBs contain other transcriptionally repressive cellular proteins (Tavalai & Stamminger, 2011), and it is possible other PML-NB proteins may antagonize HCMV replication. Crucially, HCMV encodes antagonists of PML-NB function (Table 1) whose actions lead to removal of proteins from PML-NBs and the dispersal of PML-NBs (Everett et al., 2013; Tavalai & Stamminger, 2009, 2011). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 Journal of General Virology 96 http://vir.sgmjournals.org Table 1. HCMV proteins associated with subnuclear structures Nuclear structure HCMV associated proteins Kinetic class of HCMV protein* HCMV protein functionD Requirement for Known or proposed HCMV replicationd cellular partner(s) References Nuclear pore UL48 UL77 L E Capsid transport Portal capping protein E E Nup214, Nup358 hCG1 Kobiler et al. (2012) Nup214, Nup358 hCG1 Kobiler et al. (2012) PML-NB TLR9 UL29 UL30 UL35 UL69 L L Unknown E E–L Unknown A A NE A Unknown Unknown Unknown Unknown SUMO Salsman Salsman Salsman Salsman Salsman UL82 (pp71) L A hDaxx, ATRX Kalejta & Shenk (2003), Lukashchuk et al. (2008) UL76 UL80a UL97 UL98 UL122–123 (IE1) Unknown L L E–L IE, L A E A E E Unknown Unknown Rb Unknown PML Sp100 Salsman et al. (2008) Salsman et al. (2008) Prichard et al. (2008) Salsman et al. (2008) Tavalai et al. (2011) 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 E US25 US32 E–L L Unknown Unknown NE NE Sp100 SC35 PML SUMO Unknown Tavalai et al. (2011) Ishov et al. (1997) Ishov et al. (1997) Salsman et al. (2008) Salsman et al. (2008) Nuclear speckles UL122–123 (IE2) IE, L Transcriptional transactivator E SC35 Ishov et al. (1997) pp150 bodies L Cell-cycle regulator E A2-CDK Bogdanow et al. (2013) IE, L Transcriptional transactivator Transcriptional transactivator DNA polymerase subunit ssDNA-binding protein E Unknown Ahn et al. (1999), Penfold & Mocarski (1997) A Unknown Ahn et al. (1999) E Nucleolin Ahn et al. (1999), Penfold & Mocarski (1997), Strang et al. (2012b) E Unknown UL32 (pp150) Viral replication UL122–123 (IE2) compartments UL112– UL44 E–L UL57 E al. al. al. al. al. (2008) (2008) (2008) (2008) (2008) UL84 E–L DNA replication factor E hnRNP K Penfold & Mocarski (1997) Strang et al. (2012b) Kagele et al. (2012), Gao et al. (2008) TLR5 TRL9 UL29 E L L Unknown Unknown Temperance factor Unknown Unknown A Unknown Unknown Unknown Salsman et al. (2008) Salsman et al. (2008) Salsman et al. (2008) 241 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 HCMV and subnuclear structures Nucleoli E, E–L et et et et et 242 Sharma et al. (2014) Sharma et al. (2014) Unknown Lamin A/C *From Chambers et al. (1999). E, Early; IE, immediate early; L, late; E–L, early to late. DProtein functions described in references indicated in the table and/or Fields Virology (Mocarski et al., 2007). dFrom Yu et al. (2003). E, Essential; NE, non-essential; A, augments replication. Sharma et al. (2014) Unknown E A Nuclear egress complex Viral kinase Nuclear egress complex UL53 UL97 Lamina Unknown UL50 Nuclear L E–L E Salsman et al. (2008) Unknown UL30 Unknown Unknown A Salsman et al. (2008) Salsman et al. (2008) Arcangeletti et al. (2009) Bender et al. (2014) Unknown Unknown Unknown Unknown NE A NE E Unknown Unknown Interferon antagonist Replication factor L Unknown L E UL31 UL76 UL83 (pp65) UL84 Cajal bodies Nuclear structure Table 1. cont. HCMV associated proteins Kinetic class of HCMV protein* HCMV protein functionD Requirement for Known or proposed HCMV replicationd cellular partner(s) References B. L. Strang In HCMV-infected cells, two major antagonists of PMLNB function are pp71 and IE1. pp71 degrades hDaxx by a proteasome-dependent, ubiquitin-independent mechanism (Kalejta & Shenk, 2003) and displaces ATRX from PML-NBs (Lukashchuk et al., 2008). The presence of IE1 promotes the disruption of PML-NBs, induces the proteasome-independent loss of SUMOylated PML and Sp100 proteins (Ahn & Hayward, 1997; Kang et al., 2006; Kim et al., 2011; Korioth et al., 1996; Lee et al., 2004; Tavalai et al., 2011; Wilkinson et al., 1998; Xu et al., 2001) and potentially induces the proteasome-dependent degradation of unSUMOylated Sp100 late in infection (Tavalai et al., 2011). Importantly, IE1 does not act like its well-studied counterpart expressed by HSV, ICP0. ICP0 possesses an E3 ubiquitin ligase activity directly involved in the proteasome-dependent degradation of PML protein (Boutell & Everett, 2013). IE1 has no known ubiquitin ligase or SUMOylase activity (Kang et al., 2006), but is able to exert an effect that leads to PML-NB disruption, loss of SUMOylated PML and an increase in the abundance of unmodified PML. This would suggest that IE1 can recruit a cellular protein to deSUMOylate PML within PML-NBs or SUMO is removed from PML by a cellular deSUMOylase after PML-NBs have been dispersed in the presence of IE1. The role of IE1 in Sp100 degradation is unclear. IE1 binding to Sp100 (Kim et al., 2011) may result in Sp100 degradation by an as-yetunknown mechanism. Alternatively, Sp100 degradation may be an indirect result of PML-NB modification by IE1. Other HCMV proteins may be involved in PML-NB destruction or modification (Table 1). Expression of epitope-tagged versions of US25, UL29 UL30, UL69, UL76, UL98 or TLR9 is sufficient to modestly reduce the number of PML-NBs in uninfected cells (Salsman et al., 2008). These proteins are expressed in different kinetic classes of viral transcription and have different effects on HCMV replication, plus the function of several of these proteins is unknown (Table 1). In transfection experiments epitope-tagged HCMV proteins US32, UL80a and UL35 co-localize with, but do not obviously degrade, PML-NBs in uninfected cells (Salsman et al., 2008). Epitope-tagged US32 and UL35 alter the shape and size of PML-NBs in uninfected cells. The localization and function of these proteins in relation to PML-NBs in HCMV-infected cells has yet to be elucidated. UL80a is a capsid protein. UL80a association with PML-NBs is reminiscent of PML cages trapping alphaherpesvirus varicella-zoster virus (VZV) capsids in infected cells via physical interaction between PML-NB proteins and VZV capsids (Reichelt et al., 2011). This mechanism of cellular defence to infection has not yet been observed in HCMVinfected cells. An epitope-tagged version of the putative HCMV protein UL3 has been reported to alter PML-NBs in uninfected cells (Salsman et al., 2008). However, it is now thought that the HCMV genome does not contain an ORF encoding UL3 (Gatherer et al., 2011). A final protein Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 Journal of General Virology 96 HCMV and subnuclear structures to consider in the alteration of PML-NBs in HCMVinfected cells is the HCMV viral kinase UL97. UL97 phosphorylates and inactivates the PML-NB protein retinoblastoma (Rb) (Hume et al., 2008) and expression of epitope-tagged UL97 in uninfected simian cells (Prichard et al., 2008), but not uninfected human cells (Kuny et al., 2010), notably reduces the number of PMLNBs in the cell. Clarification of the roles, if any, of the UL97 and Rb proteins in PML-NB function in HCMVinfected cells is required. Future consideration of PML-NB function in HCMVinfected cells will benefit from further exploration of the interaction of PML-NBs with other viruses, including other herpesviruses. Indeed, most detailed information concerning the interaction of PML-NBs with viral DNA genomes comes from the study of HSV infection and analysis of PML-NB antagonism by the HSV protein ICP0 (Boutell & Everett, 2013). Interaction of PML-NBs with adenoand papillomaviruses has been reported but is less well understood. Prominent findings include the ability of the adenovirus proteins E1B and E4orf3 to antagonize PML and hDaxx functions (Schreiner et al., 2010; Ullman & Hearing, 2008) and the repression of papillomavirus RNA transcription by Sp100 (Stepp et al., 2013). Future studies of adeno- and papillomavirus replication may illuminate important features of herpesvirus interactions with PML-NBs. Subnuclear structures other than PML-NBs could also be involved in repression of viral transcription. These include bodies such as the HCMV tegument protein pp150 (UL32). pp150 is a substrate of cyclin A2-CDK (Table 1) and is a sensor for cell-cycle progression during HCMV infection. By an unknown mechanism, pp150 is involved in blocking viral transcription when levels of cyclin A2-CDK are high (Bogdanow et al., 2013). It has been suggested that pp150 nuclear bodies are sites of pp150-mediated transcriptional repression and are the destination of HCMV genomes not associated with PML-NBs (Bogdanow et al., 2013; Ishov et al., 1997) (Fig. 1b). Therefore, upon engagement of the viral capsid with proteins on the cytoplasmic face of the NPC, the HCMV genome is delivered into the nucleus through the nuclear pore. Once in the nucleus, transcription from the viral genome involves cellular transcription factors and may be facilitated by interaction