Receptor LigandMediated Signaling Viral Glycoproteins

Envelope glycoproteins play an essential role in viral attachment and entry (Britt and Mach 1996; Mocarski et al. 2007; see the chapter by M.K. Isaacson et al., this volume). From a signaling standpoint, these molecules are logical players in the rapid manipulation of the host cell because they are the first viral molecules to contact a target cell. Although HCMV encodes a number of envelope glycoproteins (Britt and Mach 1996; Mocarski et al. 2007), glycoprotein B (gB/UL55; Britt and Mach 1996) and glycoprotein H (gH/UL75 and its associated partners gL/UL115, gO/UL74, and the UL131-UL128 loci; Britt and Mach 1996; Hahn et al. 2004; Wang and Shenk 2005a, b; Patrone et al. 2007) are the glycoproteins documented to be bona fide signaling molecules (Keay et al. 1995; Yurochko et al. 1997a; Boyle et al. 1999; Yurochko and Huang 1999; Simmen et al. 2001; Compton et al. 2003; Wang et al. 2003, 2005; Boehme et al. 2004, 2006; Feire et al. 2004). The gH complex was originally shown to stimulate calcium flux (Keay et al. 1995), while we have demonstrated that both gB and gH stimulate the activation of the cellular transcription factors, NFk-B and Sp1 (Yurochko et al. 1997a; Yurochko and Huang 1999). Other studies confirmed and expanded these results (Boyle et al. 1999; Simmen et al. 2001; Wang et al. 2003, 2005; Boehme et al. 2004, 2006) and together determined that HCMV fires cellular signal transduction pathways via the actions of the major viral glycoproteins, gB and gH. Viral glycoprotein-mediated signaling occurs in multiple cell types (fibroblasts, monocytes, endothelial cells, etc.), suggesting that the capacity to induce cellular signaling is part of a central theme in the viral infection strategy.

The recent identification of several cellular receptors for HCMV attachment/ entry that are found on multiple cell types supports this proposal: HCMV glycopro-teins were recently shown to interact with the epidermal growth factor receptor (EGFR; Wang et al. 2003, 2005), integrins (a2P1, a^, aVP3; Feire et al. 2004; Wang et al. 2005), and toll-like receptor 2 (TLR2; Compton et al. 2003; Boehme et al. 2006). From a signaling standpoint, the engagement of these receptors by the virus makes sense, as each receptor is biochemically integrated with the signaling machinery. EGFR dimerizes upon ligand binding and then directs downstream signaling events via the action of its intrinsic tyrosine kinase (Wang et al. 2003, 2005). Integrins do not possess intrinsic kinase activity; however, upon their engagement they interact with members of the Src family of tyrosine kinases to modulate downstream signaling events (Wang et al. 2003, 2005). Finally, like all TLRs, TLR2 is part of a signaling network involving a cascade of players (Compton et al. 2003; Boehme et al. 2006).

Mechanistically, it has been documented that gB and gH are responsible for the engagement of the various cellular receptors (EGFR, the integrins, and TLR2) and that, through this receptor/ligand interaction, they rapidly activate signal transduction pathways (Wang et al. 2003, 2005; Boehme et al. 2006). Wang et al. have reported that gB interacts with EGFR and gH interacts with cellular integrins (Wang et al. 2003, 2005), demonstrating that individual receptor/ligand events are controlled by different viral gene products. gB and gH can also interact with TLR2 (Boehme et al. 2006), while gB may additionally interact with cellular integrins (Feire et al. 2004). All three receptors appear to be present on most cell types, suggesting an evolutionarily conserved mechanism may exist for viral binding and receptor engagement during infection of multiple cell types. This possibility is supported by work showing that EGFR and/or integrins are central determinants of signaling and/or attachment/entry in fibroblasts (Wang et al. 2003, 2005), cytotro-phoblasts (Maidji E et al. 2007), endothelial cells (Bentz and Yurochko 2008) and monocytes (Yurochko et al. 1992; Chan et al., unpublished data). Nevertheless, the role these receptors play remains controversial, as it was recently reported that EGFR was not required for attachment and signaling on some fibroblast, epithelial and endothelial cell lines (Isaacson et al. 2007). Thus, it remains unclear if all three receptors are utilized on all cell types infected or if different combinations are utilized depending on the cell type. Overall, these findings suggest the following general model (discussed in more detail below): gB and gH binding to cellular receptors initiates the activation of multiple downstream players including the focal

