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Modulation of the NFκb Signalling Pathway by Human Cytomegalovirus

Meaghan H Hancock* and Jay A Nelson

Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, USA

*Corresponding Author:
Meaghan H. Hancock
Vaccine and Gene Therapy Institute
Oregon Health and Science University, Beaverton, Oregon, USA
Tel: 503-418-2784
E-mail: [email protected]

Received Date: July 18, 2017; Accepted Date: July 18, 2017; Published Date: July 31, 2017

Citation: Hancock HM, Nelson JA (2017) Modulation of the Nf-kb Signalling Pathway by Human Cytomegalovirus. Virol Curr Res 1:104.

Copyright: © 2017 Hancock MH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Many viruses trigger innate and adaptive immune responses and must circumvent the negative consequences to successfully establish infection in their hosts. Human Cytomegalovirus (HCMV) is no exception, and devotes a significant portion of its coding capacity to genes involved in immune evasion. Activation of the NFκB signalling pathway by viral binding and entry results in induction of antiviral and pro-inflammatory genes that have significant negative effects on HCMV infection. However, NFκB signalling stimulates transcription from the Major Immediate Early Promoter (MIEP) and pro-inflammatory signalling is crucial for cellular differentiation and viral reactivation from latency. Accordingly, HCMV encodes proteins that act to both stimulate and inhibit the NFκB signalling pathway. In this Review we will highlight the complex interactions between HCMV and NFκB, discussing the known agonists and antagonists encoded by the virus and suggest why manipulation of the pathway may be critical for both lytic and latent infections.


NFkB signalling pathway; Human cytomegalovirus; HCMV lifecycle

Viruses and the NFkB Signalling Pathway

The innate immune response to virus infection results in activation of the NFkB transcription factors, which regulate a vast array of antiviral and pro-inflammatory effector functions. Viruses often trigger the NFkB signalling pathway either through activation of Pattern Recognition Receptors (PRRs) or in response to membrane fusion events. In order to successfully establish an infection viruses encode genes to subvert or utilize this ubiquitous signalling pathway to their own advantage [1]. Some viruses, such as Human Immunodeficiency Virus (HIV) and Herpes Simplex Virus (HSV) utilize NFkB signalling to stimulate viral gene expression [2,3]. Oncogenic gammaherpesviruses like Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) and Epstein Barr Virus (EBV) encode proteins that activate NFkB signalling in order to utilize pro-survival signals during latency [4,5]. More commonly, viruses inhibit the NFkB signalling pathway using a diverse array of strategies [1,6]. Many RNA and DNA viruses target the PRRs and their adaptors either via downregulation or blocking their activities [7-10]. Others target downstream components of the signalling pathway [11-14] or the NFkB subunits themselves [15-18]. While strategies for manipulation of the NFkB signalling pathway using viral proteins are diverse, new approaches, most recently using viral non-coding RNAs [19-23], are regularly being discovered. NFkB signalling is a paradigm for the principles of signal transduction and transcriptional activation. Transcriptional regulation is mediated by the NFkB subunits (the transcriptional activators p65/RelA, RelB and c-Rel and the DNA binding proteins p105/p50 and p100/p52), which are abundant, potent, broadly expressed and modulate numerous important cellular functions allowing the cell to respond and adapt to environmental changes. Activation of the NFkB subunits requires phosphorylation- induced ubiquitination and proteasomal degradation of the inhibitor of NFkB proteins (most commonly IkBα, IkBβ and IkBε) that retain the NFkB subunits in the cytosol. For example,phosphorylation on the Ser32 and Ser36 residues results in degradation of IkBα via the 26S proteasome and releases the NFkB subunits to transit to the nucleus, homo- and heterodimerize and bind specific kB binding sites in the promoters of regulated genes. Canonical NFkB signalling is initiated by ligand binding to upstream cell surface receptors (including IL1β, TNFα and TLR receptors), which transduce these extracellular signals via activation of both kinases and ubiquitin ligases. Multiple upstream signalling pathways converge at the IkB kinase (IKK) complex composed of the catalytic subunits IKKα and IKKβ and the structural component IKKγ (or NEMO). Linear ubiquitination of NEMO assembles the IKK complex and activation is the result of phosphorylation of IKKα or IKKβ on serine residues in their activation loops either by upstream kinases or through trans-autophosphorylation. The activated IKK complex plays a critical role by phosphorylating the IkBs and thus activation of this complex is a highly regulated step in the NFkB signalling cascade [24]. In contrast, the non-canonical NFkB signalling pathway is induced by lymphotoxin B, B Cell Activating Factor (BAFF) or CD40 ligand and results in phosphorylation of IKKα dimers by the Nfkb Inducing Kinase (NIK). Stimulation of the non-canonical NFkB signalling pathway results in the release of RelB and p52 heterodimers [25]. Termination of the NFkB response is complex and occurs in part through a negative feedback loop resulting in NFkB-dependent expression of the IkB proteins. Newly synthesized IkB relocalizes the NFkB subunits from the DNA to the cytosol thus resulting in a selflimiting inflammatory response.