of the genome with the nuclear lamina at the periphery of the nucleus. At the periphery of the nucleus, the genome encounters nuclear speckles, which probably facilitate the splicing and export to the cytoplasm of viral RNA transcripts, and PML-NBs or pp150 bodies that restrict viral transcription. Restriction of viral transcription by PML-NBs involves the recruitment of transcriptional repressors to the viral genome and is antagonized by virally encoded proteins. Restriction of viral transcription by pp150 bodies is less well defined and is associated with progression of the infected cell through the cell cycle. http://vir.sgmjournals.org Replication of the viral genome: HCMV replication compartments, chromatin partitioning, protein quality-control compartments and nuclear speckles Once transcription of IE and E RNA transcripts is under way, viral proteins that replicate HCMV genomic DNA are produced. Viral DNA synthesis and several early and late transcriptional events occur within non-membrane-bound compartments created by the virus composed of protein and nucleic acids. The formation of these ‘viral replication compartments’ is poorly understood but allows the recruitment and concentration of factors required for viral replication to specific areas of the infected cell nucleus. Analysis of alphaherpesvirus infection implies that a herpesvirus replication compartment can develop from a single incoming viral genome (Kobiler et al., 2011). In HCMV-infected cells, replication compartments form in the vicinity of PML-NBs (Ahn et al., 1999). As compartments develop, they contain viral transcriptional transactivators (including IE2 and UL112–113 proteins), viral proteins involved in viral DNA synthesis (including UL44 and UL57) and viral DNA (Ahn et al., 1999; Penfold & Mocarski, 1997; Strang et al., 2012b) (Table 1). Early in the formation of replication compartments, factors involved in the DNA damage response, including cH2AX and ATM, localize to replication compartments (E et al., 2011) and may recognize viral genomes within compartments. It is thought that viral transcriptional transactivators drive E2F1-mediated activation of ATM signalling, which activates DNA damage response factors such as cH2AX and p53 (E et al., 2011). Activation of this pathway may be required for modulation of cell-cycle-related functions that promote viral replication. ATM signalling promotes the development of replication compartments (E et al., 2011), but it is unclear if this is an effect that directly relates to replication compartment development or an indirect effect related to events that promote viral RNA transcription or viral DNA synthesis. During HCMV replication, several compartments can form and then coalesce to form a single compartment that occupies nearly all the nucleus but does not encroach into nucleoli (Penfold & Mocarski, 1997; Strang et al., 2012a, b) (Fig. 2a, b). The mechanisms driving coalescence of HCMV replication compartments have yet to be defined. Replication compartment coalescence in HSVinfected cells occurs via non-random movement and is dependent upon nuclear actin, myosin and viral transcription (Chang et al., 2011). To provide areas in the nucleus for replication compartments to develop, the compaction and movement of chromatin is observed (O’Dowd et al., 2012; Strang et al., 2012b). This process is termed ‘chromatin partitioning’. It is unknown what drives this process, but UL98 and UL76 may be involved (Table 1). UL98 is an alkaline nuclease capable of digesting DNA (Sheaffer et al., 1997). UL76 is member of the UL24 gene family, all of which contain a Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 243 B. L. Strang conserved putative PD-(D/E)XK endonuclease motif (Knizewski et al., 2006). Although endonuclease activity of UL76 has yet to be demonstrated, indirect evidence points to a role for UL76 in the chromosome breakage required for chromatin partitioning. For example, the presence of UL76 in uninfected cells is sufficient to stimulate chromosomal aberrations (Siew et al., 2009), and in HCMV-infected cells, UL76 is involved in stimulating a DNA damage response (Costa et al., 2013). Several facets of HCMV replication compartment organization and function late in infection have recently been described, each involving the localization of HCMV protein UL44. UL44 is the putative processivity subunit of the HCMV DNA polymerase (Ertl & Powell, 1992), and several different phosphorylated forms of UL44 are present in the infected cell (Silva et al., 2011). At least one form of UL44 accumulates at the periphery of replication compartments throughout HCMV replication, and UL44 is also found at considerably lower levels within certain domains inside replication compartments, plus in the nucleus outside replication compartments and also in the cytoplasm (Hamirally et al., 2009; Penfold & Mocarski, 1997; Strang et al., 2012b). Viral DNA synthesis occurs at foci within the layer of UL44 at the periphery of replication compartments (Fig. 2b), and newly synthesized viral DNA moves into the interior of replication compartments (Strang et al., 2012b). The viral ssDNA-binding protein UL57 co-localizes with UL44 at the periphery of replication compartments and is found throughout the interior of compartments (Penfold & Mocarski, 1997; Strang et al., 2012b). UL57 at the periphery of replication compartments is likely to be directly involved in viral DNA synthesis. UL57 protein in the interior of replication compartments is associated with viral DNA (Penfold & Mocarski, 1997) and is possibly involved in UL57-mediated DNA strand exchange events, including recombination and repair of newly synthesized HCMV DNA. As well as UL44 and UL57, other factors required for viral DNA synthesis have been reported. These include the viral DNA polymerase subunit UL54, replication factor UL84 and the helicase DNA complex (UL70, UL102 and UL105) (Pari & Anders, 1993; Pari et al., 1993). However, the full repertoire of viral and cellular proteins acting in concert during DNA synthesis has yet to be described. These proteins probably include those viral and cellular proteins that associate with UL44 and/or UL84 (Gao et al., 2008; Strang et al., 2009, 2010a, b), several proteins that have illdefined roles in viral DNA synthesis (for example, UL36, IRS1 and IE2; Pari & Anders, 1993; Pari et al., 1993; Sarisky & Hayward, 1996), plus viral and cellular proteins involved in DNA damage responses and viral DNA repair (E et al., 2011; O’Dowd et al., 2012; Strang & Coen, 2010). How these and other proteins are organized within HCMV replication compartments is unclear but may involve UL44. UL44, a homodimer, is a structural homologue of 244 (a) Coalescence DNA replication (c) Cajal body (b) Replication compartment Nucleoli Perinucleolar compartment UPS compartment SC35 Fig. 2. Viral replication compartments, nuclear speckles, nucleoli and nucleoli-associated subnuclear structures. (a) Viral replication compartments form and coalesce to form a single viral replication compartment that dominates nearly the entire nuclear space. Throughout these processes, the viral protein UL44 (shown in red) accumulates at the periphery of replication compartments. (b) Viral DNA replication occurs at foci (shown in yellow) distributed throughout the layer of UL44 at the periphery of replication compartments. Ubiquitin–proteasome system (UPS) compartments (shown in blue) are found within replication compartments. SC35-containing structures thought to be nuclear speckles (shown in yellow) are found on the exterior of replication compartments. (c) Viral replication compartments do not encroach into nucleoli. In HCMV-infected cells, nucleolin (shown in green) accumulates at the periphery of nucleoli. Cajal bodies (shown in pink) and perinucleolar compartments (shown in orange) may be involved in HCMV replication and virus–host interactions. the cellular proliferating cell nuclear antigen (PCNA), which exists as a trimer (Appleton et al., 2004, 2006; Loregian et al., 2004a, b). PCNA binds multiple proteins of multiple functions at the DNA replication fork (Moldovan et al., 2007). UL44 could play a similar role and bind multiple proteins in replication compartments. Proteomic studies have indicated that UL44 associates with viral and cellular proteins found in replication compartments, including UL84 (Strang et al., 2010a). The binding of proteins to UL44 is increasingly well characterized. Studies investigating the binding of UL54 to UL44 have revealed that, like PCNA, UL44 is able to bind proteins within ‘hydrophobic pockets’; crevices located in the N termini of UL44 containing several hydrophobic amino acids into Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 Journal of General Virology 96 HCMV and subnuclear structures which proteins binding UL44 insert their binding domains (Appleton et al., 2004, 2006; Loregian et al., 2004a, b). Unexpectedly, UL44 can also associates with proteins outside