Viral Serine/Threonine Protein Kinases

Abstract

Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets.

Protein kinases are characterized by their potential to catalyze the transfer of a phosphate group from a nucleoside triphosphate (generally ATP) to an amino acid residue of a protein substrate. This phosphorylation usually results in a functional change of the target protein by interfering with its enzymatic activity, cellular location, and/or association with other proteins. Depending on the specificity of the substrate amino acids, protein kinases are subdivided in serine/threonine (S/T) or tyrosine (Tyr) kinases. In addition, rare dual-specificity kinases have also been identified. Protein kinases phosphorylate either a serine, a threonine, or a tyrosine residue when it is present in a specific stretch of other amino acids, known as the consensus sequence. The catalytic core of protein kinases consists of 12 conserved subdomains that fold into a common structure. Several strongly conserved residues were identified as indispensable for kinase activity, such as the conserved lysine in subdomain VIb, which is critical for ATP binding, and the conserved aspartate in the catalytic loop (subdomain VIb) (77). These residues are frequently used as targets for mutagenesis in the construction of kinase-negative mutants.

The human genome contains about 500 protein kinase genes, constituting approximately 2% of all human genes (127). Up to 30% of all human proteins may be modified by kinase activity, and kinases are known to regulate the majority of cellular pathways, especially those involved in signal transduction (127).

Since viruses have evolved complex interactions with their host and often mimic cellular proteins in order to usurp the cellular machinery for their own benefit, it might not be surprising that several viruses encode protein kinases. In the late 1970s, the first reports speculating about the existence of viral kinases were published (40, 206); it was only 10 years later that the first viral kinases were identified (115, 117). Over the last few years, several new insights have been gained concerning the often pleiotropic functions of viral protein kinases.

Both serine/threonine and tyrosine viral protein kinases have been identified. The best-characterized viral tyrosine kinases are the oncogenic tyrosine kinases encoded by acute transforming retroviruses. These kinases are captured forms of normal cellular genes (proto-oncogenes), and this incorporation may actually be considered an “accident de parcours” during virus replication, typically rendering the virus replication defective. These viral tyrosine kinases will not be covered in this review but have been covered elsewhere (126). Viral serine/threonine kinases, on the other hand, often display little homology to any of the known cellular kinases. They appear to be encoded exclusively by large DNA viruses and form the subject of the current review.

HERPESVIRUSES

Herpesviruses are large, enveloped viruses with a linear, double-stranded DNA genome that varies in size from 125 to 245 kb. The majority of herpesviruses have been classified in one of three subfamilies: the alpha-, beta-, and gammaherpesviruses. Eight human herpesviruses have been described: the alphaherpesviruses herpes simplex virus 1 (HSV-1) and HSV-2 and varicella-zoster virus (VZV), the betaherpesviruses human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6) and HHV-7, and the gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV).

Invariably, primary infections are followed by lifelong persistence. Specific circumstances may trigger reactivation, with or without viral shedding and/or clinical symptoms. Disease symptoms vary greatly, and include cold sores (HSV-1), genital lesions (HSV), chickenpox (VZV), shingles (VZV), congenital disease (HCMV), posttransplant diseases (HCMV), roseola (HHV-6, -7), infectious mononucleosis (EBV, HCMV), lymphoproliferative disorders (EBV), and Kaposi's sarcoma (KSHV).

All herpesviruses encode serine/threonine kinases. Two types of conserved herpesvirus serine/threonine kinases have been described. One type is conserved over the three different subfamilies. The prototypical example of this kinase is the UL13 kinase of HSV. These conserved kinases will be designated conserved herpesvirus protein kinases (CHPK) throughout the text. Another type of herpesvirus protein kinases, exemplified by the US3 kinase of HSV, is conserved in the alphaherpesvirus subfamily. In addition, serine/threonine protein kinase activity has been suggested to be present in the large subunit of the ribonucleotide reductase of HSV-2.

CHPK.

Orthologs of CHPK include UL13 of HSV, ORF47 of VZV, UL97 of HCMV, U69 of HHV-6, BGLF4 of EBV, and ORF36 of KSHV and murine herpesvirus 68 (MHV-68).

In 1989, by using sequencing assays, Chee and colleagues identified a gene encoding a putative protein kinase that appeared to be conserved in the herpesvirus family, since it was present in the alphaherpesviruses HSV-1 and VZV, the betaherpesviruses HCMV and HHV-6, and the gammaherpesvirus EBV (35). The corresponding protein was confirmed a few years later to display serine protein kinase activity for the CHPK of the alphaherpesviruses VZV and porcine pseudorabies virus (PRV) (54, 159). In vitro studies using cell cultures and primary cells showed that the importance of CHPK for virus growth in vitro varies from crucial for viral replication to unimportant, depending on both the virus and the cell (45, 184, 185). In vivo studies, however, invariably point to an important role of the kinase in virus virulence (45, 86, 150, 180). Although the kinase is conserved among herpesviruses, CHPK from different herpesvirus subfamilies show considerable variation in amino acid sequence. Although some sequence preferences for substrate specificity have been described for individual CHPK, there is no consensus phosphorylation sequence for all CHPK (10, 30, 95, 96, 100).

Despite these differences, partial functional conservation has been reported (193). Characteristics and functions of CHPK that appear to be conserved to some extent include its autophosphorylating activity, tegument incorporation, phosphorylation of cellular elongation factor 1 delta (EF-1δ), evasion of the interferon (IFN) response, and phosphorylation of ganciclovir. Specific subfamily- and virus-specific functions have also been described. Several of the CHPK funtions are shown in Fig. 1, and different substrates are shown in Table 1.

FIG. 1.

Some of the major functions associated with the conserved herpesvirus protein kinase (CHPK). Red lines indicate functions that are conserved over the three herpesvirus subfamilies. Green lines indicate functions that are common to beta- and gammaherpesviruses. Blue and black lines indicate functions that have been reported only for alpha- and betaherpesviruses, respectively.

TABLE 1.

