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Epigenetic regulation of human cytomegalovirus latency: an update

Human cytomegalovirus (HCMV) is a ubiquitous virus which infects 50–90% of the population worldwide. In immunocompetent hosts, HCMV either remains unnoticed or causes mild symptoms. Upon primary infection it establishes latent infection in a few cells. However, in certain situations where immunity is either immature or compromised, HCMV may reactivate and cause mortality and morbidity. Therefore, it is utmost important to understand how HCMV establishes latent infection and associated mechanisms responsible for its reactivation. Several mechanisms are involved in the regulation of latency including chromatin remodeling by an array of enzymes and microRNAs. Here we will describe the epigenetic regulation of HCMV latency. Further we will discuss the unique HCMV latency signature and patho-physiological relevance of latent HCMV infection.

Human cytomegalovirus (HCMV), also called human herpesvirus 5, is a member of Herpesviridae family, subfamily Betaherpesvirinae. HCMV is a ubiquitous virus which infects 50–90% of the population. The rate of infection is governed by several factors including age of the population and socioeconomic status [1] . Primary infection caused by HCMV is usually asymptomatic or with mild consequences. However, infection in organ-transplant recipients, immunocompromised individuals and newborns (congenital infection) has serious complications and associated morbidity [2,3] . HCMV has a large genome (∼240 kb) that has 165–252 open reading frames (ORFs) [4–6] . However, recent studies utilizing ribosomal profiling and mass spectrometry analysis revealed the presence of several unidentified ORFs and alternative splice sites [7] . These ORFs are expressed in a specific manner leading to two distinct infection patterns, namely, lytic and latent infection. During the course of lytic infection, HCMV expressed its genes in temporal cascades which involve immediate early (IE) genes, followed by early genes and late genes. IE genes, for example, IE1 and IE2

10.2217/EPI.14.41 © 2014 Future Medicine Ltd

Amit Kumar1 & Georges Herbein*,1 1 Department of Virology, University of Franche-Comte, CHRU Besançon, UPRES EA4266 Pathogens & Inflammation Department, SFR FED 4234, F-25030 Besançon, France *Author for correspondence: Tel.: +33 381 21 88 77 Fax: +33 381 66 56 95 [email protected]

regulate the cellular environment for productive infection [8–10] . Early gene product, for example, UL97 is involved in DNA replication [11] and late genes encode for structural components of the virion [12] . Viral progeny has been detected in several biological fluids including urine, saliva and breast milk during primary infection as well as upon reactivation suggests the possibility of several tissues as active sites of viral infection [13–15] . On the other hand, latency is one of the viral strategies of maintaining a persistent infection [16–18] . Upon primary infection HCMV establishes lifelong latency in bone-marrow-derived mononuclear cells (1 in 104 –105 infected cells) characterized by the persistence of viral genome without any virion production [19] . Furthermore, 2–13 copies of HCMV have been reported per latently infected cells [19] . Notably, HCMV exists in the circular form in latently infected cells (Figure 1) [20] . To date, there are no reports suggesting the integration of HCMV genome into the host chromatin. Although HCMV has wide cell tropism, however, the cells of myeloid origin including CD14+ peripheral blood mononuclear cells and CD34+ hematopoietic progenitor

Epigenomics (2014) 6(5), 533–546

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ISSN 1750-1911

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Review Kumar & Herbein

MRP1

Lytic infection

LUNA

miR-UL112-1

* * * Latent HCMV

pUL138 HDAC/HMTs/ PRC2

E2

IE2

* * * HCMV

1/I IE

IE1

Genome circulazation

UL138 Ribosomes miRNA-mediated inhibition

miR-US25-1/ miR-US25-2 miR-US33

US29 miR-US33

LnRNA 2.7/4.9, UL44, UL50, UL84, UL87, US17, UL28/29, UL27/38

Ribosomes

Uncharacterized latency-associated transcripts

Figure 1. Overview of mechanisms involved in regulating human cytomegalovirus latency. Once entering into the susceptible cell, HCMV is transported to the nuclear pore where it delivers viral genomic DNA into the nucleus [32] . In the nucleus HCMV DNA circularized and associates itself with cellular histone leading to the formation of ‘minichromosomes’ [33,34] . In latent phase, suppression of HCMV replication and transcription is governed by several epigenetic chromatin modifying players including HMTs and HDACs, PRC2 and microRNAs [35,36] . In addition, several latency-associated transcripts have been also shown [37–41] . HCMV: Human cytomegalovirus; HDAC: Histone deacetylase; HMT: Histone methyltransferase; PRC2: Polycomb repressive complex 2.