the hydrophobic pockets, as it has been demonstrated that the association of UL84 with UL44 is not dependent upon binding in the hydrophobic pocket (Strang et al., 2009). Rather, association of UL84 with UL44 takes place elsewhere in the N-terminal domain of UL44 and is not dependent upon homodimerization of UL44 (Strang et al., 2009). It is unclear what drives selection of proteins for binding to UL44, but there are likely determinants of protein binding in both UL44 and its interacting proteins. Indeed, it has been demonstrated that the HCMV proteins IRS1 and TRS1, which have identical N termini that are required for association with UL44, compete for UL44 binding (Strang et al., 2010b). UL44 is also indirectly involved in the recruitment of viral proteins to replication compartments. The viral uracil DNA glycosylase UL114 is required for efficient viral DNA synthesis and base excision repair (Prichard et al., 1996). Base excision repair maintains genome integrity by removing uracil from the HCMV genome, which is the result of either misincorporation of uracil during viral DNA synthesis or the spontaneous deamination of cytosine in the viral genome. Although UL114 localizes with UL44 within replication compartments (Prichard et al., 2005), it does not bind UL44, as first thought (Ranneberg-Nilsen et al., 2008). Rather, UL114 associates with the catalytic subunit of the viral DNA polymerase UL54, and UL114, UL54 and UL44 form a protein complex (Strang & Coen, 2010). Thus, UL44 indirectly recruits UL114 to replication compartments via the interaction of UL114 with UL54. There are few reports of cellular proteins localizing to HCMV replication compartments, although it is known that both p53 and replication protein A are recruited to the interior of replication compartments (Fortunato & Spector, 1998). Their roles within replication compartments are unknown but may involve modulation of cellular responses to infection and promoting efficient viral DNA synthesis. Several factors influence the architecture of HCMV replication compartments. While the N terminus of UL44 is required for binding of UL44-associated proteins, the C terminus of UL44 is required for the development of replication compartments and correct localization of UL44 in the nucleus (Silva et al., 2010). Although the C terminus of UL44 can be extensively phosphorylated by both the viral kinase UL97 and the cellular kinase cdk1, it is unlikely that these post-translational modifications affect replication compartment function, as mutational analysis of phosphorylated residues in the UL44 C terminus has little effect on viral DNA synthesis and viral replication (Silva et al., 2011). Efficient viral DNA synthesis is also required to maintain UL44 localization and compartment structure (E et al., 2011; Penfold & Mocarski, 1997; Strang et al., 2012a, b). Unexpectedly, the multifunctional cellular nucleolar protein nucleolin (Ginisty et al., 1999) is involved http://vir.sgmjournals.org in maintaining replication compartment architecture. UL44 and nucleolin associate via the N terminus of UL44 (Strang et al., 2010a) and in infected cells nucleolin colocalizes with UL44, but not viral DNA, on the exterior of replication compartments (Strang et al., 2010a, 2012a). The absence of nucleolin in the infected cell causes mislocalization of UL44 at the periphery of replication compartments and compromises efficient viral DNA synthesis (Strang et al., 2012a). Nucleolin is also required for UL84 localization with UL44 at the periphery of replication compartments, although in the absence of nucleolin, UL84 does not redistribute throughout the nucleus but relocates to the cytoplasm (Bender et al., 2014). Thus, nucleolin is required for both proper nuclear and subnuclear localization of HCMV replication compartment proteins. Furthermore, it is possible that, in infected cells, nucleolin, UL44, UL84 and either IRS1 or TRS1 interact within a large multiprotein complex (Strang et al., 2010a, b). Therefore, nucleolin may have an effect on the localization of both IRS1 and TRS1 in the infected cell. Although the organization of factors required for viral DNA synthesis in replication compartments is increasingly well understood, perhaps the most important unanswered questions involving the architecture of replication compartments involve the localization of factors required for RNA transcription from the viral genome. The location of transcriptionally active genomes within replication compartments is unknown. There is little information on the localization of viral and cellular proteins required for viral RNA transcription. In infected cells, both phosphorylated (transcriptionally active) and unphosphorylated (transcriptionally inactive) forms of RNA polymerase II concentrate within nuclear structures thought to be replication compartments (Feichtinger et al., 2011). Within these structures, unphosphorylated and phosphorylated forms of RNA polymerase II localize with the viral protein UL69 (Feichtinger et al., 2011), which is known to be required for viral RNA processing and export to the cytoplasm. The localization of RNA polymerase II in relation to UL44 has yet to be described; however, viral proteins involved in transcription and RNA metabolism, for example IE1, IE2 and UL84 (Bender et al., 2014; Silva et al., 2010), are found throughout the nucleus and co-localize with UL44 in viral replication compartments. Therefore, it is likely that viral transcription occurs throughout the interior of replication compartments and, unlike viral DNA synthesis, is not restricted to specific areas of replication compartments such as the layer of UL44 that accumulates at the periphery of replication compartments. The presence of subcompartments within HCMV replication compartments has been reported. Components of the ubiquitin–proteasome system (UPS) assemble into domains at the periphery of replication compartments (Fig. 2b), where they co-localize with UL57 (Tran et al., 2010). The accumulation of UPS proteins at the periphery of HCMV replication compartments is analogous to the formation of VICE (virus-induced chaperone enriched) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 245 B. L. Strang domains at the periphery of HSV replication compartments. VICE domains degrade misfolded proteins in a proteasome-dependent fashion to promote efficient virus replication (Livingston et al., 2009). Thus, compartments containing UPS components at the periphery of HCMV replication compartments may act as quality-control areas for HCMV proteins. It is unclear which HCMV proteins other than UL57 localize to UPS compartments. However, HCMV UL76 co-localizes with the UPS component Rpn10/ S5a in discrete areas of infected cell nuclei termed aggressomes (Lin et al., 2013). Rpn10/S5a is found in UPS compartments (Tran et al., 2010). Thus, some aggressomes may be UPS compartments associated with replication compartments that contain both UL76 and Rpn10/S5a. It is unknown what role UL76 might have in UPS compartments. Subcompartments within the interior of replication compartments that do not contain UL44 or viral DNA have been observed (Strang et al., 2012b). It is possible that these subcompartments are the aforementioned UPS compartments. The presence of SC35-containing structures on the exterior of HCMV replication compartments has been reported (Rechter et al., 2009) (Fig. 2b). It is likely that, as in HSVinfected cells (Chang et al., 2011), these are nuclear speckles that remain associated with HCMV replication compartments throughout virus replication. The directed movement of nuclear speckles with HSV replication compartments, or vice versa, is associated with efficient viral RNA transcription in HSV-infected cells, in particular promoting the export of late HSV RNA transcripts to the cytoplasm (Chang et al., 2011). The function of nuclear speckles associated with replication compartments in HCMV-infected cells has yet to be described, although it is likely that they are involved in viral RNA transcription. In summary, HCMV replication compartments are necessary for the concentration and organization of factors required for viral replication. The organization of factors required for viral DNA synthesis in replication compartments is increasingly well defined and involves UL44. Viral replication compartments are dynamic structures whose architecture is influenced by UL44 localization, viral DNA synthesis and the cellular nucleolar protein nucleolin. Nucleolin is not only required to maintain replication compartment architecture but is also involved in the subcellular localization of at least one viral protein found in replication compartments. The organization of factors required for RNA transcription from the HCMV genome in replication compartments is not well defined. However, it can be suggested that viral transcription takes place throughout the interior of replication compartments, in contrast to viral DNA synthesis, which takes place within a layer of UL44 at the periphery of replication compartments. Within replication compartments, there are subcompartments, some of which are associated with protein quality control. Nuclear speckles are associated with viral replication compartments where they probably enable viral transcription, possibly by facilitating the export of viral transcripts to the cytoplasm. 