Human CHPK and their cellular and viral substrates

Virus	CHPK	Substrate (reference[s])
		Cellular	Viral
HSV-1	UL13	IRF-3 (82)	US3 (91)
		EF-1δ (98)	gE, gI (161)
		CKIIβ (96)	ICP0 (166)
		RNA polymerase II (125)	ICP22/Us1.5 (184, 185)
		p60 (26)	UL13 (46, 47)
			VP13/14 (192)
			VP22 (45)
			UL41 (167)
HSV-2	UL13	Lamin A/C, limited (31)	US3 (31)
		EF-1δ (97)	UL13 (30)
			VP22 (70)
VZV	ORF47	Akt (186)	ORF32 (187)
			ORF47 (159)
			IE62 (160)
			IE63 (101)
			gE (99)
HCMV	UL97	Lamin A/C (130)	UL44 (109, 129)
		Rb tumor suppressor (81, 181)	pp65 (88)
		RNA polymerase II (12)	UL97 (79)
		Histone H2B (141, 210)
		IRF-3 (82)
		EF-1δ (97)
HHV-6/HHV-7	U69	Lamin A/C (111)	U69 (4)
		Rb tumor suppressor (111)
EBV	BGLF4	Lamin A/C (118)	BRMF1 (36)
		Rb tumor suppressor (111)	BGLF4 (92)
		H2AX (209)	EBNA-LP (208)
		IRF-3 (82, 225)
		EF-1δ (92)
		CKIIβ (96)
KSHV	ORF36	Lamin A/C (111)	ORF36 (76)
		Rb tumor suppressor (111)
		IRF-3 (82)

(i) Autophosphorylation and tegument incorporation.

Like many cellular serine/threonine kinases, all herpesviral CHPK orthologs appear to possess both autophosphorylation and transphosphorylation activities (95). Autophosphorylation was first demonstrated for the VZV CHPK by immunoprecipitation experiments with infected cells and by protein kinase assays (159).

The importance of autophosphorylation for the activity of CHPK orthologs is not entirely clear. For example, there have been conflicting reports on whether the autophosphorylating activity of HCMV CHPK is required for phosphorylation of exogenous substrates, such as histone 2B (10, 129, 141). Autophosphorylation has been reported to be important for the ability of the KSHV CHPK to activate JNK signaling (76).

All CHPK orthologs of HSV, VZV, HCMV, and EBV are incorporated into the virus particle (7, 168, 219). More specifically, the viral kinase is located in the tegument, a proteinaceous layer in herpesvirus virions between the capsid and the envelope.

(ii) Phosphorylation of EF-1δ.

The translation elongation factor 1 delta (EF-1δ) consists of two forms, a hypo- and a hyperphosphorylated form. In 1997, Kawaguchi et al. reported that whereas the hyperphosphorylated form of EF-1δ cells was a minor species in mock-infected cells, it became the predominant form in cells infected with HSV-1 (94). One year later, the same group identified the HSV-1 CHPK as the kinase responsible for the observed EF-1δ hyperphosphorylation (98). Hyperphosphorylation of EF-1δ was subsequently observed for members of the three different herpesvirus subfamilies, and the HCMV CHPK ortholog could compensate for the HSV CHPK to induce EF-1δ hyperphosphorylation in HSV-1-infected cells (97). EF-1δ is a subunit of EF-1, a complex of proteins which mediate the elongation of polypeptide chains during translation of mRNA. Although the physiological role of EF-1δ hyperphosphorylation is not known, this function of CHPK is likely to regulate and/or stimulate the translation process in infected cells.

The cellular cyclin-dependent kinase cdc2 also phosphorylates EF-1δ, and research on the interaction of CHPK with EF-1δ provided a first clue that CHPK orthologs to some extent mimic the function and substrate specificity of cdc2. It was demonstrated that different CHPK phosphorylate EF-1δ at the same amino acid residue (Ser-133) as for cdc2 (8, 96). Other features of CHPK, discussed below, also point toward a significant functional similarity between CHPK and cyclin-dependent kinases. However, Cano-Monreal et al. reported that the CHPK of the alphaherpesvirus HSV-2 did not phosphorylate standard cdc2 peptide substrates in vitro, suggesting that this similarity is less obvious in alphaherpesviruses (30). In support of this are the findings that all human beta- and gammaherpesvirus CHPK, except for the KSHV CHPK, are able to rescue a G₁-to-S cell cycle defect in Saccharomyces cerevisiae lacking cyclin-dependent kinase function, whereas alphaherpesvirus CHPK are unable to do so (81, 111).

(iii) Nuclear egress.

Thus far, for all CHPK, a fraction has been described to be present in the nucleus of infected cells (47, 49, 71, 72, 84, 143, 169, 226), indicating that the kinase may play important, conserved roles in the nuclear phase of infection.

A function that appears to be largely conserved is a role of CHPK in nuclear egress of progeny capsids. Herpesvirus capsids assemble in the nucleus and, because of their relatively large size, need to (locally) disrupt the dense meshwork of the nuclear lamina to gain access to the nuclear envelope to continue the process of virion assembly and egress. Disassembly and reassembly of the nuclear lamina, a key process in cell division, occurs through phosphorylation and dephosphorylation of lamins, e.g., by cyclin-dependent kinases and protein kinase C. Phosphorylation of lamins during herpesvirus infection appears to involve both cellular kinases, such as protein kinase C (144, 155, 170), but also the viral CHPK kinases. This is best documented for beta- and gammaherpesvirus CHPK. Indeed, both the CHPK of EBV and that of HCMV have been reported to phosphorylate lamins (75, 118, 130), and lack of these kinases is correlated with accumulation of capsids in the nucleus (109, 180, 233). Interestingly, the CHPK of both EBV and HCMV phosphorylate lamins on residues that are also substrates for phosphorylation by cdc2 (75, 118), further highlighting the similarities in substrate specificity between the beta- and gammaherpesvirus CHPK and cdc2. Recently, lamin A/C phosphorylation was confirmed for all human beta- and gammaherpesviruses (111).

