Biochimica et Biophysica Acta 1697 (2004) 225 – 231 www.bba-direct.com
Review
Viral and cellular kinases are potential antiviral targets and have a central role in varicella zoster virus pathogenesis Jennifer F. Moffat a,*, Michelle A. McMichael b, Stacey A. Leisenfelder a, Shannon L. Taylor a a
Department of Microbiology and Immunology, SUNY Upstate Medical University, 750 East Adams Street Syracuse, NY 13210, USA b Pennsylvania State University at Hershey, Hershey, PA, USA Received 24 September 2003; accepted 12 November 2003
Abstract Herpesviruses utilize viral and cellular kinases for replication, and these mediate essential functions that are important for viral pathogenesis. Elucidating the roles of kinases in herpesvirus infections may highlight virus – host interactions that are possible targets for kinase inhibitors with antiviral activity. Varicella zoster virus (VZV) encodes two kinases that phosphorylate viral proteins involved in regulation, assembly, and virulence. VZV infection also induces the activity of host cell cyclin-dependent kinases (cdk4 and cdk2) in nondividing cells, causing a disregulation of the cell cycle. Roscovitine and Purvalanol, kinase inhibitors that target cdks, prevent VZV replication at concentrations with few cytotoxic effects. Cdk inhibitors therefore have potential as antivirals that may extend to a broad range of viruses and have the added advantage that resistance does not arise easily. D 2003 Elsevier B.V. All rights reserved. Keywords: Varicella zoster virus; Herpesvirus; Cyclin-dependent kinase; Kinase inhibitor; Purvalanol; Roscovitine; Cell cycle
1. Introduction Herpesviruses have co-evolved with humans, leading to finely tuned interactions between virus and cell proteins. The high degree of specificity of some of these interactions can lead to a narrow virus host range or tissue tropism. For example, human cytomegalovirus (HCMV) infects only humans and cultured human cells, and herpes simplex virus (HSV) and varicella zoster virus (VZV) are characterized by their tropism for neurons and epithelial cells. Cellular factors determine host range and tissue tropism by mediating multiple phases of infection, including virus attachment and entry, DNA replication, mRNA transcription, protein synthesis and trafficking, and virus assembly. Many of these processes are tightly regulated by phosphorylation, and both viral and cellular kinases contribute essential functions for virus replication. The role of kinases in
Abbreviations: Cdk, cyclin-dependent kinase; CK2, casein kinase II; HFF, human foreskin fibroblast; HCMV, human cytomegalovirus; HSV-1, herpes simplex virus type 1; Olo, Olomoucine; Purv, Purvalanol; Rosco, Roscovitine; SCID-hu, severe combined immunodeficient mouse with human tissue xenograft; VZV, varicella zoster virus * Corresponding author. Tel.: +1-315-464-5454; fax: +1-315-464-4417. E-mail address:
[email protected] (J.F. Moffat). 1570-9639/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2003.11.026
VZV pathogenesis is a new area of investigation that has revealed some of the complex interactions between virus and host. The hypothesis of researchers in this field is that both viral and cellular kinases determine host range and pathogenesis. Kinase inhibitors may not only help us understand the virus – host interaction, but also may serve as novel antiviral agents. 1.1. Varicella zoster virus VZV is a 125-kb, double-stranded DNA virus that encodes 69 unique open reading frames. VZV proteins manipulate the cellular environment, mediate virus replication at the expense of the host, produce virion progeny, and establish and maintain the latent state. During primary infection, VZV causes varicella, or chicken pox, and replicates in mucosal epithelial cells, T cells, neurons, and dermal fibroblasts and epidermal keratinocytes in skin. Lifetime latency is established in ganglia and reactivation later in life leads to the painful condition known as zoster, also called shingles. VZV infects mainly non-dividing cells, thus the virus must induce the cellular DNA synthesis machinery it needs to replicate its own genome. VZV encodes two serine-threonine protein kinases that are important for replication in vivo, but are not required for
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2. VZV kinases and their role in infection
kinases and glycoproteins in pathogenesis [17]. In the absence of ORF47 protein, VZV was unable to grow in either human thymus T cells or in full-thickness skin implants, indicating a requirement for ORF47 in vivo [18,19]. When ORF47 protein contained alterations in the key ATP binding site at amino acid residues D282Y283S284, or the C-terminal region where the active site is located was deleted, some VZV replication occurred in skin, although it was limited to small lesions in the epidermis [14]. The discrepancy between the in vivo requirement for the entire ORF47 protein and the partial dispensability of the kinase activity was clarified by use of mutants in the N-terminus that preserved the active site but were unable to bind to IE62, an ORF47 substrate and essential viral transactivator. These data indicate that ORF47 is multifunctional, with kinase activity as well as protein –protein binding properties that play essential roles in VZV pathogenesis.