cells (HPCs) have been widely recognized as the site of HCMV latency [21–24] . Of note, reactivation of latent HCMV in immunosuppressed or immunocompromised individuals is the main cause of morbidity associated with HCMV infection. Molecular mechanisms involved in reactivating HCMV latency are poorly understood. However, in the recent past, besides genetic factors, studies unveiling the epigenetic regulation of HCMV latency have begun to emerge. Epigenetics refers to the heritable changes modulating the gene transcription without any change in the DNA sequences [25] . Epigenetic mechanisms globally govern the cell development and differentiation and maintenance of genomic stability in eukaryotes [26] . Aberrant performance of cellular epigenetic machinery often leads to the pathological consequence including cancer [27] . Enzymes involved in DNA methylation, histone modification (acetylation/deacetylation, methylation/ demethylation, sumoylation), chromatin remodeling and noncoding RNAs especially microRNA (miRNA)

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are important players involved in epigenetic regulations [16,28–31] . In this review we will describe how HCMV gene expression is modulated by epigenetic mechanisms and how HCMV uses cellular epigenetic machinery for the establishment of successful persistent infection. In addition, we will discuss the implication of HCMV latency/reactivation in the development of several HCMV-linked diseases. Epigenetic mechanisms involved in HCMV latency Establishment of HCMV latency

Upon entry into the susceptible cell type, HCMV capsid containing viral genome is transported to the nuclear pore where it delivers viral genomic DNA into the nucleus [32] (Figure 1) . Inside the nucleus, HCMV DNA becomes rapidly circularized and associates itself with cell-encoded histone leading the formation of chromatin scaffold (reviewed in [33,34] (Figure 1) . In productive lytic infection de novo expression of IE proteins

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Epigenetic regulation of human cytomegalovirus latency

(IE1, IE2) has been reported from major IE enhancer/ promoter (MIEP) [42] . Expression of IE proteins is followed by early and late proteins. The expression of IE proteins decides whether the virus will undergo lytic or latent infection. Several reports suggest that in latent phase suppression of MIEP is governed by various epigenetic chromatin modifying players including methyltransferases and histone deacetylases (Figure 2) [43– 47] . HCMV establishes latent infection in a few cells in vivo. Therefore, it is difficult to study the latency in natural infection scenario. As a result, we need to rely upon information pertaining to establishment of HCMV latency that has been derived from the experimental models. These experimental models include more close representatives of natural latent infection; for example, ex vivo infected culture of CD14+ monocytes and CD34+ HPCs and less authentic differentiated or undifferentiated THP-1 cells. For instance, Ioudinkova and colleagues studied the methylation and acetylation pattern of local chromatin structure of MIEP, early and late gene of HCMV in nondifferentiated THP-1 cells (a model for HCMV latency) and differentiated THP-1 cells (a model for HCMV lytic phase). Using chromatin immunoprecipitation, they observed that in productive phase heavy acetylation but not dimethylation of H3K9 was associated with MIEP and viral genes including DNA polymerase, pp65 and pp150. In contrast, in latently infected THP-1 cells neither acetylation nor dimethylation of H3K9 was observed, whereas HCMV genes coding for DNA polymerase, pp65 and pp150, were found to be dimethylated [44] . Their data suggest the involvement Histone

of chromatin-structure-meditated regulation of lytic and latent phases of HCMV. However, this hypothesis is under intense study and needs further validation. Involvement of post-translational histone modifications has also been reported in other members of Herpesviridae. It seems that viruses utilize the histonemodifying machinery in the same way as utilized by their host for homeostasis and tissue-specific expression. For instance, in cases of latent herpes simplex virus Type 1 infection, histones associated with the promoter of the latency-associated transcripts are the only region having transcriptional permissive structure. These latency-associated transcript regions have H3K4me2 and H3K9, K14 acetylation [52] . Similarly, different kinds of chromatin modification marks have been reported in histone-associated latent genome of Kaposi’s sarcoma-associated herpesvirus virus (KSHV) and Epstein–Barr virus (EBV) [53] . EBV and KSHV also methylated their promoter to favor latency [53] , which is not well known in HCMV. Polycomb repressive complex 2 (PRC2) is a methyltransferase which favors tissue-specific differentiation [54,55] and triggers pluripotency in embryonic stem cells [56] . PRC2 is an integral cellular component indispensable for the cell development and homeostasis. PRC2 is known to catalyzed histone H3 trimethylation on lysine 27 residues of heterochromatin [56] . PRC2 is made of SET domain containing EZH2 which has catalytic methyltransferase activity along with zinc finger protein SUZ12 and WD40 repeat protein EED (Figure 2) . PRC2 also influences the latency of HCMV by methylation at H3K9 residue Histone