A role for CHPK of alphaherpesviruses in nuclear egress is less clear, possibly because these viruses encode a second viral kinase, US3, which appears to fulfill at least part of this function (see below). The CHPK of HSV-1 is still thought to have some function in nuclear egress, albeit indirectly, by phosphorylating the viral US3 kinase and thereby regulating the function of US3 in nuclear egress (91). In support of a combined role of CHPK and US3, deletion of both in the genome of the porcine alphaherpesvirus PRV resulted in strongly reduced production of infectious virus (54). A direct effect of alphaherpesvirus CHPK on lamins and the nuclear lamina is either absent or very limited. A recent study reported a failure to detect phosphorylation of lamin A or disruption of the nuclear lamina by HSV-1 and VZV CHPK (111). For HSV-2 CHPK, a noticeable, but limited, effect on lamin phosphorylation and nuclear lamina integrity was observed, in line with an earlier report (31, 111).

(iv) Virus replication.

Besides their role in viral nuclear egress, CHPK have been reported to play other functions in the nucleus that may affect virus replication efficiency.

In 2008, two groups independently showed that the HCMV CHPK directly phosphorylates and thereby inactivates the retinoblastoma tumor suppressor (Rb), again similar to one of the functions of cdc2 (81, 181). Recently, Rb phosphorylation was demonstrated for all human beta- and gammaherpesvirus CHPK, but not the alphaherpesvirus counterparts (111). Nevertheless, the HSV-1 CHPK has also been reported to affect regulation of the cell cycle via an increase in cellular cdc2 activity (2).

HSV-1 CHPK also promotes the expression of a subset of viral genes, including ICP22 and several late genes (2, 184, 185), although this effect appears to be independent of the kinase activity of the protein (207). Interestingly, the CHPK of the betaherpesvirus HCMV could compensate for the loss of HSV CHPK with regard to its role in viral gene expression (162), demonstrating that there is some functional overlap between alpha- and betaherpesvirus CHPK members.

Deletion of the CHPK in HCMV results in reduced viral gene expression (109, 233). In vitro kinase assays showed that the HCMV CHPK is able to directly phosphorylate RNA polymerase II, which is also phosphorylated in HCMV-infected cells (12). Inhibition of this phosphorylation by the addition of roscovitine resulted in defective immediate-early gene expression, suggesting a causal relationship between RNA polymerase II phosphorylation and increased viral gene expression (205). However, it is unclear whether the effect of roscovitine can be attributed solely to the CHPK. Roscovitine is a purine derivative that also inhibits several cellular cyclin-dependent kinases by competing with ATP binding, and as such is being used in clinical trials as a treatment for various tumors (110, 135, 196).

Reduced viral gene expression observed in the absence of the HCMV CHPK has also been attributed to the modest reduction in viral DNA accumulation observed with a CHPK-deleted virus (109, 233). This defect in DNA accumulation may correlate with the finding that this CHPK phosphorylates the viral DNA polymerase processivity factor UL44, an essential component of the replication complex (109, 129). Like the HCMV CHPK, the CHPK of EBV phosphorylates the DNA processivity factor BMRF1, which is homologous to HCMV UL44 (36, 65). Phosphorylation of BMRF1 may affect its transactivation activity (234). EBV CHPK also phosphorylates the EBV coactivator EBNA-LP, one of the EBV proteins involved in B-cell transformation. Phosphorylation of EBNA-LP is important for its function, but phosphorylation does not critically rely on CHPK, since cellular cdc2 is able to phosphorylate EBNA-LP on the same residue (93).

Effects of CHPK on viral gene expression may also be related to phosphorylation of histones. In vitro, HCMV CHPK phosphorylates histone H2B, which has also been hypothesized to affect gene expression (11). More recently, CHPK of EBV and MHV-68 were found to induce phosphorylation of histone H2AX, in contrast to the CHPK counterparts of the related virus KSHV and the more distantly related virus HSV (209). Phosphorylated H2AX is involved in the cellular DNA damage response (28). Lack of MHV-68 CHPK or H2AX resulted in inefficient MHV-68 replication in primary macrophages, indicating that a virus-activated DNA damage response facilitates MHV-68 lytic replication (209). Recently, the lack of CHPK or H2AX was also correlated with decreased levels of MHV-68 latency in mice (210). Clearly, more research is needed to clarify the role of CHPK in viral gene expression, DNA replication, and viral pathogenesis.

(v) Evasion of the interferon response.

A potential role for CHPK in evasion of the IFN response was first suggested for HSV-1. In 2001, Shibaki et al. reported that a CHPK-null virus was rapidly cleared upon intraperitoneal inoculation of mice. The authors showed that this correlated with increased levels of type I IFN induced by the mutant virus and greater sensitivity of the mutant toward the antiviral effects of type I IFN (200). Later, HSV-1 was found to induce the expression of a negative regulator of IFN, suppressor of cytokine signaling 3 (SOCS-3). Deletion of the CHPK resulted in less efficient induction of SOCS-3 (236).

Recently, the involvement of CHPK orthologs in counteracting the interferon response was further substantiated and was found to be conserved over the three different herpesvirus subfamilies. The CHPK of HSV-1, HCMV, EBV, KSHV, and MHV-68 were all found to subvert the type I IFN response by suppressing the activity of interferon regulatory factor 3 (IRF-3) (82, 225). The viral kinase interacts with activated IRF-3, thereby interfering with IRF-3 recruitment to the IFN promoter (82, 225). Although the interaction of EBV CHPK with IRF-3 leads to phosphorylation of IRF-3 (225), the kinase activity of the CHPK does not appear to be absolutely required for its effect on the IFN response (82).

(vi) Phosphorylation of viral tegument and membrane proteins.

CHPK-mediated phosphorylation of tegument proteins (other than the CHPK itself) and envelope proteins is best documented for alphaherpesviruses.