246 Structures associated with HCMV replication: nucleoli, Cajal bodies and perinucleolar compartments To date, little attention has been paid to the role of nucleoli and associated subnuclear structures such as Cajal bodies and perinucleolar compartments in HCMV-infected cells. It is increasing clear, however, that these subnuclear structures may have important roles in HCMV genome replication and virus–host interactions. Nucleoli are dynamic non-membrane-bound protein and RNA structures that form on rRNA genes. A nucleolus has a tripartite structure in which the fibrillar centre is surrounded by a dense fibrillar component (containing the majority of nucleolin found in nucleoli) that is surrounded by the granular component. In HCMVinfected cells, nucleoli remain intact (Strang et al., 2012c), and visualization of nucleolin in HCMV-infected cells suggests changes in the size of nucleoli upon infection (Strang et al., 2012a). Also, striking changes to nucleolar composition and architecture occur, as, following infection, nucleolin levels in nucleoli increase and nucleolin accumulates at the periphery of nucleoli (Strang et al., 2012a) (Fig. 2c). These changes in nucleolar composition and architecture may relate to the recruitment of nucleolin to replication compartments, a nucleolar stress response to infection or the production of ribosomes in the HCMVinfected cell. The recruitment of nucleolin to replication compartments may be related to the interaction of nucleolin with viral proteins found in replication compartments. It has been observed that the HCMV protein UL84 co-localizes with nucleolin in nucleoli and at the periphery of replication compartments (Bender et al., 2014). Therefore, it is possible that UL84 recruits nucleolin to replication compartments from nucleoli, which results in changes to nucleolar composition and architecture. Nucleoli can act as sensors of environmental, genotoxic or osmotic stress, and pathogen invasion (Boisvert et al., 2007; Boulon et al., 2010; Hiscox, 2002; Hiscox et al., 2010). These stresses result in a variety of outcomes including changes in nucleolar morphology and the nucleolar proteome, plus pleiotropic effects of p53 activation (Boulon et al., 2010). The changes in the nucleolar proteome and architecture observed in HCMV-infected cells could relate to a nucleolar stress response. The nucleolar localization of nucleolin in HCMV-infected cells is akin to that seen in coronavirus-infected cells. It has been suggested that localization of nucleolin in coronavirusinfected cells is related to ‘nucleolar capping’ (Dove et al., 2006). Capping is associated with a nucleolar stress response caused by factors such as transcriptional inhibition or DNA damage. These stresses cause condensation and segregation of the fibrillar centre and the granular component. This is accompanied by the formation of ‘caps’ of nucleoplasmic RNA-binding proteins on the body of nucleoli (Boulon et al., 2010; Shav-Tal et al., 2005). It has been reported that nucleolar capping is observed in HCMV-infected cells (Gaddy et al., 2010). However, it is Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 Journal of General Virology 96 HCMV and subnuclear structures unclear if the reported nucleolar caps are actually associated with nucleoli. Furthermore, analysis of nucleoli in HCMV-infected cells by electron microscopy does not suggest the presence of nucleolar caps (Strang et al., 2012c). It is unknown whether HCMV infection stimulates p53-mediated nucleolar stress responses, which include specific changes to the protein translational profile of the cell (Boulon et al., 2010). Finally, the principal role of nucleoli in uninfected cells is to produce ribosomes. Levels of certain rRNAs increase in infected cells (Tanaka et al., 1975). Therefore, ribosome production during HCMV replication may not be compromised. However, the increase in rRNA production in infected cells could be related to changes in nucleolar composition and architecture. An integral part of any of the possibilities discussed could be the action of HCMV proteins in nucleoli. Several viral proteins can localize to nucleoli. It has been reported that HCMV pp65 and UL84 proteins are a nucleolar antigens (Arcangeletti et al., 2009; Bender et al., 2014). Also, transfection experiments have indicated that several epitopetagged versions of HCMV proteins (TLR5, TLR7, US33, UL29, UL31 and UL76) localize to the nucleolus in uninfected cells (Salsman et al., 2008). These proteins are expressed in different kinetic classes of viral RNA transcription and have different effects on HCMV replication, plus the function of several of these proteins is unknown (Table 1). Also, it has been reported that epitope-tagged versions of the putative HCMV proteins TLR7 and UL108 localize to nucleoli in uninfected cells (Salsman et al., 2008). However, it is now thought that the HCMV genome does not possess ORFs encoding TLR7 or UL108 (Gatherer et al., 2011). Cajal bodies and perinucleolar compartments are subnuclear structures associated with nucleoli (Fig. 2c) and both may be involved in viral RNA production. Cajal bodies are non-membrane-bound protein and RNA structures involved in transcription and maturation of ribonuclear particles (Ogg & Lamond, 2002). A role for Cajal bodies in HCMV replication has yet to be described. However, transfection of epitope-tagged HCMV UL30 leads to disruption and/or loss of Cajal bodies in uninfected cells (Table 1). Thus, it has been suggested (Salsman et al., 2008) that UL30 may allow HCMV to inhibit transcription and ribonuclear particle maturation. Cajal bodies are thought to play a role in adenovirus replication, wherein adenovirus infection results in fragmentation of Cajal bodies (James et al., 2010). Also, Cajal bodies are required for efficient production of several late adenoviral RNA transcripts (James et al., 2010). Thus, it is possible, as in adenovirusinfected cells, that Cajal bodies are disrupted upon HCMV infection and have a role in HCMV RNA transcription. However, Cajal bodies are generally absent in slowly dividing or non-dividing cells (Ogg & Lamond, 2002) and may not be present in the fibroblast, vascular and neuronal cell types within which HCMV replicates during productive infection. It has been suggested that the putative HCMV protein UL3 is also involved in modification of Cajal bodies (Salsman et al., 2008). However, as mentioned above, it is http://vir.sgmjournals.org now thought that an ORF encoding UL3 is not present in the HCMV genome (Gatherer et al., 2011). Perinucleolar compartments are thought to be involved in RNA polymerase II and III mediated RNA transcription. In HCMV-infected cells, at least one perinucleolar compartment protein, polyryrimidine tract-binding protein (PTB), is relocalized from the nucleus to the cytoplasm or degraded in the nucleus (Gaddy et al., 2010). It has been suggested (Gaddy et al., 2010) that loss of PTB from perinucleolar compartments is related to negative regulation of viral splicing by PTB (Cosme et al., 2009). In summary, upon HCMV infection of the cell, nucleoli remain intact but changes to nucleolar composition and architecture are observed. These changes may be related to a cellular nucleolar stress response, the recruitment of nucleolin to HCMV replication compartments or the presence of viral proteins within nucleoli. Subnuclear structures associated with nucleoli, including Cajal bodies and perinucleolar compartments, may be involved in viral RNA production in HCMV-infected cells. Production of DNA-filled capsids: a viral structure putatively involved in capsid assembly and genome packaging Late viral transcription produces proteins required to form viral capsids. The assembly of HCMV capsids and packaging of genomes into them is increasingly well understood (Gibson, 2008; McVoy et al., 2000; Scheffczik et al., 2002; Wang et al., 2012). However, it is unclear where within viral replication compartments capsid assembly and genome packaging take place. These questions have not been extensively investigated. There are few data regarding the localization of HCMV capsid proteins within the infected cell nucleus. However, it has been reported that a ‘sponge-like’ web of UL80 proteins forms throughout the infected cell nucleus, dependent upon UL80 self-interaction (Loveland et al., 2007; Nguyen et al., 2008). UL80 proteins include the capsid assembly protein precursor (UL80.5) and the maturational protease precursor (UL80a), which interact with the major HCMV capsid protein UL86 (Wood et al., 1997). It is possible that the web