ERF CRE YY1 NF-κB

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H3K4me

EZH2

IE mRNAs

IE1

ERF CRE YY1 NF-κB

WWWWWWWWWWWWWWWWWWW H3K27me3

H3K9/14 act

MIEP

WWWWWWWWWWWWWWWWWWW

WWWWWWWWWWWWWWWWWWW MIEP

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ERF H3K9me

SUZ12 EED PRC2

YY1

H3K9me

HMTs HDACs

H3K27me3

X

IE2

Latent infection

Lytic infection

Figure 2. Transition of lytic to latent infection by chromatin remodeling of major immediate early enhancer/promoter. Major immediate early enhancer/promoter (MIEP) has binding sites for several transcriptional regulators including ERF, CRE, YY1 and NFκB [42] . In lytic phase, histones associated with MIEP are in euchromatin kind of configuration characterized by an increase in H3K4 methylation and H3K9/14 acetylation [16,30,35] . On the other hand, in latent phase histones are methylated at K9 (H3K9me) and K27 (H3K27me3) leading to the formation of transcriptional repressive structure [16,30,35] . These events are governed by several players including HMTs, HDACs and PRC2. Role of YY1 [48] and ERF [49,50] have been shown in promoting latency. Besides above-mentioned factors several new mechanisms have been recently shown that regulate the transition of latent into lytic infection [51] . CRE: Cyclic AMP response elements; ERF: Ets-2 repressor factor; PRC2: Polycomb repressive complex 2; YY1: Yin Yang 1.


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Review Kumar & Herbein [57–59] .

For instance, recent work from Kulesza laboratory demonstrates the influence of PRC2 in favoring latency by suppressing the HCMV lytic genes’ expression in HCMV model cell lines for latency (THP 1 and NT2D1) (Figure 2) . Inhibition of PRC2 resulted in an increase in the expression of HCMV viral transcripts and decrease in H3K27me3 status has been demonstrated (Figure 2) . Similar findings have been previously reported from Kulesza laboratory in murine cytomegalovirus (MCMV) [57] . Role of miRNA in HCMV latency

Cellular miRNAs are ∼22-nucleotide long singlestranded effector molecules of eukaryotic RNA interference machinery. miRNAs are known to govern several cellular processes including differentiation, organogenesis and cell growth, and also exert anti-viral effects [60] . Besides eukaryotic cells, viruses also possess miRNAs which govern several important aspects of viral life cycle [31,61–62] . Till date more than 15 miRNAs have been identified in HCMV genomes [63] . The role of miRNA in the maintenance of latency in EBV [64] and KSHV [65,66] has been reported. Similarly, HCMV-encoded miRNAs are known to play important role in switching lytic infection into latent mode in experimental culture system only. Whether these miRNAs also play a role in regulating HCMV latency in natural infection scenario is not known and needs extensive experimentation. For example, one of the best characterized HCMV miRNA miR-UL112–1 has been shown to express during the early phase of viral life cycle (Figure 1) [67] . miR-UL112–1 is known to regulate the expression of IE1 gene (Table 1) [68,69] . The expression of miR-UL112–1 is reported to be inversely correlated with the expression of IE1 [67] . The pre-mature expression of miR-UL112–1 dramatically reduces the expression of IE1 and viral replication [68] . In addition, the involve-