The CHPK of VZV is essential for infection of differentiated human T cells and skin xenografts in SCID-hu mice (150). The VZV CHPK phosphorylates the tegument proteins IE62 and IE63 (101, 102, 160), and phosphorylation of IE62 has been correlated with CHPK-mediated virulence in SCID-hu mice. IE62 is the major immediate-early transactivating protein, and the interaction between ORF47 and IE62, rather than IE62 phosphorylation per se, appears to be a pivotal element in CHPK-mediated virulence in SCID-hu mice (19).

The VZV CHPK also phosphorylates the viral glycoprotein gE (99). Kenyon et al. reported that ORF47-mediated phosphorylation of gE may be associated with the surprising increase in virus spread observed for a CHPK-null VZV recombinant in MeWo cells. Phosphorylation by the CHPK redirected gE to the plasma membrane instead of the trans-Golgi network, where herpesvirus budding is thought to occur, possibly explaining the suppressive effect on viral spread (99).

The HSV CHPK also phosphorylates gE and its complexing partner gI (159). In addition, the CHPK of HSV phosphorylates the tegument proteins VP13/14 and VP22, which may facilitate their release from the capsid during entry (45, 70, 152). Alphaherpesvirus CHPK-mediated phosphorylation of tegument and/or cellular proteins in combination with phosphorylation by the alphaherpesvirus kinase US3 (see below) may regulate the directionality of microtubule-based virus transport in neuronal axons (39). HSV CHPK also phosphorylates the virion host shutoff protein (UL41), a viral tegument protein involved in shutting down host translation. Inactivation of CHPK resulted in the production of virions that did not possess host shutoff activity, although they did incorporate UL41 (167).

For HCMV, the CHPK interacts with and phosphorylates the tegument protein pp65 in vitro (88). Although the biological consequences of this phosphorylation are unclear, in the absence of CHPK activity, HCMV-infected cells produce large nuclear aggregates that contain substantial quantities of pp65 (179). The association of HCMV CPHK with pp65 most likely explains the protein kinase activity associated with immunoprecipitates of pp65 (25, 88), although pp65 itself has also been suggested to display kinase activity (201, 235).

(vii) Phosphorylation of GCV.

The remarkable success of the nucleoside analogue acyclovir (ACV) in antiviral therapy against HSV and VZV is based on its selectivity: the viral thymidine kinase converts ACV to its monophosphate form and cellular kinases then further process it to its active triphosphate form. Ganciclovir (GCV) was synthesized in 1980 at Syntex Laboratories as an analogue of acyclovir with an improved structural resemblance to natural nucleosides. GCV turned out to display potent antiviral activity against HCMV. This was somewhat surprising, since HCMV does not encode a thymidine kinase-like enzyme. In 1992, the CHPK of HCMV was found to be responsible for the conversion of GCV to its monophosphate form (123, 202). Hence, the HCMV CHPK possesses the unusual ability to act as both a protein kinase and a nucleoside kinase. The lack of homology to any known nucleoside kinase and the inability of the HCMV CHPK to phosphorylate natural nucleosides indicate that it is not a natural nucleoside kinase (143). Besides GCV, this CHPK also phosphorylates ACV and penciclovir (238). The ability of CHPK orthologs to phosphorylate GCV is conserved with various levels of efficiency in some, but not all, betaherpesviruses and gammaherpesviruses, but apparently not in alphaherpesviruses (4, 29, 136, 142, 193, 222).

US3 Protein kinase orthologs.

In the second part of the 1980s, the US3 orthologs of HSV-1 and -2 and VZV were reported to show homology to eukaryotic protein kinases (133) and soon thereafter, by using antisera and a kinase assay, HSV US3 was identified as a bona fide viral protein kinase (67). The US3 kinase is conserved throughout the alphaherpesvirus subfamily but is not present in other herpesvirus genomes. The consensus target sequence for US3 of the porcine alphaherpesvirus PRV was characterized as R_nX(S/T)ZZ, where n is greater than or equal to 2; X can be absent or Arg, Ala, Val, Pro, or Ser; and Z can be any amino acid except an acidic residue (116, 183) and was found to be broadly similar for HSV-1 and -2 and VZV (48, 58, 183) and the cellular protein kinase A (17, 89). Recent findings indicate that HSV-1 US3 is more promiscuous than previously thought and suggest the existence of more substrates than originally predicted (153). US3 autophosphorylation at a site that corresponds to serine 147 in HSV-1 has been reported to affect US3 function and localization in both HSV-1 and HSV-2 (90, 151). Several possible US3 phosphorylation substrates have been proposed, but few have been confirmed as biologically relevant thus far.

Based on studies showing that recombinant US3 mutant viruses have modestly impaired growth properties in cell cultures but are severely attenuated in animal models, US3 was identified as a positive regulator of viral replication and viral pathogenicity (134, 191, 218). Several lines of evidence suggest the involvement of US3 in a variety of functions, some of which are conserved among the alphaherpesviruses, while others appear to be unique for a specific virus. The kinase activity of US3 was shown to be crucial for most of its functions. Different functions of US3 are shown in Fig. 2.

FIG. 2.

Major functions associated with US3 kinase orthologs of alphaherpesviruses. Proteins in blue represent US3 substrates that may be involved in the different functions of US3.

(i) Nuclear egress.

In 1995, a first report was published pointing at the involvement of US3 in nuclear egress (221). Alphaherpesviruses, and herpesviruses in general, use a unique system to transport progeny nucleocapsids out of the nucleus and into the cytoplasm. Nucleocapsids undergo primary envelopment by budding in the inner nuclear membrane, followed by fusion with the outer nuclear membrane, releasing the virion into the cytoplasm (reviewed in reference 140). In cells infected with US3-null PRV, HSV-1, and Marek's disease virus (MDV), virions aggregate aberrantly within the perinuclear space in large invaginations (105, 191, 198, 221), suggesting a conserved role for the US3 kinase in the de-envelopment step during nuclear egress. This defect in capsid nuclear export is, however, not absolute, since extracellular virus titers are typically only mildly reduced in the absence of US3 (39, 191, 194, 215, 218).