of UL80 proteins could act as a scaffold for capsid assembly and genome packaging within viral replication compartments. Packaging of viral DNA into nascent viral capsids probably takes place within replication compartments. The viral protein responsible for DNA binding during the packaging process, UL56, is found within replication compartments. UL56 localization to replication compartments is dependent on viral DNA synthesis, and UL56 may interact with UL44 within replication compartments (Giesen et al., 2000). Furthermore, it has been speculated that packaging of viral DNA into capsids is responsible for the movement of newly synthesized viral DNA from the periphery of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 247 B. L. Strang replication compartments to the interior of replication compartments (Strang et al., 2012b). Therefore, further study is required to understand the relationship between the production of DNA-containing viral capsids and viral replication compartments. It can be proposed, however, that viral capsid proteins play a role in organizing capsid production by creating a subnuclear structure on which capsid assembly and DNA packaging might take place. Exit of capsids from the nucleus: the nuclear lamina To escape the nucleus, DNA-containing capsids must first travel from within viral replication compartments to the nuclear periphery. It is unknown what facilitates this process in HCMV-infected cells. In HSV-infected cells, directed capsid movement in the nucleus is dependent upon myosin and actin (Forest et al., 2005). It is possible that HCMV also utilizes these cellular cargo transporters to facilitate capsid movement. Recently, it has been reported that mutation of HCMV UL84 leads to a defect in virus replication associated with a defect in nuclear egress of capsids and the clustering of capsids around nucleoli (Strang et al., 2012c). This would suggest that UL84 is, directly or indirectly, involved in the efficient movement of HCMV capsids within the nucleus. It has yet to be determined what relevance the mislocalization of capsids to nucleoli has on the movement of capsids in the nucleus. At the periphery of the nucleus, capsids must traverse the physical barrier of the nuclear lamina and pass though the nuclear membrane. There have been several recent advances in our understanding of these processes. HCMV proteins mediate events at the nuclear lamina required for nuclear egress. The viral nuclear egress complex (UL50 and UL53) recruits the viral kinase UL97 to the nuclear lamina in HCMV-infected cells (Sharma et al., 2014) (Table 1). Phosphorylation of the lamina component lamin A/C by UL97 promotes lamina disruption, akin to that observed during cell division, resulting in thinning and the appearance of gaps in the lamina (Hamirally et al., 2009). This should facilitate capsid movement through the lamina to the nuclear membrane. The requirement for other viral (including c-ORF29 and UL45) and cellular (including emerin, p32 and protein kinase C) proteins implicated in nuclear egress in this model remains unclear (Milbradt et al., 2014; Miller et al., 2010). It is unknown what mechanism selects only DNA-containing capsids for nuclear egress in HCMV-infected cells. In HSV-infected cells, this process is governed by the interaction of the nuclear egress complex and capsid proteins (Yang & Baines, 2011). A similar mechanism may operate in HCMV-infected cells. Once through the lamina, capsids must traverse the nuclear membrane in a process now thought to be similar to the passage of large ribonucleoprotein particles from the nucleus 248 to the cytoplasm (Jokhi et al., 2013; Speese et al., 2012). Briefly, capsids must bud through the inner leaflet of the nuclear membrane into the intramembrane space. A fusion event with the outer leaflet of the membrane grants capsids access to the cytoplasm. Capsids then engage the cytoplasmic membrane envelopment processes via a cytoplasmic viral assembly compartment (Alwine, 2012), which leads to virion exit from the cell. It is thought that the development of the cytoplasmic assembly compartment next to the nucleus deforms the nuclear membrane. This creates a concave surface on the nucleus, giving it a ‘kidney bean’ shape (Azzeh et al., 2006). To gain direct access to the cytoplasmic assembly compartment, capsids probably exit the nucleus through the concave face of the nuclear membrane. In summary, the process that facilitates the movement of DNA-containing capsids to the nuclear periphery is ill defined but probably involves cellular cargo transporters. Once at the nuclear membrane, a virally encoded nuclear egress complex possibly selects DNA-containing capsids for nuclear egress. This nuclear egress complex then facilitates capsid movement through the nuclear lamina by promoting modification of lamina components which results in thinning and the appearance of gaps in the lamina. The capsid moves through the inner and outer leaflets of the nuclear membrane via the intramembrane space. Once in the cytoplasm, capsids participate in a complex membrane envelopment process via a viral cytoplasmic assembly compartment that leads to virion exit from the cell. There may be a relationship between the construction of the viral cytoplasmic assembly compartment, deformation of the nucleus and exit of capsids through the nuclear membrane proximal to assembly compartments. Further questions and future perspectives As discussed here, there have been notable advances in our understanding of HCMV replication and virus– host interactions by examining subnuclear structures. However, a full examination of the questions that remain is limited by several factors. These include our inability to fully dissect viral gene expression or protein function and explore the composition of viral and cellular subnuclear structures. To address these issues, technological development is required. Of particular importance to interrogating the role of virally encoded factors will be the continued development of robust systems to produce recombinant HCMV viruses, including viruses with lethal mutations in their genomes. The study of viral genomes containing lethal mutations can currently be achieved by studying cells transfected with bacmids containing mutated HCMV genomes, for example Silva et al. (2010), although this methodology has its limitations. Examination of viral cellular subnuclear structure composition requires the development of well-characterized antibodies recognizing unmodified and post-translationally modified forms of viral and cellular proteins. Recombinant viruses and antibodies can then be coupled with increasingly sophisticated Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 Journal of General Virology 96 HCMV and subnuclear structures molecular and cellular techniques. These include live-cell imaging, super-resolution microscopy, correlative light electron microscopic analysis (Sharma et al., 2014), chemical biology techniques (for example, ‘click chemistry’; Strang et al., 2012b) and proteomic analysis of subnuclear structures (Andersen et al., 2002; Hiscox et al., 2010). A number of the observations discussed above rely on the study of transfection experiments using epitope-tagged versions of viral proteins in uninfected cells (Kuny et al., 2010; Prichard et al., 2008; Salsman et al., 2008). It is unclear what effect overexpression during transfection or epitope tagging might have on protein function. Thus, data based on transfection experiments should be interpreted cautiously. Furthermore, certain aspects of this review rely on the study of HSV. However, several features of HSV replication differ from HCMV replication, probably due to evolutionary divergence among herpesviruses. These include antagonism of the PML response (Everett et al., 2013), nucleolar organization (Lymberopoulos & Pearson, 2007; Strang et al., 2012a), the localization of nucleolin (Lymberopoulos & Pearson, 2007; Strang et al., 2012a) and replication compartment organization (Liptak et al., 1996; Strang et al., 2012b). There are areas where examination of HSV, or other herpesviruses, may not benefit HCMV research. These points underscore the need to generate reagents and methodologies to examine events that occur in HCMV-infected cells. The use of laboratory viral strains, the cell types studied and virus latency must be considered in future experiments. Most of the data outlined in this review were derived from the study of HCMV laboratory strains. The genomic complexity of HCMV is still being addressed. Primary strains of HCMV contain several more ORFs than laboratory strains of HCMV (Dolan et al., 2004). Also, recent highresolution transcriptome mapping (Gatherer et al., 2011) and ribosome profiling (Stern-Ginossar et al., 2012) have revealed the presence of many previously unrecognized viral RNA transcripts and non-canonical ORFs. Further investigation is required to discover how recently identified HCMV factors might