ment of miR-UL112–1 in promoting HCMV latency has been suggested, however, needs experimental validation (Figure 1) [69] . Recent data from Mandelboim’s laboratory further characterized two HCMV-encoded miRNAs, namely, miR-US25–1 and miR-US25–2. These miRNAs have been known to reduce replication of several viruses including HCMV [70] . Whether these miRNAs can promote HCMV latency or target cellular factors essential for HCMV replication needs extensive investigations [70] . Identification of HCMVspecific miRNA in latent in vivo reservoirs can further support these findings. Recently, Shen and colleagues studied the expression profile of HCMV miRNA in permissive, semi-permissive and latent in vitro models of HCMV infection. They identified miR-US33 which inhibits lytic infection upon ectopic expression and suggested the role of miR-US33 as a molecular switch which can regulate the transition of lytic into latent infection (Table 1) (Figure 1) [71] . In addition, the role of host-encoded miRNA hsa-miR-92a has been implicated in HCMV latency. Upon induction of latency in CD34+ HPCs, expression of hsa-miR-92a is downregulated resulting in the upregulation of its target GATA-2 and subsequently cellular cytokine IL-10 (cIL10) (Figure 3) [72] . cIL-10 is a multifunctional cytokine that exerts immunosuppressive and anti-inflammatory effects [73] . It is produced by variety of immune cells. Increased expression of cIL-10 may enhance the survival of latently infected HCMV cells (Figure 3) [72] . Of note, expression of miRNA is regulated by epigenetic machinery and in turn miRNAs regulate the expression of cellular factors involved in epigenetic regulation [78] . These miRNAs are termed as ‘epimicroRNA’ [29,79] . Identification of novel HCMVencoded ‘epi-miRNA’ can further shed light on the mechanism of latency govern by miRNAs. In addition, HCMV miRNA regulating latency can be

Table 1. Human cytomegalovirus gene products involved in latency. Viral transcript/protein

Target

Mechanism

Latency (L)/ Reactivation (R)

Ref.

pUL138

MRP1

Degradation of MRP1

Not known

pUL133

Not known

Reduces viral replication by unknown mechanism

L

[75]

pUL111A

Not known

Suppress pro-inflammatory cytokines production; L restricts the differentiation of myeloid progenitors cells into mature DC

[76]

UL81-82 ast (LUNA)

IE1 mRNA

Transcriptional control of IE1

L

[37,38]

miR-UL112-1

IE1 & UL144 mRNA

Translational suppression

L

[68,77]

miR-US33

US29 mRNA

Inhibits lytic infection

L

[71]

miR-US25-1

IE1 & pp65 mRNA

Inhibits viral replication

L

[70]

miR-US25-2

IE1 & pp65 mRNA

inhibits viral replication

L

[70]

[74]

DC: Dendritic cell; IE: Immediate early; L: Latency; MRP1: Multidrug resistance associated protein-1.

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utilized as a therapeutic tool against latent HCMV reservoirs. HCMV latency signature

Although not all the potential latent sites in vivo are well characterized, monocytes appear to be one of the predominant cells where HCMV establishes latency in vivo [23] . HCMV has been recovered from monocytes isolated from healthy donors upon reactivation [20,80–81] . In addition to monocytes, latent HCMV has been detected in granulocyte-macrophage progenitor cells [82] and myeloid progenitor cells [22,83] . The differentiation of CD14+ or CD34+ cells into macrophages or dendritic cells can shift latent infection into lytic phase [22,84–87] . CD14+ or CD34+ cells have been widely used as a model for the HCMV latency establishment and reactivation [16,88–90] . HCMV has been shown to productively replicate (with hallmark of latency infection) in CD34+/CD38- (primitive hematopoietic cell) [91] . On the other hand, in mature CD34+/c-kit+ cells HCMV infection leads to the expression of limited sets of proteins [91] . Role of cellular environment in regulating latency has also been postulated [91] .

Review

Most of the information regarding HCMV biology has been based on the laboratory strains. These laboratory strains have been extensively passaged in fibroblasts. Data indicate that extensive passaging of HCMV in fibroblasts lead to the deletion of 13–15 kb of genome which is otherwise present in the low passage or clinical strains. This deleted region called ‘ULb’ region plays important role in immune invasion and favors the establishment of latency [85] . UL138 is one of the ORF within the ‘ULb’ region [85] . Goodrum et al. demonstrate the requirement of UL138 ORF for latent infection in CD34+ cells [85] . In addition, recently the role of pUL138 protein in favoring latency in HPCs has been shown [92] . Petrucelli and colleagues observed only partial loss of latency-associated characteristics in HPCs infected with recombinant HCMV (HCMVUL138Stop and UL138-null). Their data further indicate the requirement of other UL138 transcripts in favoring latency in HPCs. Additionally, pUL138 localizes in Golgi bodies of the infected cells and does not downregulate the expression of gene from MIEP locus [92] . Role of pUL138 in establishing latency seems to be cell-type specific. For instance, in endothelial cells UL133-UL138 locus is required for the efficient repliHCMV