Human Cytomegalovirus Modulation of the NFkB Signalling Pathway

Herpesviruses have co-evolved with their hosts over millions of years in order to succeed in establishing a life-long infection in the face of constant immune surveillance. In order to persist for the lifetime of the host, herpesviruses have evolved myriad strategies to utilize and evade the host innate and adaptive immune responses. Human cytomegalovirus (HCMV/HHV-5) is a member of the beta herpesvirus family with high prevalence in the human population; in the United States 50-90% of adults are seropositive and seropositivity is closer to 100% in developing countries [26]. While HCMV infection is generally subclinical in healthy individuals, serious disease can arise when the host immune system is compromised and viral reactivation occurs. HCMV replicates in numerous cell types including macrophages, dendritic cells, fibroblasts, epithelial and endothelial cells as well as smooth muscle cells, neuronal cells, hepatocytes and trophoblasts. In these cell types, HCMV undergoes a lytic replication cycle involving viral binding and entry of the capsid into the cytoplasm releasing tegument proteins that act to immediately disarm intrinsic cellular immuneresponses. After injection of the viral DNA into the nucleus, cellular transcriptional trans activators act to stimulate transcription from the Major Immediate Early Promoter (MIEP), which results in the transcription of multiple Immediate Early (IE) genes including the major isoforms IE protein 72 (IE72/IE1) and IE86/ IE2. Expression of IE1 and IE2 is critical for the efficient launch of the lytic replication cycle [27,28]. The MIEP enhancer region is highly complex, containing an array of positive and negative cis-acting elements including binding sites for numerous cellular transcription factors suchas CREB/ATF, AP-1, Elk-1, SRF and NFkB [29]. These cisactingelements work both cooperatively and independently to initiate RNA polymerase II transcription from the MIEP thus ensuring activation of the promoter by a variety of cellular signalling pathways regardless of the differentiation and activation state of the cell. IE proteins help to stimulate expression of Early (E) phase proteins, many of which are involved in DNA replication. E proteins also help to stimulate Late (L) gene expression, whose products are involved in virion assembly and release. HCMV replicates poorly in less differentiated cell types such as CD14+ monocytes and CD34+ Hematopoietic Progenitor Cells (HPCs). In these cells most viral genes are not expressed and the viral genome is maintained in the absence of progeny virus production. The limited viral proteins and non-coding RNAs expressed during latency play important roles in suppressing viral gene expression and regulating intracellular signalling pathways [30]. To uncover how HCMV successfully evades host innate and adaptive immunity in such a diverse array of cell types and during fundamentally disparate lifecycles an understanding of the role of both viral proteins and non-coding RNAs in manipulating cellular signal transduction pathways is required. The role of NFkB signalling in the HCMV lifecycle is exceedingly complex and evidence suggests that the virus activates both canonical and non-canonical signalling pathways. In turn, HCMV encodes both agonists and antagonists of NFkB signalling in order to aid in viral replication and dissemination, establishment of latency and reactivation. Early work examining regulation of the MIEP identified multiple 18 nucleotide repeats within the MIEP enhancer region containing consensus NFkB binding sites [31-33]. It was postulated that induction of the NFkB signalling pathway at early times after infection could enhance expression from the MIEP and thus help initiate the lytic cascade of gene expression [32,34,35]. It was shown that TNFα, a potent inducer of the NFkB signalling pathway, enhances expression from the MIEP via increased binding of p50 and p65 to the 18 nucleotide repeat [36]. In fact, later work demonstrated that activation of the NFkB signalling cascade is initiated by viral binding [35,37] mediated by gB and gH interacting with their cognate receptors