Since these initial reports, US3 has been implicated in different steps of the nuclear egress pathway. First, lamin A/C and emerin, key elements of the nuclear lamina network, can be phosphorylated by HSV-1 US3 (114, 153). This leads to the disruption of the nuclear lamina, which represents a barrier for the virions to reach the inner nuclear membrane. Second, infection with US3-null HSV-1 or PRV results in an altered distribution of the viral UL34 and UL31 proteins, both of which are crucial regulators of primary envelopment of nucleocapsids at the nuclear membrane, from a roughly continuous distribution to a distribution in discrete aggregates in the inner nuclear membrane (105, 190). This relocalization of the envelopment machinery was recently found to be regulated by phosphorylation of the N terminus of UL31 by HSV-1 US3 (154). A third potential involvement of US3 in nuclear egress has also been put forward. HSV-1 US3 phosphorylates the cytoplasmic domain of the envelope glycoprotein gB (83). The gB protein is one of the viral proteins that is essential to mediate fusion between envelope and host membrane during viral entry. For HSV-1, gB, together with gH, has been suggested to be involved in the fusion between the primary enveloped virion in the perinuclear space and the outer nuclear membrane (232). Recent evidence indicates that phosphorylation of gB by US3 drives this process (232). The latter may not be a conserved function of alphaherpesviruses since, for PRV, neither gB nor gH appears to be present at the nuclear membranes and neither protein functions in the nuclear egress of virions (74, 104).

(ii) Antiapoptotic activity.

US3 possesses antiapoptotic properties in HSV, PRV, and MDV, but possibly not in bovine herpesvirus 1 (BoHV-1) (9, 69, 119, 197, 204). The kinase activity of HSV-1, PRV, and MDV US3 is necessary for this US3 function (33, 52), and several phosphorylation targets implicated in the HSV-1 US3-mediated suppression of apoptosis have been identified, including Bad, Bid, and procaspase 3, pointing at the involvement of US3 in different antiapoptotic pathways (18, 32). This may explain how US3 is able to inhibit apoptosis induced by very diverse apoptotic stimuli, including herpesvirus infection itself, cytotoxic T lymphocytes and granzyme B, overexpression of BclX, staurosporine, and sorbitol (9, 32, 33, 78, 87, 119, 157, 165).

(iii) Actin rearrangements.

The US3 kinase induces drastic rearrangements of the cytoskeleton, including disassembly of actin stress fibers and the formation of actin- and microtubule-containing cellular projections, which are implicated in intercellular spread (63). The kinase activity of US3 is essential for these processes in HSV-2, PRV, and BoHV-1, but not in MDV, suggesting different or multiple cytoskeleton-affecting activities of US3 (27, 66, 156, 197, 215). Recent evidence with PRV US3 indicates that the kinase interferes with actin-controlling Rho-GTPase signaling to cause the cytoskeletal alterations. PRV US3 phosphorylates and thereby activates p21-activated kinases (PAKs), key regulators of Rho GTPase signaling (216).

(iv) Gene expression.

Over the last couple of years, it has become evident that US3 also affects gene expression. US3 orthologs of HSV-1, HSV-2, PRV, and VZV phosphorylate HDAC1 and -2, thereby inhibiting the deacetylation of histones, which otherwise silence gene expression (151, 176, 223, 224). US3-mediated phosphorylation of HDAC2 occurs at a C-terminally located conserved serine residue (224). HSV-1 US3 is able to phosphorylate HDAC-1 and -2 directly (177, 178), although there are indications that VZV, PRV, and HSV-1 phosphorylate HDAC indirectly by activating a cellular kinase pathway (223, 224). For PRV, hyperphosphorylation of HDAC2 was still observed in cells infected with a US3-null recombinant (224). This is in contrast to findings for HSV-1 and VZV and indicates virus-specific US3-independent mechanisms of HDAC hyperphosphorylation. Inhibition of HDAC activity increased plaquing efficiency of US3-null virus for PRV and VZV, but not for HSV-1, pointing to virus- and cell-dependent differences in the functional significance of US3-mediated HDAC modification (223, 224).

An additional role in gene expression has been described for the US3 ortholog of VZV, ORF66. Both ORF66 and the CHPK of VZV have a unique viral substrate, IE62, a nuclear transcription regulatory protein. After phosphorylation by ORF66, IE62 accumulates in the cytoplasm, where it is incorporated in newly formed virions. This points at a role for the VZV US3 ortholog in reducing nuclear import of IE62 and thereby regulating gene expression (58, 60).

(v) Immune evasion.

Apart from their ability to protect cells from apoptotic cell death, US3 orthologs have been linked to other aspects of evasion of the host immune response. The VZV ORF66 was reported to be involved in downregulating the surface expression of the major histocompatibility complex class I (MHC-I) in VZV-infected cells and ORF66-transfected cells (1, 59), suggesting a role for ORF66 in immune evasion. The kinase activity of ORF66 protein kinase was beneficial but not absolutely required for MHC-I downregulation (59). Recently, PRV US3 was also shown to be able to downregulate MHC-I surface expression. However, this ability was highly cell-type dependent. The underlying mechanism is unclear but appears different from that for VZV ORF66 since transfection of PRV US3 did not affect MHC-I downregulation (53). Still, the potential effect of US3 orthologs on MHC-I surface expression appears to be cell-type dependent and not always very robust; therefore, further investigation may be needed to determine its importance.

For VZV- and PRV-infected cells, the lack of US3 reduced the capacity of the virus to interfere with induction of the IFN signaling pathway following exposure to IFN (175, 195). Recently, Peri et al. reported that US3-null HSV-1 induces a strong activation of IRF-3 and type I IFN mRNA expression and that HSV-1 US3 downregulates the intracellular expression of TLR3 and the type I interferon-inducible protein MxA, which is known to posses antiviral activity (171). In addition, HSV-1 US3 phosphorylates the gamma interferon (IFN-γ) receptor, which interferes with IFN-γ-dependent gene expression (120).

Ribonucleotide reductase.