cIL-10

Increased cell survival

Genome circulazation Host chromosomes

* * * HCMV

GATA-2

-2 TA GA

Ribosomes

hsa-miR-92a reduced expression in latency

* * * Latent HCMV

HDAC/HMTs/ PRC2

cIL-10

miRNAmediated inhibition hsa-miR-92a

Figure 3. Role of host-encoded microRNA (hsa-miR-92a) in regulation of human cytomegalovirus latency. Expression of hsa-miR-92a is downregulated upon induction of HCMV latency in CD34+ HPCs. One of the cellular targets of hsa-miR-92a is GATA-2 (an activator of cIL-10). Downregulation of hsa-miR-92a expression ultimately results in the increased expression of cIL-10 and increased cell survival [72] . HCMV: Human cytomegalovirus; HPC: Hematopoietic progenitor cell.



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Review Kumar & Herbein cation of the virus, whereas in fibroblast this locus is dispensable [75] . Recently, Umashankar and colleagues demonstrated that pUL135 (encoded by ULb′ region) has ability to rescue pUL138-mediated viral latency in fibroblasts and CD34+ HPCs [93] . Their data indicate the presence of a molecular switch (pUL135-pUL138) in the ULb′ region of the virus. Whether such switch also operates in natural infection needs to be addressed [93] . Besides pUL138, the role of pUL133 (a gene product of UL133-UL138 locus) in suppressing HCMV replication in CD34+ HPCs has been shown (Table 1) [75,94] . Recently, Weekes and colleagues using ‘plasma membrane profiling’ coupled with mass spectrometry showed the pUL138-mediated degradation and loss of functionality of a cell surface multidrug resistance associated protein-1 (MRP1) (Figure 1) (Table 1) [74] . Importantly, they also demonstrated the depletion of virus from latent reservoir using cytotoxic substrate for MRP1 [74] . The possibility of using MRP1-specific drugs in selectively eliminating the latent HCMV reservoirs has been suggested. Beside the UL133-UL138 locus encoded proteins, a pro-latency transcript UL81–82 antisense transcript (UL81–82ast, also called LUNA) has been found in the cDNA library of monocytes (Figure 1) . The expression of UL81–82ast has been detected during early infection of fibroblast and vanishes with the transcription of UL82 gene. It seems that UL81–82ast regulates HCMV latency by suppressing the expression of genes from MIEP locus (Table 1) [37,38] . Information regarding the transcriptome of cells undergoing lytic or latent infection has now begun to emerge. Recently, Gatherer and colleagues revealed the transcriptional signature of HCMV-infected cells undergoing lytic cycle using deep sequencing [95] . Similarly, using next generation sequencing transcriptional profile of latently infected CD14+ or CD34+ cells in vitro and CD14+ or CD34+ cells isolated from HCMV seropositive patients [39] have been recently generated. Their data revealed the presence of two long noncoding RNA (RNA 2.7 and RNA 4.9) and mRNA coding for UL44, UL50, UL84, UL87, UL95, UL138 and LUNA in latent infection. Chromatin immunoprecipitation coupled with next generation sequencing showed the interaction of UL44 and UL84 with the latent HCMV genome in CD14+ cells. In addition, the interaction of RNA 4.9 with PRC as well as with MIEP suggests the involvement of RNA 4.9 in inducing latency (Figure 1) [39] . In CD34+ infected cells, in addition to the transcripts observed in latently infected CD14+ cells, US17, UL28/29, UL37/38, UL133/135 and UL114 have been also detected (Figure 1) [39] . Some of these transcripts are characteristically unique to the