in human fibroblasts [38,39] and monocytes [40] at least in part via interactions with TLR2 [41,42]. The signalling initiated by viral binding results in depletion of preformed cytosolic stores of p50 and p65. Subsequently, de novo synthesis of p50 and p65 occurs through a positive feedback signalling [34] and transactivation by IE proteins [37] involving regulation of the SP1 transcription factor [43]. In addition, Casein Kinase II (CKII) packaged in the virion has been proposed to rapidly phosphorylate IkBα following viral entry, allowing for an additional means of releasing the NFkB subunits which may be necessary for infection of diverse cell types [44]. Interestingly, studies of NFkB activation in primary Monocyte-Derived Macrophages (MDMs) determined that although canonical p50/p65 heterodimers are present at the MIEP very early after viral infection [40,45], complexes composed of p52 and Bcl-3 are found at the MIEP at 5 days post-infection, suggesting context dependent changes in NFkB signalling in different cellular environments [45]. Similar stimuli are known to activate distinct NFkB complexes in cell-type dependent manners [46,47], but how and why the non-canonical NFkB signalling pathway is activated in MDMs remains unclear. p52/Bcl-3 heterodimers are not as efficient at stimulating expression from MIEP reporter constructs [45]; therefore one possibility is that non-canonical NFkB signalling may act to limit MIEP expression in MDMs. This early work clearly indicated that viral binding and entry induces activation of NFkB signalling and results in expression from the MIEP. However, the MIEP contains numerous binding sites for additional cellular transcriptional activators and repressors and thus the relative importance of NFkB in the overall stimulation of the MIEP and ultimately virus replication was unclear. Additionally, activation of NFkB signalling results in induction of numerous cellular genes, including cell adhesion molecules, complement and acute phase proteins as well as pro-inflammatory cytokines and chemokines which can have antiviral effects on HCMV replication. Thus, the contribution of the NFkB signalling pathway to full viral replication has been studied extensively in vitro - with conflicting results. Growth curves of AD169 and Toledo HCMV strains in human fibroblasts overexpressing a Dominant Negative (DN) mutant of IkBα, suggested that blocking NFkB signalling in fibroblasts was neutral to viral replication [48]. Additionally, when an NFkB site-mutated HCMV MIEP replaces its MCMV counterpart in the MCMV genome the resulting virus replicates with Wild Type (WT) kinetics in fibroblasts [48]. In contrast, using pharmacological inhibition of the NFkB pathway, as well as the IkBα DN mutant, it was suggested that blocking NFkB signalling resulted in a modest increase in AD169 replication, and prevented exogenous TNFα and IFNγ from negatively affecting virus replication [49]. In addition, this study utilized a constitutively active mutant of IKKβ and showed that constitutive activation of canonical NFkB signalling inhibited viral replication through the production of IFNβ. In order to directly test the requirement of NFkB signalling in regulation of the MIEP during viral infection, Gustems et al. [50] constructed an HCMV AD169 mutant containing pointmutations in all 4 NFkB binding sites within the MIEP and showed no deleterious effects on IE expression or viral replication in human fibroblasts. This work indicated that in the context of lytic AD169 infection of fibroblasts, transactivation of the MIEP can be accomplished through the additional transcription factor binding sites found within the enhancer region [29]. In fact, our work and that of others (unpublished observations, [51,52]) suggest that AD169 does not trigger or modulate the NFkB signalling pathway in the same manner as clinical strains of HCMV and may account for the relative resistance of AD169 replication to inhibition of the NFkB signalling pathway. In contrast to the studies described above, work by several groups [53-59], using both AD169 and clinical strains of HCMV and various NFkB inhibitors as well as DN IkBα, IKKα and IKKβ constructs