2.1. VZV ORF47 kinase
2.2. VZV ORF66 kinase
Proteins related to ORF47 are found in all herpesviruses, and include the UL13 kinase of HSV-1 [5,6] and the UL97 kinase of HCMV [7]. The active sites of these viral kinases have a motif with limited homology to casein kinase II (CK2). ORF47 phosphorylates serines and threonines in the context of acidic residues glutamate and aspartate (S/T-X-D/ E-D/E), and these acid patches are found on the viral targets. ORF47 has been cloned and characterized, and its viral substrates in vitro include itself, the important transactivators IE62 and IE63, the essential glycoprotein E, and the protein encoded by ORF32 [8 – 12]. Cellular targets are likely but are unidentified. There are distinct differences between ORF47 and CK2 in that heparin and DRB (5,6dichloro-1-h-D-ribofuranosylbenzimidazole) inhibit CK2, while having no effect on ORF47 activity in vitro. Other unique features are that ORF47 activity is stimulated by manganese and the polyamine spermidine, and both ATP and GTP can serve as donors in the phosphoryltransferase reaction. Advances in molecular genetic techniques for VZV allowed the construction and isolation of recombinant mutant viruses that lacked ORF47 expression entirely, or encoded point mutations that abolished kinase activity [13,14]. Isolation of these mutants was contingent on the ability of the recombinant viruses to grow in cultured cells, thus ORF47 is not an essential protein. In the human melanoma cell line commonly used to cultivate VZV, ORF47 mutants had no growth defects. This was surprising since ORF47 phosphorylates essential virus proteins and is a major component of the tegument, an amorphous protein structure that lies between the viral capsid and envelope [15]. The importance of ORF47 became evident when mutants were studied in human tissues in a mouse model of VZV pathogenesis [16]. This model consists of human thymus or skin tissue implanted in immunodeficient SCID mice (SCIDhu), and has been invaluable for studying the role of VZV
The VZV kinase encoded by ORF66 is member of a group of kinases found only in the alphaherpesvirinae, and includes the US3 kinases encoded by HSV and the swine pathogen pseudorabies virus (PRV) [4,20 – 22]. The US3 kinases have roles in protection of infected cells from apoptosis and in virus assembly, but neither of these functions has been shown for ORF66 [23 – 27]. The US3 kinases do not appear to be required for growth in cultured cells for any alphaherpesvirus studied, but all viruses deleted for US3 are attenuated in animal models. The recombinant VZV mutant strain that lacked ORF66 was tested in the SCID-hu model and normal levels of virus were recovered from skin implants, whereas there was a reduction in virus growth in T cells [18,19]. ORF66 protein is found in both the nucleus and cytoplasm of infected cells and when expressed from transfected plasmids; however, it is highly insoluble, forms aggregates, and has not been purified [28]. An unexpected finding was that IE62 protein was phosphorylated by ORF66 in infected cells and in vitro (P.R. Kinchington, personal communication). Further analysis with ORF66 mutants disrupted in the catalytic or ATPbinding sites, using IE62 peptides as substrates, determined that ORF66 specifically phosphorylates IE62 on domains that are separate from those targeted by ORF47 kinase and cellular CK2 [28]. ORF66 targeted serines and threonines that were preceded by three or four basic amino acids such as arginine and lysine. Recent studies demonstrated an important function of ORF66 in mediating the cytoplasmic localization of IE62 late in infection as a prerequisite for incorporation into virion particles [29]. One model proposes that this could be a key feature of VZV latency, since ORF66 and IE62 are both detected in the cytoplasm of latently infected human neurons [30]. ORF66 may bind to and phosphorylate IE62, retaining it in the neuron cytoplasm in order to prevent IE62 from initiating VZV gene transcription that could lead to reactivation.