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latent infection (e.g., RNA2.7, RNA4.9, UL133/135). On the other hand, other transcripts presence may be due to the leakiness in latent system or remnants of lytic infection. Therefore, further experiments are required to elucidate their actual role in latency. Pathophysiological relevance of HCMV latency HCMV although a neglected virus in immunocompetent host, however, shows its wide spectrum in pathophysiological scenarios where either immunity is immature or suppressed. These pathophysiological conditions include viral infection (e.g., HIV, EBV and HHV-6), congenital infection, HCMV infection in organ-transplant patients and cancer. In these cases, HCMV infection can be life-threatening upon reactivation. Therefore, it is utmost important to understand how HCMV reactivates in the above-mentioned scenario. On the basis of variation in viral glycoprotein, HCMV has been classified into several genotypes [96,97] . More than 50 glycoproteins have been identified in HCMV strains [97,98] . Several studies have been performed to find a correlation between HCMV genotypes and its implication in latency and pathogenicity. For instance, we studied the occurrence of HCMV genotypes based on glycoprotein B (gB) in immunocompromised patients. We found that the severity and morbidity was associated with mixed gB genotype infection rather than a single gB genotype infection [99] . Similar findings suggesting the putative role of mixed genotype infection in reactivation of HCMV in immunocompromised patients have been reported [100] . Several attempts have been made to find any association of a specific HCMV genotype with HCMV reactivation in immunocompromised patients, hematopoietic stem cell transplantation (HSCT) recipients and congenital infections [77,101–103] . However, so far no correlation has been found. HCMV congenital infection

HCMV infection is one of the frequent congenital viral infections responsible for morbidity and mortality in newborns [104] . Besides deafness and serious neurological disorders, HCMV may also cause hepatitis, thrombocytopenia and pneumonitis in congenitally infected newborns [2,105] . Outcome of congenital viral infection has been found to be associated with the time of infection of mother and newborn [2,77] . Interestingly, AravBoger and colleagues developed an artificial neural network (ANN) to determine the outcome of congenital HCMV infection based on the different HCMV sequences (UL144, UL146, UL147 and US28) isolated



from mothers and infants which display different congenital outcomes. UL144 codes for a truncated tumor necrosis factor receptor. UL144 polymorphism has been associated with the congenital HCMV outcome [77] . UL146 and UL147 encode for α-chemokines. In conventional phylogenetic analysis, UL146 and UL147 do not bear any relationship with the disease outcome. However, upon feeding the sequence information in ANN they found UL144 and UL146 genes as the most reliable sequences in predicting the severity of congenital infection [105] . Similarly, the expression of latency and lytic-infection-associated genes can be determined and robust ANN can be developed which may give an idea of whether HCMV-induced congenital damages are the outcome of lytic infection or reactivation of latent infection. Notably, MCMV infection in newborn mice (MCMV congenital animal model) displays high viral titer and productive infection in most organs of the mice. In addition, lytic infection in the brain parenchyma during early post-natal phase has been associated with severe neurological damages in mice [106] . Recently, Belzile and colleagues used the primitive pre-rosette neural stem cells (pNSCs) as model for fetal nervous system and studied the impact of HCMV infection in pNSCs [107] . They observed nonprogressive replication of HCMV in pNSCs; however, upon differentiation into neurons viral replication became progressive. Despite the abortive infection of HCMV in pNSCs, they were able to detect the genome of HCMV up to 1 month in pNSCs cultures. Their data suggest primitive neural stem cells as a site of viral latency [107] . The mechanism responsible for HCMV neuron-specific latency can provide us therapeutic strategies for keeping latent HCMV silent for longer period of time. HCMV reactivation in transplant recipients

HCMV infection in transplant recipients including solid organ transplantation (SOT) and HSCT is one of the frequent causes of virus-induced allograft rejection. In addition to allograft rejection, other associated morbidities include decreased graft survival age and increased in susceptibility to various opportunistic pathogens [108] . Severity of the outcomes of HCMV infection in SOT depends upon the sero-status of the donor and recipient [16] . The highest morbidity is associated in cases where donor is seropositive (D+) and recipient is sero-negative (R-) [108–110] . Due to the lack of feasible animal model for HCMV [111] , most of the data elucidating the reactivation of HCMV in transplant recipients have been derived from the MCMV animal model. HCMV and MCMV exhibit several similarities in terms of genomic organization, establishment of latency and reactivation.