demonstrate that IE and subsequent gene expression as well as viral yields are reduced when NFkB signalling is blocked in fibroblasts and endothelial cells. Intriguingly, expression of the IkBα DN protein had the greatest deleterious effect on MIEP transactivation compared to DN IKKα and IKKβ constructs [59]. These observations suggest that there are multiple signalling pathways activated by HCMV infection that converge at the phosphorylation of IkBα, some of which do not include activation of the IKK complex, such as direct phosphorylation of IkBα by tegument-associated CKII [44]. These studies also indicated that IKKα plays a more important role in MIEP transaction than IKKβ [59] and hints at the involvement of the noncanonical NFkB signalling pathway in fibroblasts as has been observed in MDMs [45]. Interestingly, when the later phase of NFkB signalling that occurs as a result of IE1 transactivation of the p50 and p65 promoters [37] was blocked by addition of pharmacological inhibitors, viral replication was still impaired [58], suggesting an essential role for sustained NFkB signalling during HCMV infection. The apparently contradictory observations about the importance of NFkB signalling during viral infection could be at least partially resolved by studies which examined the role of NFkB signalling in replicating and growth arrested cells [55]. Using DN IKKβ constructs and viruses lacking the NFkB target sequences within the MIEP the authors demonstrate that virus replication is only restricted in growth arrested, and not proliferating fibroblasts and endothelial cells. These data suggest that the differentiation and activation state of the infected cell plays a significant role in NFkB-mediated MIEP transactivation and lytic replication. Further experimentation to address the contradictory requirement of NFkB signalling to the HCMV lifecycle is required to resolve this essential question. Finally, the role of NFkB signalling in regulating gene expression at other stages of the HCMV lifecycle has not been thoroughly investigated. US3 contains NFkB binding sites [60,61] that may contribute to the requirement of NFkB at later times in the infection cycle and additional kB binding sites exist within the HCMV genome [55]. Whether NFkB signalling and transactivation of the MIEP is essential to virus replication both in vitro and in vivo remains an ongoing question, but microarray data indicates that expression of NFkB-inducible genes is more robust when viral gene expression is inhibited [62], suggesting that some viral gene products act to dampen the NFkB response. It was first reported that different lab-adapted and clinical strains of HCMV could block signalling through the canonical NFkB pathway initiated by IL1β or TNFα at or above the point of convergence of the NFkB signalling pathways [63,64]. IkBα phosphorylation and degradation was abrogated and expression of several pro- inflammatory cytokines was prevented in infected fibroblasts and endothelial cells treated with IL1β or TNFα after 72 h of infection [63,64]. Similarly, phosphorylation and degradation of IkBα was not detected at 5 days post-infection in MDMs [45]. In fact, IkBα transcript [40] and protein levels [45] are increased during infection of MDMs, suggesting that canonical NFkB signalling is also actively blocked in this cell type at later times of infection [65]. The antagonism of NFkB signalling requires expression of both early [64] and late gene products [63,64]. Interestingly, when infected cells are treated with IL1β a near-complete block in IkBα degradation is observed, while treatment of infected cells with TNFα resulted in residual IkBα phosphorylation and degradation, suggesting that HCMV antagonism of the NFkB signalling pathway is dependent upon which upstream signalling pathway triggers IkBα phosphorylation [63]. Using AD169 mutants the ability to block TNFα-mediated NFkB signalling could be genetically separated from blocking IL1β-mediated signalling [64]. To date, the viral gene product(s) necessary for this late block in NFkB signalling have not been identified, but several gene products have been implicated in interfering with the NFkB signalling pathway.