Alphaherpesviruses, like other herpesviruses, encode a ribonucleotide reductase that is involved in generating deoxyribonucleotides. Therefore, these viruses do not depend on cellular S-phase enzymes, allowing them to replicate in nondividing cells, including neuronal cells (34). The ribonucleotide reductase consists of two subunits, R1 and R2. The large R1 subunit (ICP10) of the HSV-2 ribonucleotide reductase has been suggested to contain protein kinase activity, which may be responsible for the antiapoptotic activity associated with ICP10 (173). Inhibition of apoptosis by ICP10 involves a c-Raf-1-dependent mechanism and induction of the antiapoptotic protein Bag-1 by the activated ERK survival pathway. ICP10 is also responsible for an increased activation/stability of the transcription factor CREB and stabilization of the antiapoptotic protein Bcl-2 (172). This antiapoptotic function could be exploited in cancer therapy, based on the observation that an HSV-2-based oncolytic virus with a deletion of the putative protein kinase domain of the ICP10 gene is a potent inducer of apoptotic death in tumor cells (41, 68). Although ICP10 indisputably displays antiapoptotic activity, there has been some controversy as to whether the protein contains intrinsic protein kinase activity. Based on biochemical studies, it has been suggested that the observed kinase activity is caused by contaminating cellular kinases, in particular casein kinase II (34, 44, 112).

POXVIRUSES

The Poxviridae family can be subdivided into the Entomopoxvirinae, which infect insects, and the Chordopoxvirinae, which infect vertebrates. All poxviruses are double-stranded DNA viruses with an envelope and a very large genome, ranging from 130 to 360 kb. All poxviruses encode serine/threonine kinases. Unlike the vast majority of DNA viruses, poxviruses replicate in the cytoplasm since they encode all necessary replication enzymes and therefore do not rely on cellular nuclear enzymes. Poxviruses share this property with all members of a proposed clade of very large DNA viruses of eukaryotes, the nucleo-cytoplasmic large DNA viruses (NCLDV). Poxviruses and African swine fever virus are the only NCLDV with mammalian hosts (106). Several poxviruses have been reported to infect humans. Most notable is the smallpox virus (variola virus), for which humans are the only known natural host. Smallpox has caused millions of deaths and has had a major impact on human history (132). Although smallpox was eradicated in 1980, the virus is still considered to be a threat as a potential bioterrorist weapon (6). Other poxviruses able to infect humans include vaccinia virus (origin unknown; used for immunization against smallpox), cowpox virus (causes ulcerative lesions on the hands of dairy workers), molluscum contagiosum virus (causes infectious warty papules of the skin), monkeypox virus (causes rare smallpox disease of children in central Africa), pseudocowpox virus (causes nonulcerating nodules on the hands of dairy workers), and Orf virus (causes single lesions on the skin of people handling sheep and goats). Major functions associated with conserved poxviral kinases are shown in Fig. 3.

FIG. 3.

Major functions associated with B1 and F10 kinases of poxviruses. Proteins in orange represent phosphorylation substrates that may be involved in the different functions of B1 and F10.

B1 kinase.

Almost 30 years ago, a wide array of temperature-sensitive mutants of vaccinia virus, the Condit collection, was constructed (42, 43). The ts2 and ts25 mutants were found to be arrested at the stage of DNA replication, and the mutations were located in the B1 gene (42, 189). Sequencing of this region of the genome revealed that the B1 gene displays strong homology to serine/threonine kinases (80, 212). Soon thereafter, B1 was indeed confirmed to encode a catalytically active viral serine/threonine kinase of 34 kDa, which was expressed early in infection, associated with cytoplasmic virus factories, and incorporated as a minor component in the virion (14, 122). Interestingly, based on sequence homology, identification of B1 resulted in the subsequent discovery of a novel class of cellular serine/threonine kinases that share up to 40% amino acid identity with B1, the vaccinia virus B1 kinase-related kinases (VRKs) (158, 163). VRK orthologs are present in mammals, fruit flies, and nematodes, but apparently not in yeast. They reside predominantly in the nucleus, where they fulfill several crucial roles in successful DNA replication, mitosis, and gene expression (reviewed in reference 103).

Nearly all known poxviruses encode B1 orthologs; the exception is molluscum contagiosum virus (199). Although not proven as of yet, it is possible that the molluscum contagiosum virus is able to compensate for the lack of B1 by usurping related host kinases, such as VRKs (108). In line with this idea, expression of the VRK1 genes of humans and, to a lesser extent, mice in the B1-defective ts2 mutant of vaccinia virus rescues the inhibition of DNA replication, virus production, and plaque formation observed with this mutant (24).

The phenotypes associated with temperature-sensitive mutants of B1 show that the kinase is critically involved in at least two key stages of the virus cycle: DNA replication and production of intermediate viral proteins (108, 189).

(i) DNA replication.

In 2007, a paper by Wiebe and Traktman provided fascinating insights into the mechanism of B1-mediated vaccinia virus DNA replication and, at the same time, uncovered a novel aspect of the innate, cellular antiviral response (229). The paper showed that B1 acts on barrier-to-autointegration factor (BAF). BAF is a DNA-binding protein, known to participate in nuclear reorganization and reassembly during mitosis by bridging chromatin to the nuclear membrane (reviewed in reference 128). The B1-related VRKs had already previously been shown to be able to phosphorylate BAF, thereby abrogating the DNA-binding abilities of BAF (164). In cells infected with vaccinia virus, but not with the B1-defective ts2 mutant, BAF phosphorylation was increased and BAF was prevented from association with virus DNA replication sites. Importantly, RNA interference-mediated silencing of BAF rescued the defect in virus growth of the ts2 mutant. Hence, cytoplasmic BAF likely constitutes a novel component of the innate viral response, acting by binding and thereby sequestering foreign, cytoplasmic DNA. B1 allows vaccinia virus to overcome this defense barrier of the cell by phosphorylating BAF, thereby interfering with its DNA-binding abilities.

(ii) Production of intermediate viral proteins.