Review

Therefore, causes of mortality and morbidity in D+/Rhave been tested in murine model. Allogeneic kidney transplant induces the expression of MCMV IE genes in murine model [112] . The correlation between induction of IE genes and increase in the expression of TNFα, interleukin-2 and activation of NF-kB has been also established [112] . Interestingly, Zhang and colleagues used an alternative animal model to study HCMV reactivation in response to transplantation. They used TNF-receptordeficient transgenic mice having beta-galactosidase gene driven by the HCMV (MIEP) promoter. They observed the activation of MIEP in both allogeneic as well as syngeneic transplantation cases without the involvement of TNF-α [113] . Taken together, data suggest the possibilities of several independent pathways leading to the reactivation of HCMV in SOTs. Besides SOTs, reactivation of HCMV has been associated with severe morbidity in patients undergoing HSCT [114–116] . The donor age and immunological parameters also play an important role in the pathogenesis of HCMV infection [117,118] . Importantly, antiHCMV drugs CMX001 has been shown to reduce the incidence of HCMV infection in HSCT recipients [119] . However, the precise mechanisms of reactivation of HCMV in SOT and HSCT are poorly understood and need further investigations. HCMV reactivation & viral coinfections

HCMV upon reactivation in HIV infected individuals can cause retinitis, neurological damage, gastrointestinal problems, hepatitis, pneumonitis and adrenalitis [120,121] . How HIV infection promotes HCMV reactivation is not completely understood. At least neither direct nor indirect involvement of HIV transcripts or gene products has been shown in reactivating latent HCMV. HIV infection notably changes the expression profile of several cytokines including TNF-α [122] . In addition, the role of TNF-α has been shown in reactivating latent HCMV by nuclear localization of NF-κB which in turn binds to the NF-κB sites present in core and enhancer region of MIEP (Figure 2) [17] . These findings have been also shown in MCMV [123] . Recent work from Hengel and Hagemeier laboratories demonstrated the role of HCMVlatency-associated protein pUL138 in upregulating the expression of TNFR1 on the surface of infected cells that could make latent cells hypersensitive to TNF-α [124,125] . In addition, the comparative cytokine profile of aqueous humor of AIDS patients (with or without HCMV) revealed the upregulation of several factors including G-CSF, fractalkine, PDGF-AA, MCP-1 and Flt-3L [126] . Not surprisingly, the role of G-CSF has been shown in reactivating HCMV in a humanized


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Review Kumar & Herbein mice model [127] . Taken together, these data indicate the important role of cytokines in regulating HCMV latency in HIV-HCMV coinfected individuals. In addition to HIV, a few studies suggest the role of HCMV in reactivation of EBV in vitro as well as in vivo [128–130] . Similarly, fewer studies examine the impact of EBV infection in reactivation of HCMV. Strikingly, Barton and colleagues showed that latent murine gammaherpesvirus 68 (MHV-68)

or MCMV confers resistance to bacterial pathogens Listeria monocytogenes and Yersinia pestis in murine model. MHV-68 and MCMV are genetically similar to the EBV and HCMV, respectively. In addition, the mechanism of activation was found to be systemic activation of macrophages and prolonged secretion of INF-γ [131] . The dual latency of HCMV and EBV during childhood has been suggested to contribute to the expansion of NKG2C (+) natural killer cells

Executive summary Epigenetic mechanisms involved in human cytomegalovirus latency • Latency is one of the sophisticated viral strategies which favor persistence. • Besides monocytes, human cytomegalovirus (HCMV) establishes latency in early progenitor cells including granulocyte-macrophage progenitor cells and myeloid progenitor cells. • Expression of immediate early (IE) proteins from major IE enhancer/promoter locus is one of the hallmarks of lytic infection. • In latent infection, major IE enhancer/promoter locus is repressed by heterochromatin formation due to the action of enzymes involved in chromatin remodeling including methyltransferases and histone deacetylases. • In addition, polycomb repressive complex 2 plays an important role in the maintenance of HCMV latency.

Role of microRNA in HCMV latency • Cellular and viral microRNA also participate in the establishment and maintenance of viral latency. • More than 15 microRNAs are known to be encoded by HCMV. • Role of viral-encoded miR-UL112–1, miR-US25–1, miR-US25–2 and miR-US33 have been postulated in regulating latent infection. • In addition, host encoded hsa-miR-92a favors lytic infection. • For better understanding of the link between epigenetic machinery and microRNA, extensive search for HCMV-encoded ‘epi-microRNA’ is required.

HCMV latency signature • A ∼5 kb segment of ULb′ unique to low-passage HCMV strains may be required for the establishment of latency. • A limited number of latency associated viral transcripts have been identified including UL81–82 antisense (LUNA), UL138 and UL133. • Loss of MRP1 is governed by pUL138. Use of MRP1 cytotoxic substrates has been suggested in eliminating latent HCMV reservoirs. • In CD14+ cells, several latency-specific transcripts have been identified including long noncoding RNA (RNA2.7, RNA4.9) and mRNA coding for UL144, UL44, UL50, UL84, UL87, UL95, UL138 and LUNA. • In addition, in CD34+ cells beside transcripts identified in CD14+ cells, mRNA coding for US17, UL28/29, UL37/38, UL133/135 and UL114 have been also found.