HCMV-Encoded Antagonists of the NFkB Signalling Pathways

Illustrates the HCMV proteins and non-coding RNAs that interfere with the NFkB signalling pathway (Figure 1). Viral proteins involved in blocking NFkB signalling.


Figure 1: HCMV-encoded antagonists of the NFkB signalling pathway. NFkB signalling can be induced by activation of a variety of cell surface receptors as well as HCMV binding and entry. Upstream signalling cascades culminate at the activation of the IKK complex. Several HCMV proteins and miRNAs (shown in red) block activation of the IKK complex or downstream binding of the NFkB transcription factors to their cognate sequences.

The tegument protein Pp65 was the first HCMV protein shown to interfere with NFkB signalling [66]. Using DNA arrays, it was demonstrated that Pp65-deficient viruses induced anti-viral and proinflammatory genes to a greater extent than WT virus and exogenous expression of Pp65 could block type I IFN signalling. Pp65-deficient viruses induce NFkB subunit binding to a greater extent than WT, but have no effect on IRF3 binding, suggesting that Pp65 interferes specifically with the NFkB signalling pathway. The immediate early protein IE86 also blocks NFkB signalling in infected cells [67-69]. IE86 attenuates the production of IFNβ during HCMV infection either by preventing NFkB subunit binding to the IFN promoter [68] or by blocking interactions between the subunits and other transcriptional activators [70]. In addition, expression of IE86 blocks NFkBdependent gene expression in response to external stimuli, such as Sendai virus and TNFα treatment indicating that IE86 alone is sufficient to block NFkB signalling [67]. These studies examined the effects of IE86 in isolation or at early times post-infection, well before the late block to NFkB signalling observed in studies by Jarvis et al. [63] and Montag et al. [64]. Thus HCMV likely encodes multiple gene products from different kinetic classes that block NFkB signalling. It remains an intriguing question as to why HCMV encodes an inhibitor of canonical NFkB signalling that is expressed with Ikinetics when the MIEP is transactivated by NFkB subunit binding. Perhaps this is a mechanism of negative feedback utilized by the virus to prevent overactivation of NFkB signalling and pro-inflammatory cytokine production, given the functional redundancy of transcription factor binding to the MIEP. HCMV cmv-IL-10 (UL111a) is a functional homolog of cellular IL-10, itself a potent inhibitor of pro-inflammatory responses. Like cellular IL-10, recombinant cmv-IL-10 treatment of THP-1 cells can block NFkB signalling at or above the level of IkBα degradation, although the exact mechanism for the inhibition has not been further elucidated [71]. The tegument protein UL26 has most recently been demonstrated to possess NFkB inhibiting functions [52]. Expression of UL26 can block TNFα and Sendai-virus-induced IKK activation, IkBα degradation and IL6 production, suggesting that it functions at or above the point of convergence of multiple NFkB signalling pathways and may contribute to the late block in NFkB signalling observed in HCMV-infected cells [63,64]. An UL26-mutant virus induces canonical NFkB signalling with similar kinetics to WT infection, suggesting tegument-associated UL26 does not block early induction of the pathway. Interestingly, the UL26 mutant virus induces higher expression of the RelB NFkB subunit, especially at later time of infection, suggesting that UL26 may play a role in suppressing noncanonical NFkB signalling.

HCMV Non-coding RNAs Involved in Blocking NFkB Signalling

Along with viral proteins, HCMV also expresses non-coding RNAs that interfere with different aspects of NFkB signalling. MicroRNAs (miRNAs) are small, ~22 nucleotide RNAs that act to posttranscriptionally regulate gene expression. miRNAs normally interact with short regions of complementarity in the 3’ UTR of targeted transcripts which results in recruitment of cellular protein complexes that ultimately lead to translations repression and/or mRNA degradation [72]. Thus, by targeting regions of complementarity in genes involved in the NFkB signalling pathway, HCMV miRNAs could participate in the late block to NFkB signalling observed in HCMV infected cells [63,64]. In fact, most HCMV miRNAs are expressed with early kinetics, accumulate throughout the course of lytic infection [73,74] and are abundant at the late stages of infection. Additionally, several HCMV miRNAs are expressed during latency in CD34+ HPCs [75] and could act to modulate NFkB signalling when most viral proteins are no longer expressed. HCMV miR-sUS5-1 and miRUL112- 3p have recently been demonstrated to block NFkB signalling induced by IL1β and TNFα at late times post-infection [20]. Both miRNAs target IKKα and IKKβ, limit the phosphorylation and degradation of IkBα and attenuate the downstream expression of the pro-inflammatory cytokines RANTES, IL6 and TNFα in fibroblasts, endothelial cells and THP-1 cells. Infection of cells with an HCMV TB40/E mutant lacking expression of miR-US5-1 and miR-UL112-3p results in higher levels of IKKα and IKKβ proteins compared to WTinfected cells, allows for partial IkBα degradation following exogenous IL1β or TNFα treatment and increased secretion of pro-inflammatory cytokines compared to WT infected cells. By replacing the miRNA sequences with shRNAs targeting IKKα and IKKβ, the expression and secretion of pro- inflammatory cytokines could be reduced to WT levels, indicating that the mutant phenotype was due to the loss of IKK complex targeting [20]. In addition, miR-UL112- 3p also targets the TLR2 receptor, thereby blocking TLR2-induced IRAK1 activation and subsequent expression of pro-inflammatory cytokines [21]. Given that TLR2 signalling results in activation of the IKK complex, it is likely that at least some of the observed effects of miR-UL112-3p on proinflammatory cytokine expression is also due to its effects on IKKα and IKKβ expression [20]. miR-US5-1 and miR-UL112 also work in concert with a third HCMV miRNA, miR-US5-2, to interfere with the endocytic recycling compartment and severely attenuate the secretion of pro-inflammatory cytokines [76]. Additionally, miR-UL112-3p may target IL-32, an inducer of NFkB signalling [77]. Finally, HCMV miRUL148D targets RANTES [78] and ACVR1B of the activin signalling axis which promotes increased IL6 secretion upon activin stimulation [75]. These studies underscore how HCMV miRNAs can interfere with NFkB signalling at numerous steps to limit the deleterious effects of pro-inflammatory cytokine production.