influence viral or cellular subnuclear structures. These factors include newly identified transcripts from the US33 and TRL9 loci (Gatherer et al., 2011), which may localize to the nucleolus of HCMV-infected cells (Salsman et al., 2008) (Table 1). Also, the HCMV transcriptome contains a number of long (Gatherer et al., 2011) and short (Grey et al., 2005) non-coding RNAs. As yet, there are no data indicating that HCMV non-coding RNAs are involved in the function of viral or cellular subnuclear structures. The HCMV transcriptome varies between cell types (Towler et al., 2012), suggesting cell-type-specific requirements for viral factors. Transcripts differentially expressed between cell lines tested include those encoding UL44 (involved in viral replication compartment architecture), UL98 (suggested to be involved in chromatin partitioning), TRL5 (thought to be localized to the nucleolus), UL80 http://vir.sgmjournals.org proteins (thought to be involved in capsid assembly within replication compartments) plus UL97 and UL50 (involved in capsid egress through the nuclear lamina) (Towler et al., 2012) (Table 1). Thus, the function of viral and cellular subnuclear structures could vary among cell types. Virus latency is a hallmark of herpesvirus infection. However, our understanding of the establishment, maintenance and reactivation from latency of HCMV is incomplete. Recent in vitro models of virus infection in haematopoietic cells (for example, CD14+ and CD34+ cells) have illuminated important aspects of HCMV latency. The loss of hDaxx in CD34+ cells is required for transcription of IE viral genes upon infection by laboratory, but not primary, strains of HCMV (Saffert et al., 2010). This would argue that PMLNBs repress HCMV transcription from laboratory HCMV strains and that primary HCMV strains possess as-yetunidentified functions that control viral transcription during latency. Co-infection of CD34+ cells with both laboratory and primary strains of HCMV blocks the ability of histone deacetylases to activate IE gene transcription from the laboratory strain of HCMV (Saffert et al., 2010). Thus, transcriptional repression of primary HCMV strains may relate to chromatin modification by histone deacetylases. Also, it is unclear if hDaxx has a major role in repression of HCMV gene transcription during latency. In the embryonic carcinoma cell line NT2D1, which can recapitulate the differentiation-dependent regulation of IE gene expression observed during latency, repression of IE gene expression has been reported in the presence (Saffert & Kalejta, 2007) and absence (Groves & Sinclair, 2007) of hDaxx in undifferentiated NT2D1 cells. This may be due to differences in the virus strains used in each study. Further experiments are required to ascertain the relationship between virus strain, cell type, cell differentiation and gene expression during HCMV latency. There are few other data regarding the involvement of other subnuclear structures in HCMV latency. Furthermore, the latent HCMV transcriptome in either CD14+ or CD34+ cells (Rossetto et al., 2013) does not produce the high levels of transcripts encoding HCMV proteins thought to localized to cellular subnuclear structures during productive HCMV replication. Thus, if cellular subnuclear structures are involved in HCMV latency, different repertoires of viral factors interact with subnuclear structures during productive and latent HCMV infections. Acknowledgements Thanks to Andrew Davison, Wade Gibson, David Pasdeloup and Frazer Rixon for insightful discussions, plus Chris Boutell and Mayuri Sharma for comments on the manuscript. I would also like to thank the anonymous reviewers for their constructive comments, which have greatly improved this manuscript. I sincerely apologize to colleagues whose important work was not discussed or cited due to space limitations. This work was supported by St George’s, University of London, London, UK. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 249 B. L. Strang References expression and viral replication of human cytomegalovirus. J Virol 83, 2839–2850. Ahn, J. H. & Hayward, G. S. (1997). The major immediate-early Costa, H., Nascimento, R., Sinclair, J. & Parkhouse, R. M. (2013). proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells. J Virol 71, 4599–4613. Human cytomegalovirus gene UL76 induces IL-8 expression through activation of the DNA damage response. PLoS Pathog 9, e1003609. Ahn, J. H., Jang, W. J. & Hayward, G. S. (1999). The human cytomegalovirus IE2 and UL112-113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein-associated nuclear bodies (PODs or ND10). J Virol 73, 10458–10471. Alwine, J. C. (2012). The human cytomegalovirus assembly compartment: a masterpiece of viral manipulation of cellular processes that facilitates assembly and egress. PLoS Pathog 8, e1002878. Andersen, J. S., Lyon, C. E., Fox, A. H., Leung, A. K., Lam, Y. W., Steen, H., Mann, M. & Lamond, A. I. (2002). Directed proteomic analysis of the human nucleolus. Curr Biol 12, 1–11. Appleton, B. A., Loregian, A., Filman, D. J., Coen, D. M. & Hogle, J. M. (2004). The cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer. Mol Cell 15, 233–244. Appleton, B. A., Brooks, J., Loregian, A., Filman, D. J., Coen, D. M. & Hogle, J. M. (2006). Crystal structure of the cytomegalovirus DNA polymerase subunit UL44 in complex with the C terminus from the catalytic subunit. Differences in structure and function relative to unliganded UL44. J Biol Chem 281, 5224–5232. Dolan, A., Cunningham, C., Hector, R. D., Hassan-Walker, A. F., Lee, L., Addison, C., Dargan, D. J., McGeoch, D. J., Gatherer, D. & other authors (2004). Genetic content of wild-type human cytomegalo- virus. J Gen Virol 85, 1301–1312. Dove, B. K., You, J. H., Reed, M. L., Emmett, S. R., Brooks, G. & Hiscox, J. A. (2006). Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus. Cell Microbiol 8, 1147–1157. E, X., Pickering, M. T., Debatis, M., Castillo, J., Lagadinos, A., Wang, S., Lu, S. & Kowalik, T. F. (2011). An E2F1-mediated DNA damage response contributes to the replication of human cytomegalovirus. PLoS Pathog 7, e1001342. Ertl, P. F. & Powell, K. L. (1992). Physical and functional interaction of human cytomegalovirus DNA polymerase and its accessory protein (ICP36) expressed in insect cells. J Virol 66, 4126–4133. Everett, R. D., Boutell, C. & Hale, B. G. (2013). Interplay between viruses and host sumoylation pathways. Nat Rev Microbiol 11, 400– 411. Feichtinger, S., Stamminger, T., Müller, R., Graf, L., Klebl, B., Eickhoff, J. & Marschall, M. (2011). Recruitment of cyclin-dependent Arcangeletti, M. C., Rodighiero, I., De Conto, F., Gatti, R., Orlandini, G., Ferraglia, F., Motta, F., Covan, S., Razin, S. V. & other authors (2009). Modulatory effect of rRNA synthesis and ppUL83 nucleolar kinase 9 to nuclear compartments during cytomegalovirus late replication: importance of an interaction between viral pUL69 and cyclin T1. J Gen Virol 92, 1519–1531. compartmentalization on human cytomegalovirus gene expression in vitro. J Cell Biochem 108, 415–423. Forest, T., Barnard, S. & Baines, J. D. (2005). Active intranuclear Azzeh, M., Honigman, A., Taraboulos, A., Rouvinski, A. & Wolf, D. G. (2006). Structural changes in human cytomegalovirus cytoplasmic Fortunato, E. A. & Spector, D. H. (1998). p53 and RPA are sequestered assembly sites in the absence of UL97 kinase activity. Virology 354, 69–79. Bender, B. J., Coen, D. M. & Strang, B. L. (2014). Dynamic and nucleolin-dependent localization of human cytomegalovirus UL84 to the periphery of viral replication compartments and nucleoli. J Virol 88, 11738–11747. movement of herpesvirus capsids. Nat Cell Biol 7, 429–431. in viral replication centers in the nuclei of cells infected with human cytomegalovirus. J Virol 72, 2033–2039. Gaddy, C. E., Wong, D. S., Markowitz-Shulman, A. & Colberg-Poley, A. M. (2010). Regulation of the subcellular distribution of key cellular RNA-processing factors during permissive human cytomegalovirus infection. J Gen Virol 91, 1547–1559. Bogdanow, B., Weisbach, H., von Einem, J., Straschewski, S., Voigt, S., Winkler, M., Hagemeier, C. & Wiebusch, L. (2013). Human Gao, Y., Colletti, K. & Pari, G. S. (2008). Identification of human cytomegalovirus UL84 virus- and cell-encoded binding partners by using proteomics analysis. J Virol 82, 96–104. cytomegalovirus tegument protein pp150 acts as a cyclin A2-CDKdependent sensor of the host cell cycle and differentiation state. Proc Natl Acad Sci U S A 110, 17510–17515. Gatherer, D., Seirafian, S., Cunningham, C., Holton, M., Dargan, D. J., Baluchova, K., Hector, R. D., Galbraith, J., Herzyk, P. & other authors (2011). High-resolution human cytomegalovirus transcriptome. Proc Boisvert, F. M., van Koningsbruggen, S., Navascués, J. & Lamond, A. I. (2007). The multifunctional nucleolus. Nat Rev Mol Cell Biol 8, Gibson, W. (2008). Structure and formation of the cytomegalovirus 574–585. Natl Acad Sci U S A 108, 19755–19760. virion. Curr