Pathophysiology relevance of HCMV latency • HCMV congenital infection is one of the most frequent infections in newborns and may lead to loss of vision or hearing and several other neurological disorders. • Artificial neural network has been developed, which can suggest the outcome of congenital infection based on sequences of UL144 and UL146 isolated from mother and newborn. • Primitive neural stem cells have been suggested as a site for HCMV latency. • In addition, HCMV reactivation in solid organ transplantation, hematopoietic stem cell transplantation and umbilical cord blood transplantation have serious complications. Increased expressions of cytokines like TNF-α and IL-2 have been reported in transplant recipients, which may promote NF-κB nuclear localization and ultimately induction of IE genes. Similar observations have been made in HCMV reactivation in viral infections including HIV. • Exact mechanism of HCMV reactivation is poorly understood and needs extensive investigations. • Role of HCMV has been postulated in initiating or promoting tumor growth. • In contrast, HCMV can promote the expansion of natural killer cells which modulates tumor development in transplant recipients. • Information derived from reactivation of HCMV should be utilized in developing specific therapeutics for eliminating the latent HCMV reservoir. • This could improve the life of patients undergoing transplant or having other viral infections.

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[132] . However, whether such symbiotic relationship also exists in the situation of EBV and HCMV coinfected immunocompromised cases requires further investigations. Another member of Herpesviridae, human herpesvirus-6 (HHV-6) reactivation has been associated with the HCMV infection in transplant cases [133–135] . The coinfection of HCMV and HHV-6 often results in severe morbidity in SOTs [136,137] . Prospective studies are required to investigate the influence of other herpesvirus on reactivation of HCMV in patients with clinical complications.

HCMV & cancer

In the recent past, several studies indicate the presence of HCMV antigen and nucleic acid in cancerous tissue [138–143] . There is ongoing debate on the issue of oncogenicity of HCMV. HCMV gene products such as US28 have been reported to induce tumor formation in murine models [144] . For instance, we and others observed increased HCMV seroprevalence and detection of HCMV antigens and DNA in several tissues of cancer patients [141,143] . In multiple instances, several reports indicate the prevalence of HCMV gene products in tumor tissue and peripheral blood of glioblastoma patients [138–139,145–147] . However, contrasting findings are also available [148] . Besides the suggestive role of HCMV in initiating and promoting cancer, several contrasting but interesting studies indicate the role of HCMV in the relapse of cancer [149] . A recent study suggests that the exposure of HCMV in both pre- and post-renal transplantation can increase the risk of cancer development [150] . Notably, NK cells are the important players in controlling viral infection and tumor growth [151] . Recent studies have shown the role of HCMV reactivation in promoting the expansion of NK cells expressing NKG2C after SOT [152] , umbilical cord blood transplantation [153,154] and HSCT [155] . To what extent NK cells can control reactivation of References

HCMV in transplantation recipients and responsible mechanism needs further investigations [156] . Taken together the role of HCMV in cancer progression and relapse is a highly volatile issue and exchange of information and technologies between laboratories with contrasting results is necessary to derive some significant conclusions. Conclusion & future prospective Regulation of HCMV latency is an important area of ongoing research. The complex interplay of host machinery and viral factors involved modulating the latent and lytic phases of HCMV have begun to emerge. In addition, the lack of reliable animal models and latent HCMV cell model lines make us to rely upon the information for parallel resources. Hence emphasis should be given in developing novel humanized animal models to study HCMV biology. Furthermore, the reactivation of HCMV in various pathophysiological scenarios presents HCMV in its gravest form. Therefore, concrete information derived from the epigenetic regulation of HCMV latency should be used to develop specific strategies to control the reactivation and elimination of latent HCMV reservoirs. Financial & competing interests disclosure This work was supported by grants from the University of Franche-Comté (UFC), the Région Franche-Comté (RECHFON12–000013) and Europe (FEDER, Fonds Européens de Développement Régional N°2014–0045) to Georges Herbein. Amit Kumar is a recipient of a postdoctoral fellowship of the Région Franche-Comté (N° 2012C-06102). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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