HCMV-encoded Agonists of NFkB Signalling PathwaysParadoxically, while encoding numerous proteins and non-coding RNAs that block NFkB signalling in fibroblasts, endothelial cells and monocytes, HCMV also encodes several agonists of NFkB signalling. It has long been postulated that certain NFkB- responsive genes and the effects of activation of the NFkB signalling pathway could also be beneficial to viral replication and spread, especially in vivo [79]. Proinflammatory cytokines and chemokines recruit cells to the site of lytic infection that can be used for dissemination and seeding new viral infections [80]. Additionally, anti-apoptotic genes induced by NFkB signalling may help to prolong the life of the cell for efficient virus production [81]. Finally, an intriguing possibility is that HCMV encodes proteins that help to enhance NFkB signalling specifically in latently infected cells in order to augment transactivation of the MIEP to promote reactivation of the virus from latency. Figure 2 highlights the proteins that act to stimulate signalling through the NFkB pathway.


Figure 2: HCMV-encoded agonists of the NFkB signaling pathway. HCMV encodes three cell surface proteins (US28, UL138 and UL144, shown in red) that can activate or enhance NFkB signaling. In addition, HCMV UL76 and IE1 can activate NFkB signaling through unknown mechanisms.

In contrast to the NFkB-inhibiting functions of IE2, IE1 transactivates numerous cellular and viral genes utilizing the NFkB signalling pathway. Although many of its ascribed functions are due to positive feedback on the MIEP, IE1 alone induces NFkB signalling in several cell types [32]. IE1 transactivates the p65 promoter [37,38], IL6 promoter [82], TNFα promoter [83], and the IL8 promoter [84] through the NFkB signalling pathway. Interestingly, it was determined that IE1 selectively induces RelB/p50 subunits rather than the canonical p65/p50 complexes in smooth muscle cells and fibroblasts [85]. UL144 is a transmembrane protein with properties similar to the TNF Receptor (TNFR) family that potently activates the NFkB signalling pathway and expression of the chemokine CCL22 in a TRAF6- and TRIM23-dependent manner [86,87]. In light of the ability of IE86 to block NFkB subunit binding, Poole et al. [88] determined that UL144- mediated activation of CCL22 was insensitive to IE86 expression during infection suggesting that the ability of IE86 to block NFkB subunit binding is promoter- and context-dependent. UL76, a putative endonuclease, induces the NFkB signalling pathway through activation of ATM and the DNA damage response. Activation of ATM ultimately results in the phosphorylation of NEMO leading to p65 translocation to the IL8 promoter, increased IL8 expression and enhancement of HCMV replication [89]. IL8 is an important chemokine for neutrophil attraction, which the authors postulate may be important for viral replication and dissemination [90,91]. US28 is a 7-transmembrane chemokine receptor that activates multiple cellular signalling pathways in ligand-dependent and -independent manners that is expressed during latency in CD34+ HPCs. US28 constitutively activates NFkB signalling utilizing Gq/11 protein-dependent pathways [92]. US28 has been postulated to play a role inactivation of the MIEP through its NFkB signalling activity [93] and activation of the NFkB signalling pathway by US28 has been linked to increased COX2 expression and angiogenesis in endothelial cells [94]. UL138 was described in two reports to enhance TNFR1 expression on the cell surface [95,96]. UL138 physically interacts with TNFR1, prolonging its half-life and signalling capacity [96]. Interestingly, in comparing a UL138 mutant virus to AD169 strains lacking the ULb’ region, additional TNF-regulating factors were postulated [96]. It is possible that during latent infection of CD34+ HPCs, UL138 acts to enhance the TNF- responsiveness of infected cells. Given the importance of TNF signalling to HCMV reactivation [65,97], and the role of NFkB signalling in enhancing MIEP expression [32,33,36,37,59], it is intriguing to postulate that the virus modulates NFkB signalling to regulate reactivation from latency.