The mechanism underlying the role of B1 in expression of viral intermediate proteins (108) is less clear. Likely important in this respect is that B1 phosphorylates viral late gene transcription factor 4 (VLTF-4; H5) (15, 16). Although H5 is a late gene transcription factor, B1-mediated phosphorylation of H5 may also affect intermediate protein synthesis, since H5 is the only VLTF that is expressed prior to DNA replication (107). Since H5 is also involved in transcription elongation (via interaction with A18 and G2), DNA replication (via interaction with A20), and virion morphogenesis, phosphorylation by B1 may also affect these aspects of the virus cycle (23, 50, 131). However, arguing against a direct involvement of B1-mediated H5 phosphorylation in DNA replication is the finding that rescue of DNA replication in the ts2 mutant by expression of the B1-like VRK1 was not associated with H5 phosphorylation (24). An alternative role for the B1/H5 interaction may lie in evasion of the innate antiviral response, since both proteins appear to be involved in inhibiting CD1d-mediated antigen presentation to natural killer T (NKT) cells (228).

Besides the effects of B1 on BAF and H5, the viral kinase has been shown to phosphorylate two ribosomal proteins, Sa and S2, which may have consequences on the efficiency of viral versus cellular translation (13). In vitro kinase assays have shown that the A30 protein, which is involved in virion morphogenesis, may also be a B1 substrate (139).

F10 kinase.

In 1994, a second kinase, encoded by the F10 gene, was identified in the vaccinia virus genome. The F10 kinase has a molecular weight of 50 kDa, and as is the case for B1, the kinase is incorporated into virions and is essential for viability of the virus. The majority of kinase activity packaged in the vaccinia virion core is the product of the F10 gene (121). F10 is strongly conserved in poxvirus genomes, with orthologs occurring not only in chordopoxviruses but also in entomopoxviruses (57). Unlike B1, F10 shows very little similarity to cellular protein kinases and has a very atypical structure, which probably explains the relatively late discovery of this viral kinase. Kinase subdomains II (ATP-binding pocket) and VI (catalytic loop) are the only typical kinase domains that are obviously recognizable in F10 (182). F10 has been characterized as a dual-specificity kinase, a relatively rare type of kinase that is able to phosphorylate not only serine/threonine but also tyrosine residues (51).

Experiments using temperature-sensitive mutants from the Condit collection showed that lack of F10 did not prevent genome replication or gene expression, but halted the virus at the earliest points of virion morphogenesis (213, 227). Subsequent studies revealed that F10 phosphorylates, and thereby influences several key viral factors involved in wrapping of electron-dense virosomes by membrane crescents to form the immature virion. F10 phosphorylates two viral membrane proteins that are critical in early morphogenesis, A14 and A17, which is thought to be required to form the membranes associated with immature virions (20, 51, 138). Together with viral proteins A30 and G7, F10 makes part of a viral assembly complex, which stimulates F10-mediated A17 phosphorylation (203). Additional components of this viral assembly complex were subsequently identified and include J1, A15, D2, and D3 (37, 203). A30, G7, and F10 itself are phosphorylated in an F10-mediated manner in this complex (139, 203). In vitro kinase assays confirmed A30 as a potential substrate for F10, whereas G7 was not phosphorylated by F10 in vitro, leaving the possibility that F10-mediated G7 phosphorylation occurs via a cellular kinase or occurs only in vivo (139).

In vitro kinase assays showed that F10 can phosphorylate E8R and thereby abrogates the DNA-binding capacity of this protein (55). Although there are indications that F10-mediated E8R phosphorylation may occur inside virus particles, thereby potentially facilitating DNA exit from the core during virus entry, this remains to be formally demonstrated (55).

BACULOVIRUSES

Members of the family Baculoviridae are large, double-stranded, enveloped DNA viruses that infect mainly insects. In 1983, Miller and coworkers reported the presence of serine/threonine kinase activity associated with both the extracellular and the occluded forms of a baculovirus (145). Until now, only one protein kinase gene has been identified in all baculovirus genomes sequenced: PK1. Sequences of PK1 of baculoviruses Spodoptera litura nucleopolyhedrovirus (SpltNPV) (148), Choristoneura fumiferana granulovirus (ChfuGV) (73), Lymantria dispar nuclear polyhedrosis virus (LdNPV) (22), Helicoverpa armigera single nucleopolyhedrovirus (HaSNVP) (237), and Autographa californica nucleopolyhedrovirus (AcNPV) (188) were analyzed and were found to contain typical serine/threonine kinase domains. Recombinant PK1 of AcNPV, SpltNPV, and LdNPV phosphorylates exogenous substrates, such as histone H1 and myelin basic protein (MBP) (22, 148, 188). Based on genome sequence analyses, the existence of a viral protein kinase in Anagrapha falcifera multinuclear polyhedrosis virus (AfMNPV) and Neodiprion lecontei nucleopolyhedrovirus (NeleNPV) was also predicted (64, 113). Baculovirus kinase activity was first suggested to be essential for release of viral DNA from virions during entry (230, 231). More recently, PK1 has been shown to regulate transcription from very late promoters, such as the polyhedrin (polh) and p10 promoters. This was first reported in a study of the temperature-sensitive AcNPV mutant (62) and was recently confirmed for both AcNPV and SpltNPV (146-149). AcNPV PK1 also plays a role in the phosphorylation of the LEF8 protein, which is a component of the very late gene transcription initiation complex (147).

OTHER VIRUSES

Apart from the above-mentioned viruses, viral kinases have been described or predicted based on homology to kinase domains in several other large DNA viral families, including the asfavirus, nimavirus, phycodnavirus, iridovirus, and mimivirus families. In irido- and phycodnavirus families, multiple copies of protein kinase genes were identified (85, 211). A comparison of nine sequenced iridoviruses revealed the presence of two conserved S/T kinases (56).

Phylogenetic trees have been constructed based on the pox-, herpes-, asfa-, nima-, phycodna-, irido-, and baculovirus protein kinase sequences to obtain new insights in the evolution of the PK genes and/or taxonomic classification of viruses (124, 217), which confirm broadly the evolutionary relationships of the different virus families. Although the existence and apparent conservation of viral protein kinases in the above-mentioned viral families point toward potentially important roles during their replication, further research is needed to address their exact biological roles.