Top Microbiol Immunol 325, 187–204. Boulon, S., Westman, B. J., Hutten, S., Boisvert, F. M. & Lamond, A. I. (2010). The nucleolus under stress. Mol Cell 40, 216–227. Giesen, K., Radsak, K. & Bogner, E. (2000). Targeting of the gene Boutell, C. & Everett, R. D. (2013). Regulation of alphaherpesvirus product encoded by ORF UL56 of human cytomegalovirus into viral replication centers. FEBS Lett 471, 215–218. infections by the ICP0 family of proteins. J Gen Virol 94, 465–481. Ginisty, H., Sicard, H., Roger, B. & Bouvet, P. (1999). Structure and Chambers, J., Angulo, A., Amaratunga, D., Guo, H., Jiang, Y., Wan, J. S., Bittner, A., Frueh, K., Jackson, M. R. & other authors (1999). Glass, M. & Everett, R. D. (2013). Components of promyelocytic DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression. J Virol 73, 5757–5766. Chang, L., Godinez, W. J., Kim, I. H., Tektonidis, M., de Lanerolle, P., Eils, R., Rohr, K. & Knipe, D. M. (2011). Herpesviral replication compartments move and coalesce at nuclear speckles to enhance export of viral late mRNA. Proc Natl Acad Sci U S A 108, E136–E144. Cosme, R. S., Yamamura, Y. & Tang, Q. (2009). Roles of polypyrimidine tract binding proteins in major immediate-early gene 250 functions of nucleolin. J Cell Sci 112, 761–772. leukemia nuclear bodies (ND10) act cooperatively to repress herpesvirus infection. J Virol 87, 2174–2185. Grey, F., Antoniewicz, A., Allen, E., Saugstad, J., McShea, A., Carrington, J. C. & Nelson, J. (2005). Identification and character- ization of human cytomegalovirus-encoded microRNAs. J Virol 79, 12095–12099. Groves, I. J. & Sinclair, J. H. (2007). Knockdown of hDaxx in normally non-permissive undifferentiated cells does not permit human cytomegalovirus immediate-early gene expression. J Gen Virol 88, 2935–2940. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 Journal of General Virology 96 HCMV and subnuclear structures Hamirally, S., Kamil, J. P., Ndassa-Colday, Y. M., Lin, A. J., Jahng, W. J., Baek, M. C., Noton, S., Silva, L. A., Simpson-Holley, M. & other authors (2009). Viral mimicry of Cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress. PLoS Pathog 5, e1000275. Kuny, C. V., Chinchilla, K., Culbertson, M. R. & Kalejta, R. F. (2010). Cyclin-dependent kinase-like function is shared by the beta- and gamma-subset of the conserved herpesvirus protein kinases. PLoS Pathog 6, e1001092. Lamond, A. I. & Spector, D. L. (2003). Nuclear speckles: a model for Hiscox, J. A. (2002). The nucleolus – a gateway to viral infection? Arch nuclear organelles. Nat Rev Mol Cell Biol 4, 605–612. Virol 147, 1077–1089. Lee, H. R., Kim, D. J., Lee, J. M., Choi, C. Y., Ahn, B. Y., Hayward, G. S. & Ahn, J. H. (2004). Ability of the human cytomegalovirus IE1 protein Hiscox, J. A., Whitehouse, A. & Matthews, D. A. (2010). Nucleolar Hume, A. J., Finkel, J. S., Kamil, J. P., Coen, D. M., Culbertson, M. R. & Kalejta, R. F. (2008). Phosphorylation of retinoblastoma protein by proteomics and viral infection. Proteomics 10, 4077–4086. to modulate sumoylation of PML correlates with its functional activities in transcriptional regulation and infectivity in cultured fibroblast cells. J Virol 78, 6527–6542. viral protein with cyclin-dependent kinase function. Science 320, 797– 799. Lin, S. R., Jiang, M. J., Wang, H. H., Hu, C. H., Hsu, M. S., Hsi, E., Duh, C. Y. & Wang, S. K. (2013). Human cytomegalovirus UL76 elicits Ishov, A. M., Stenberg, R. M. & Maul, G. G. (1997). Human novel aggresome formation via interaction with S5a of the ubiquitin proteasome system. J Virol 87, 11562–11578. cytomegalovirus immediate early interaction with host nuclear structures: definition of an immediate transcript environment. J Cell Biol 138, 5–16. Liptak, L. M., Uprichard, S. L. & Knipe, D. M. (1996). Functional order of assembly of herpes simplex virus DNA replication proteins into prereplicative site structures. J Virol 70, 1759–1767. Ishov, A. M., Sotnikov, A. G., Negorev, D., Vladimirova, O. V., Neff, N., Kamitani, T., Yeh, E. T., Strauss, J. F., III & Maul, G. G. (1999). PML is Livingston, C. M., Ifrim, M. F., Cowan, A. E. & Weller, S. K. (2009). critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1. J Cell Biol 147, 221–234. Virus-Induced Chaperone-Enriched (VICE) domains function as nuclear protein quality control centers during HSV-1 infection. PLoS Pathog 5, e1000619. Ishov, A. M., Vladimirova, O. V. & Maul, G. G. (2004). Loregian, A., Appleton, B. A., Hogle, J. M. & Coen, D. M. (2004a). Heterochromatin and ND10 are cell-cycle regulated and phosphorylation-dependent alternate nuclear sites of the transcription repressor Daxx and SWI/SNF protein ATRX. J Cell Sci 117, 3807– 3820. Specific residues in the connector loop of the human cytomegalovirus DNA polymerase accessory protein UL44 are crucial for interaction with the UL54 catalytic subunit. J Virol 78, 9084–9092. James, N. J., Howell, G. J., Walker, J. H. & Blair, G. E. (2010). The role Residues of human cytomegalovirus DNA polymerase catalytic subunit UL54 that are necessary and sufficient for interaction with the accessory protein UL44. J Virol 78, 158–167. of Cajal bodies in the expression of late phase adenovirus proteins. Virology 399, 299–311. Jokhi, V., Ashley, J., Nunnari, J., Noma, A., Ito, N., Wakabayashi-Ito, N., Moore, M. J. & Budnik, V. (2013). Torsin mediates primary Loregian, A., Appleton, B. A., Hogle, J. M. & Coen, D. M. (2004b). Loveland, A. N., Nguyen, N. L., Brignole, E. J. & Gibson, W. (2007). envelopment of large ribonucleoprotein granules at the nuclear envelope. Cell Rep 3, 988–995. The amino-conserved domain of human cytomegalovirus UL80a proteins is required for key interactions during early stages of capsid formation and virus production. J Virol 81, 620–628. Kagele, D., Rossetto, C. C., Elorza, M. & Pari, G. S. (2013). Analysis of Lukashchuk, V., McFarlane, S., Everett, R. D. & Preston, C. M. (2008). the interactions of viral and cellular factors with human cytomegalovirus lytic origin of replication, oriLyt. Virology 424, 106–114. Kalejta, R. F. & Shenk, T. (2003). Proteasome-dependent, ubiquitin- Human cytomegalovirus protein pp71 displaces the chromatinassociated factor ATRX from nuclear domain 10 at early stages of infection. J Virol 82, 12543–12554. independent degradation of the Rb family of tumor suppressors by the human cytomegalovirus pp71 protein. Proc Natl Acad Sci U S A 100, 3263–3268. Lymberopoulos, M. H. & Pearson, A. (2007). Involvement of UL24 in herpes-simplex-virus-1-induced dispersal of nucleolin. Virology 363, 397–409. Kang, H., Kim, E. T., Lee, H. R., Park, J. J., Go, Y. Y., Choi, C. Y. & Ahn, J. H. (2006). Inhibition of SUMO-independent PML oligomerization McVoy, M. A. & Adler, S. P. (1994). Human cytomegalovirus DNA by the human cytomegalovirus IE1 protein. J Gen Virol 87, 2181– 2190. Kim, Y. E., Lee, J. H., Kim, E. T., Shin, H. J., Gu, S. Y., Seol, H. S., Ling, P. D., Lee, C. H. & Ahn, J. H. (2011). Human cytomegalovirus infection causes degradation of Sp100 proteins that suppress viral gene expression. J Virol 85, 11928–11937. Knizewski, L., Kinch, L., Grishin, N. V., Rychlewski, L. & Ginalski, K. (2006). Human herpesvirus 1 UL24 gene encodes a potential PD-(D/ replicates after early circularization by concatemer formation, and inversion occurs within the concatemer. J Virol 68, 1040–1051. McVoy, M. A., Nixon, D. E., Hur, J. K. & Adler, S. P. (2000). The ends on herpesvirus DNA replicative concatemers contain pac2 cis cleavage/packaging elements and their formation is controlled by terminal cis sequences. J Virol 74, 1587–1592. Milbradt, J., Kraut, A., Hutterer, C., Sonntag, E., Schmeiser, C., Ferro, M., Wagner, S., Lenac, T., Claus, C. & other authors (2014). E)XK endonuclease. J Virol 80, 2575–2577. Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus. Mol Cell Proteomics 13, 2132–2146. Kobiler, O., Brodersen, P., Taylor, M. P., Ludmir, E. B. & Enquist, L. W. (2011). Herpesvirus replication compartments originate with single Miller, M. S., Furlong, W. E., Pennell, L., Geadah, M. & Hertel, L. (2010). RASCAL is a new human cytomegalovirus-encoded protein incoming viral genomes. MBio 2, e00278-11. that localizes to the nuclear lamina and in cytoplasmic vesicles at late times postinfection. J Virol 84, 6483–6496. Kobiler, O., Drayman, N., Butin-Israeli, V. & Oppenheim, A. (2012). Virus strategies for passing the nuclear envelope barrier. Nucleus 3, 526–539. Mocarski, E. S., Shenk, T. & Pass, R. F. (2007). Cytomegaloviruses. In Fields Virology, 5th edn, vol. 2, pp. 2701–2772. Edited by D. M. Knipe & P. M. Howley. New York, NY: Lippincott, Williams & Wilkins. Korioth, F., Maul, G. G., Plachter, B., Stamminger, T. & Frey, J. (1996). The nuclear domain 10 (ND10) is disrupted by the human Moldovan, G. L., Pfander, B. & Jentsch, S. (2007). PCNA, the maestro cytomegalovirus gene product IE1. Exp Cell Res 229, 155–158. of the replication fork. Cell 129, 665–679. http://vir.