While HCMV has evolved to utilize the NFkB signalling pathway to launch its lytic replication cycle it has also had to evolve to control the antiviral responses thus induced. Evidence suggests that NFkB signalling that is tightly controlled by the virus at early times postinfection is beneficial to viral replication. However, the virus has evolved mechanisms to block any strong NFkB signalling induced by extrinsic signals that could be detrimental to viral replication [20,52,63,64]. Moreover, HCMV modulates both canonical and noncanonical NFkB signalling. At early times activation of the canonical pathway predominates [37,38], but evidence of both activation [45,85,87] and suppression [52] of the non-canonical signalling pathway at later times post-infection has also been demonstrated. Activation of the non-canonical NFkB pathways by exogenous stimuli results in IFNβ production [98] suggesting extrinsic activation of noncanonical signalling, like extrinsic activation of canonical signalling [20,52,63,64] can be detrimental to virus replication. The intricate modulation of these different arms of the NFkB pathways may allow HCMV to enhance the pro-viral effects, while limiting the antiviral effects of NFkB signalling. On the surface, the apparently contradictory roles of NFkB signalling during HCMV infection are confusing, but likely underlie the complexity of the HCMV replication cycle in the host. During lytic infection, NFkB signalling is used to enhance MIEP expression and viral replication, prolong the life of the infected cell while aiding in viral dissemination by recruiting additional cell types to the site of infection. During HCMV infection of monocytes, NFkB signalling helps to initiate a differentiation program resulting in a unique macrophage phenotype [99,100]. Additionally, NFkB-mediated up-regulation of ICAM-1 and ICAM-3 is essential for monocyte motility and firm adhesion to endothelial cells [101], a function key to the ability of monocytes to disseminate and seed new viral infections. Interestingly, HCMV-infected MDMs do not basally express high levels of NFkB-dependent cytokines, but can potentiate cytokine expression induced by lipopolysaccharide [102], suggesting that infected MDMs are poised to reactivate virus upon pro-inflammatory cytokine expression. Allogeneic T cell stimulation produces high levels of IL-6, TNFα and IFNγ and results in HCMV reactivation in monocytes from the peripheral blood [97]. Neutralization of TNFα or IFNγ prevents HCMV reactivation, suggesting that a highly inflammatory environment is critical for viral reactivation [65]. Thus, the virus must maintain a careful balancing act to manipulate the outcomes of NFkB activation for its own benefit depending on the cell type infected. The role of NFkB signalling in latent HCMV infection of CD34+ cells has not been investigated. Whether viral binding and entry stimulates NFkB signalling in CD34+ HPCs as it does in other cell types is an intriguing question. NFkB signalling pathway components are transcriptionally upregulated in HPCs protected from FAS-mediated apoptosis [103], suggesting that HCMV-induced NFkB signalling may help protect and prolong the life of infected HPCs [104]. Noncanonical NFkB signalling, which is induced by HCMV infection [45,85,87], has been implicated in CD34+ HPC differentiation towards the myeloid lineage [105]. In addition, TNFα-mediated activation of NFkB signalling in HPCs prevents erythropoiesis [106,107], which is markedly suppressed during HCMV infection of HPCs [108]. NFkB signalling is also critical for CD34+ -derived myeloid DC differentiation and function [109], which may highlight a critical link between NFkB signalling, myeloid differentiation and viral reactivation. UL138 and US28, two viral gene products essential for latency in CD34+ HPCs [110,111], stimulate the NFkB signalling pathway and thus may play a role in both transactivation of the MIEP and cellular differentiation in order to promote reactivation. HCMV miRNAs are also expressed during latency, and at least some HCMV miRNAs act to block NFkB signalling [20,21]. One possibility is that viral proteins help to poise the latently infected cell for reactivation, but viral miRNAs act as fine-tuners of the NFkB response, blocking any low-level signals that would result in sub-optimal differentiation and viral reactivation. The mechanistic details of how HCMV limits the antiviral effects while enhancing the pro-viral facets of NFkB signalling remain a mystery. What is clear is that both viral proteins and non-coding RNAs participate in altering the intracellular signalling pathways in HCMV-infected cells in order to successfully establish life-long infections in vivo

Funding Information

NIH grants AI21064 to Jay A. Nelson. The funders had no role in study design, data collection and interpretation or decision to submit work for publication.


We wish to thank Patrizia Caposio and Jessica Smith for insightful comments during the preparation of this manuscript and are grateful to Andrew Townsend for technical assistance.


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