CONCLUDING THOUGHTS

One of the curious aspects of viral serine/threonine kinases is that they appear to be encoded exclusively by large and evolutionarily old DNA viruses, such as herpesviruses, poxviruses, and baculoviruses.

Trying to explain this requires some speculation. One possibility would be that viral kinases merely are an “optional feature” of viruses, encoded only by those viral species that have a large enough genome to be able to afford them. Alternatively, they may be evolutionary relics, present in many of the evolutionarily ancient, complex DNA viruses and selected away in viruses of more recent origin. The fact that all studied viral kinases constitute important virulence factors argues against this possibility in favor of a third possibility: viral serine/threonine kinases may actually be one of the key elements of these old, large DNA viruses and may have aided them in still being around after millions of years of host evolution.

Indeed, evolutionarily very distinct members of the herpesviruses, with natural hosts ranging from koi to humans, are predicted to encode viral kinases (5). Throughout the tremendous evolution of their hosts, herpesviruses have successfully adapted themselves and kept their ability to establish lifelong infections. Apparently, viral kinases were not selected away in the evolutionary process but instead may have aided in the adaptation of these viruses to the increasing complexity of their host.

Intrinsic to the limited size of their genome, in order to be able to replicate and spread in a host, viruses have to do a lot with a little. Hence, it comes as no surprise that many viral proteins are multifunctional. This is especially true for viral kinases, which have the ability to phosphorylate, and thereby alter, several viral and cellular targets, which may result in a plethora of different effects. Some of these effects are intentional and beneficial for viral replication, and others may actually be side effects that are biologically irrelevant and thereby artifactual. One of the main tasks in the field of viral kinase research will be to better discriminate these possibilities. This is especially challenging since the major roles of viral kinases in pathogenesis do not appear to translate easily to obvious phenotypes in vitro. Indeed, as indicated above, many kinases, including the herpesvirus kinases, are often not absolutely required for growth in cell culture, although lack of viral kinase activity results in strongly reduced virulence. One important issue may be that, in contrast to the typical immortalized cell cultures, many of the viral target cells in vivo comprise highly differentiated, nonproliferating cells. These in vivo target cells are therefore less suitable for viral replication, which may increase the importance of viral kinases, especially since many of these kinases have been reported to promote viral replication and gene expression. As an example to support this idea, mutation of the CHPK kinase of VZV does not substantially affect viral growth in cell culture, whereas it is essential for replication in human T cells and human thymus/liver xenografts in SCID-hu mice (150). Also indicative of the caution needed when interpreting results from immortalized cell lines is that SV40 T antigens have been reported to induce lamin A/C phosphorylation in 293T cells, thereby compensating for the defect in viral egress observed for a CHPK-null EBV recombinant in 293 cells (137).

In order to meet the need to better define the biological importance of individual functions of viral kinases, several approaches may be followed. Studies of important primary target cells and/or organ cultures may highlight which kinase-dependent phenotypes manifest themselves in these cells. Since many of the kinase targets are cellular proteins, homologous studies, using primary cells of the natural host of the virus, will be particularly informative. In addition, a search for mutations in the viral kinases that abrogate their interaction with specific, but not all, substrates may be vital in revealing the importance of individual functions of viral kinases. Alternatively, complementing kinase deficiency with cellular or viral factors that restore only selected functions of the kinase should also yield important data in this respect. For example, replacing the US3 gene in alphaherpesviruses with specific antiapoptotic factors will allow us to better define the importance of the antiapoptotic activity of this kinase in virus biology.

There are several indications that viral serine/threonine protein kinases may be promising future targets for the development of antiviral drugs. First, although sometimes not associated with obvious phenotypes in cell culture, as described above, all viral protein kinases identified appear to play important and frequently essential roles in virus virulence. Second, and perhaps equally important, most viral serine/threonine protein kinases are evolutionarily very distinct from cellular protein kinases, which should facilitate the selection of drugs that do not interfere with cellular kinase activity.

A first proof of principle that viral kinases may represent interesting drug targets is maribavir. Maribavir is a drug directed against the UL97 kinase of HCMV and displays potent antiviral activity against HCMV (21). It is important to keep in mind that the drug of preference used against HCMV is GCV and that UL97 plays an important role in activating GCV by phosphorylating it to its monophosphate form (123, 202). Hence, the two treatment strategies may interfere with each other (38, 61). Maribavir successfully passed phase I and phase II clinical trials (214). Although this demonstrates the potential of viral kinases as therapeutic targets, unfortunately, maribavir very recently did not pass phase III clinical trials conducted by Viropharma. Despite this mishap, viral serine/threonine protein kinases should still be considered one of the promising avenues for the design of novel antiviral drugs (3). One example may be the B1 kinase, which is of critical importance in the poxvirus replication cycle. A first indication for the potential of B1 as a target for antiviral therapy was demonstrated, since interfering RNAs that target B1 effectively reduced vaccinia and monkeypox virus yields and plaques in in vitro assays, especially when administered together with the proven antiviral drug cidofovir (220).

An aspect of viral kinases that may warrant further research is their effect on cellular signaling networks and cascades. Phosphorylation and dephosphorylation arguably represent the most important regulatory switches of signaling proteins, affecting virtually every aspect of the biology of a cell and the host organism (174). Up to now, relatively little information has been available on the potential effect of viral serine/threonine kinases on cellular signaling cascades. This is even more relevant when considering that most, if not all, of these viral kinases are incorporated in the virus particle. Although largely unexplored at this time, there is thus the possibility that viral kinases may affect cellular signaling cascades at the very early stages of infection, during viral entry, thereby preparing and perhaps steering the otherwise hostile cellular environment to their own benefit and needs.

Obviously, there is a need for more research on viral protein kinases to fully address their invariably multifunctional and pivotal roles in viral replication, spread, and immune evasion. This information, together with rational drug design strategies, is needed to explore the therapeutic potential of these viral enzymes.