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 251 B. L. Strang Nguyen, N. L., Loveland, A. N. & Gibson, W. (2008). Nuclear localization sequences in cytomegalovirus capsid assembly proteins (UL80 proteins) are required for virus production: inactivating NLS1, NLS2, or both affects replication to strikingly different extents. J Virol 82, 5381–5389. O’Dowd, J. M., Zavala, A. G., Brown, C. J., Mori, T. & Fortunato, E. A. (2012). HCMV-infected cells maintain efficient nucleotide excision repair of the viral genome while abrogating repair of the host genome. PLoS Pathog 8, e1003038. Ogg, S. C. & Lamond, A. I. (2002). Cajal bodies and coilin – moving towards function. J Cell Biol 159, 17–21. Pari, G. S. & Anders, D. G. (1993). Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication. J Virol 67, 6979–6988. subcellular localization and alteration of PML nuclear bodies. PLoS Pathog 4, e1000100. Sarisky, R. T. & Hayward, G. S. (1996). Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLytdependent DNA replication and formation of replication compartments in cotransfection assays. J Virol 70, 7398–7413. Scheffczik, H., Savva, C. G., Holzenburg, A., Kolesnikova, L. & Bogner, E. (2002). The terminase subunits pUL56 and pUL89 of human cytomegalovirus are DNA-metabolizing proteins with toroidal structure. Nucleic Acids Res 30, 1695–1703. Schreiner, S., Wimmer, P., Sirma, H., Everett, R. D., Blanchette, P., Groitl, P. & Dobner, T. (2010). Proteasome-dependent degradation of Daxx by the viral E1B-55K protein in human adenovirus-infected cells. J Virol 84, 7029–7038. Pari, G. S., Kacica, M. A. & Anders, D. G. (1993). Open reading frames Seeler, J. S., Marchio, A., Sitterlin, D., Transy, C. & Dejean, A. (1998). Interaction of SP100 with HP1 proteins: a link between UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis. J Virol 67, 2575–2582. the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment. Proc Natl Acad Sci U S A 95, 7316– 7321. Penfold, M. E. & Mocarski, E. S. (1997). Formation of cytomegalo- Seeler, J. S., Marchio, A., Losson, R., Desterro, J. M., Hay, R. T., Chambon, P. & Dejean, A. (2001). Common properties of nuclear virus DNA replication compartments defined by localization of viral proteins and DNA synthesis. Virology 239, 46–61. Prichard, M. N., Duke, G. M. & Mocarski, E. S. (1996). Human cytomegalovirus uracil DNA glycosylase is required for the normal temporal regulation of both DNA synthesis and viral replication. J Virol 70, 3018–3025. Prichard, M. N., Lawlor, H., Duke, G. M., Mo, C., Wang, Z., Dixon, M., Kemble, G. & Kern, E. R. (2005). Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA. Virol J 2, 55. Prichard, M. N., Sztul, E., Daily, S. L., Perry, A. L., Frederick, S. L., Gill, R. B., Hartline, C. B., Streblow, D. N., Varnum, S. M. & other authors (2008). Human cytomegalovirus UL97 kinase activity is required for body protein SP100 and TIF1alpha chromatin factor: role of SUMO modification. Mol Cell Biol 21, 3314–3324. Sharma, M., Kamil, J. P., Coughlin, M., Reim, N. I. & Coen, D. M. (2014). Human cytomegalovirus UL50 and UL53 recruit viral protein kinase UL97, not protein kinase C, for disruption of nuclear lamina and nuclear egress in infected cells. J Virol 88, 249–262. Shav-Tal, Y., Blechman, J., Darzacq, X., Montagna, C., Dye, B. T., Patton, J. G., Singer, R. H. & Zipori, D. (2005). Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition. Mol Biol Cell 16, 2395–2413. Sheaffer, A. K., Weinheimer, S. P. & Tenney, D. J. (1997). The human cytomegalovirus UL98 gene encodes the conserved herpesvirus alkaline nuclease. J Gen Virol 78, 2953–2961. the hyperphosphorylation of retinoblastoma protein and inhibits the formation of nuclear aggresomes. J Virol 82, 5054–5067. Siew, V. K., Duh, C. Y. & Wang, S. K. (2009). Human cytomegalovirus Ranneberg-Nilsen, T., Dale, H. A., Luna, L., Slettebakk, R., Sundheim, O., Rollag, H. & Bjørås, M. (2008). Characterization of Silva, L., Cliffe, A., Chang, L. & Knipe, D. M. (2008). Role for A-type human cytomegalovirus uracil DNA glycosylase (UL114) and its interaction with polymerase processivity factor (UL44). J Mol Biol 381, 276–288. Rechter, S., Scott, G. M., Eickhoff, J., Zielke, K., Auerochs, S., Müller, R., Stamminger, T., Rawlinson, W. D. & Marschall, M. (2009). Cyclin- dependent kinases phosphorylate the cytomegalovirus RNA export protein pUL69 and modulate its nuclear localization and activity. J Biol Chem 284, 8605–8613. Reichelt, M., Wang, L., Sommer, M., Perrino, J., Nour, A. M., Sen, N., Baiker, A., Zerboni, L. & Arvin, A. M. (2011). Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus. PLoS Pathog 7, e1001266. Rossetto, C. C., Tarrant-Elorza, M. & Pari, G. S. (2013). Cis and trans acting factors involved in human cytomegalovirus experimental and natural latent infection of CD14 + monocytes and CD34 + cells. PLoS Pathog 9, e1003366. Saffert, R. T. & Kalejta, R. F. (2007). Human cytomegalovirus gene UL76 induces chromosome aberrations. J Biomed Sci 16, 107. lamins in herpesviral DNA targeting and heterochromatin modulation. PLoS Pathog 4, e1000071. Silva, L. A., Loregian, A., Pari, G. S., Strang, B. L. & Coen, D. M. (2010). The carboxy-terminal segment of the human cytomegalovirus DNA polymerase accessory subunit UL44 is crucial for viral replication. J Virol 84, 11563–11568. Silva, L. A., Strang, B. L., Lin, E. W., Kamil, J. P. & Coen, D. M. (2011). Sites and roles of phosphorylation of the human cytomegalovirus DNA polymerase subunit UL44. Virology 417, 268–280. Speese, S. D., Ashley, J., Jokhi, V., Nunnari, J., Barria, R., Li, Y., Ataman, B., Koon, A., Chang, Y. T. & other authors (2012). Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling. Cell 149, 832–846. Stepp, W. H., Meyers, J. M. & McBride, A. A. (2013). Sp100 provides intrinsic immunity against human papillomavirus infection. MBio 4, e00845-e13. expression is silenced by Daxx-mediated intrinsic immune defense in model latent infections established in vitro. J Virol 81, 9109–9120. Stern-Ginossar, N., Weisburd, B., Michalski, A., Le, V. T., Hein, M. Y., Huang, S. X., Ma, M., Shen, B., Qian, S. B. & other authors (2012). Decoding human cytomegalovirus. Science 338, 1088– Saffert, R. T., Penkert, R. R. & Kalejta, R. F. (2010). Cellular and viral 1093. control over the initial events of human cytomegalovirus experimental latency in CD34+ cells. J Virol 84, 5594–5604. Sternsdorf, T., Jensen, K., Reich, B. & Will, H. (1999). The nuclear dot Salsman, J., Zimmerman, N., Chen, T., Domagala, M. & Frappier, L. (2008). Genome-wide screen of three herpesviruses for protein 252 protein sp100, characterization of domains necessary for dimerization, subcellular localization, and modification by small ubiquitin-like modifiers. J Biol Chem 274, 12555–12566. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 Journal of General Virology 96 HCMV and subnuclear structures 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., 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. & Coen, D. M. (2010a). Nucleolin associates 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. with the human cytomegalovirus DNA polymerase accessory subunit UL44 and is necessary for efficient viral replication. J Virol 84, 1771– 1784. 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. Strang, B. L., Geballe, A. P. & Coen, D. M. (2010b). Association of Wang, J. B., Zhu, Y., McVoy, M. A. & Parris, D. S. (2012). Changes in human cytomegalovirus proteins IRS1 and TRS1 with the viral DNA polymerase accessory subunit UL44. J Gen Virol 91, 2167–2175. subcellular localization reveal interactions between human cytomegalovirus terminase subunits. Virol J 9, 315. Strang, B. L., Boulant, S., Kirchhausen, T. & Coen, D. M. (2012a). Wilkinson, G. W., Kelly, C., Sinclair, J. H. & Rickards, C. (1998). Host cell nucleolin is required to maintain the architecture of human cytomegalovirus replication compartments. MBio 3, 301–311. 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, 2089–2095. 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. Disruption of PML-associated nuclear bodies mediated by the human cytomegalovirus major immediate early gene product. J Gen Virol 79, 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 PML-mediated 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. & Wang, W. (2003). The ATRX syndrome Tavalai, N. & Stamminger, T. (2009). Interplay between herpesvirus infection and host defense by PML nuclear bodies. Viruses 1, 1240– 1264. 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. Tavalai, N. & Stamminger, T. (2011). Intrinsic cellular defense Yang, K. & Baines, J. D. (2011). Selection of HSV capsids for mechanisms targeting human cytomegalovirus. Virus Res 157, 128–133. envelopment involves interaction between capsid surface components pUL31, pUL17, and pUL25. Proc Natl Acad Sci U S A 108, 14276–14281. 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. http://vir.sgmjournals.org 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 11 Aug 2017 15:49:36 253