Cellular Activation Viral Entry

Viral & Cellular Gene Expression

Fig. 1 HCMV binding to cognate receptors initiates signaling cascades. Binding of the envelope glycoproteins, gB and gH, to the cellular receptors, EGFR, integrins and TLR2 begin the outside-in signaling process observed in cells following infection. These known HCMV receptors are integrated with cellular signal transduction pathways; thus viral ligand engagement is the stimulus to fire downstream signaling processes. The initial receptor/ligand-directed signaling modulates a number of pathways, of which a few examples are shown in the drawing. The consequences of this outside-in signaling modulated by the viral glycoproteins include viral entry, cellular activation and transcriptional regulation of cellular and viral genes

Cellular Activation Viral Entry

Viral & Cellular Gene Expression

Fig. 1 HCMV binding to cognate receptors initiates signaling cascades. Binding of the envelope glycoproteins, gB and gH, to the cellular receptors, EGFR, integrins and TLR2 begin the outside-in signaling process observed in cells following infection. These known HCMV receptors are integrated with cellular signal transduction pathways; thus viral ligand engagement is the stimulus to fire downstream signaling processes. The initial receptor/ligand-directed signaling modulates a number of pathways, of which a few examples are shown in the drawing. The consequences of this outside-in signaling modulated by the viral glycoproteins include viral entry, cellular activation and transcriptional regulation of cellular and viral genes adhesion kinase (FAK), the IKK cascade, the MAPK pathway, and the PI(3)K pathway to promote both viral entry and cellular changes such as the activation of NFk-B and other transcription factors required for the transactivation of key cellular and/or viral genes (Fig. 1).

Captured Cellular Enzymes

The virion has long been known to harbor enzymatic activity (Mar et al. 1981), although the nature of this signaling potential has been unresolved. The signaling potential present in the virion imparts the virus with another mechanism to rapidly mediate distinct cellular changes following infection. Two distinct signaling capabilities are present in the virion: (1) HCMV captures cellular enzymes that directly modify the host cell signaling capabilities following viral fusion (discussed in this section) and (2) tegument proteins found in the mature virion can directly modulate host cell biochemical pathways (discussed in the next section).

The virion contains at least four distinct functional enzyme activities of host cell origin (Michelson et al. 1996; Gallina et al. 1999; Nogalski et al. 2007). A recent mass spectrometry analysis of the HCMV proteome revealed that additional cellular modulators may exist in the virion (Varnum et al. 2004). Michelson et al. first showed that HCMV virions contain serine/threonine protein phosphatase activity due to the cellular protein phosphatases PP1 and PP2A (Michelson et al. 1996). This work provided key evidence that HCMV captures cellular enzymes capable of manipulating phosphorylation. Kinases are also present in the HCMV virion. Gallina et al. showed that HCMV possess serine/threonine kinase activity due to the cellular kinase, (polo-like kinase 1 (Plk1; Gallina et al. 1999)). Plk1 was shown to interact with the major tegument protein, UL83/pp65, identifying a mechanism in which cellular products could be captured by the virus during maturation through a specific interaction with viral tegument proteins. We identified a second serine/ threonine kinase, casein kinase II (CKII), that is also incorporated into the mature virion (Nogalski et al. 2007). The virion CKII possesses potent IkB kinase activity and promotes the efficient transactivation of the major IE promoter (MIEP). Why would the virus have evolved a mechanism to capture cellular enzymes? Reversible phosphorylation via the reciprocal action of kinases and phosphatases is an effective and rapid mechanism for modulating cellular function (Arena et al. 2005); thus this biochemical process is an attractive target for a virus that needs to rapidly modulate the host cell for viral infection, survival and persistence. The release of captured enzymes may allow an increase in the local concentration of those enzymes in the viral microenvironment (Nogalski et al. 2007). It is also possible the virion-associated enzymes have a different subcellular localization and thus potentially different targets (Gallina et al. 1999). Additionally, because the virus infects multiple cell types with different biological characteristics, the evolution of multiple mechanisms to drive the rapid activation of the cell may ensure sufficient and appropriate activation of each cell type following infection.

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