growth in cultured cells. These kinases, ORF47 and ORF66, were identified based on homology to cellular kinases after the VZV genome was completely sequenced [1– 4]. Phosphorylation of VZV proteins by viral and cellular kinases is important for replication, but these interactions are not fully defined since they are complex and may be dependent on the cell or tissue being studied. It is not known whether the kinases targeting VZV glycoproteins or the phosphorylation sites are the same in cultured cells as in vivo. In addition, cyclin-dependent kinases are disregulated in VZV-infected cells and appear to be required for VZV replication. The contribution of kinases to VZV pathogenesis will be discussed, with special emphasis on the emerging use of kinase inhibitors.
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2.3. Phosphorylation of viral glycoproteins Phosphorylation by viral and cellular kinases is necessary for the function of two VZV glycoproteins, gE and gI, that form a heterodimer in infected cells. The VZV gE/I complex is essential in cultured cells and in vivo for its role in cell – cell spread and virus envelopment [31 –34]. Movement of gE from the trans-Golgi to the plasma membrane, endocytosis, and return to the trans-Golgi are mediated by phosphorylation-sensitive sites on the cytoplasmic tail [35]. These sorting signals are regulated by kinases that phosphorylate serine/threonine at a CK2 consensus site and tyrosine at an YXXL motif [35 – 39]. Studies are underway to determine whether deletion of the kinase recognition sites in the gE tail have an effect on gE trafficking and virus replication in the SCID-hu model. The phenotypes of these gE mutants may be obscured by the presence of gI, which also encodes endocytosis motifs and phosphorylation sites
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on its cytoplasmic tail [40,41]. The gI endodomain contains an SPP triplet at amino acid 343 that is recognized by cdk1 and cdk2 [42]. It is possible that CK2 and cdks interact at the gE/I endodomain, since the recognition sites are in close proximity and cdk1 is known to phosphorylate CK2 [43,44]. The ORF47 kinase also binds to the CK2 site on gE and it will be very interesting to learn if it is a target of cdks bound to gI. One hypothesis is that CK2 can substitute for ORF47 in certain cell types, thus ORF47 is essential in cells and tissues where CK2 activity is low. Other unresolved issues are whether ORF66 kinase, which has limited homology to cdk1, can phosphorylate gI at the cdk site, and if ORF47 is a substrate of ORF66. The importance of phosphorylation to the function of gE/I and IE62 in cultured cells is clear, and VZV uses cellular kinases and two viral kinases to regulate them. However, additional kinase– substrate interactions that are critical to VZV replication remain unidentified.
Fig. 1. VZV infection induces cdk2 and cdk4 activity and alters their localization in fibroblasts. HFF cells were infected with VZV, or mock infected, for 7 days, and then cell lysates were prepared in radioimmunoprecipitation (RIPA) buffer (upper panel). The Western blots for cdk4, cdk2, cyclin E and h-actin showed increases in the amount of hypophosphorylated cdk2 and in cyclin E. Cdk2 and cdk4 were immunoprecipitated and kinase assays performed using histone H1 or a GST-pRb fusion protein as substrates, respectively. Only VZV-infected fibroblasts had detectable kinase activity that phosphorylated the substrate. HFFs were grown on glass chamber slides and infected with VZV for 2 days (lower panel). VZV proteins were detected with a high titer polyclonal human serum and a FITC-conjugated anti-human antibody (green). Cdk4 and cdk2 were detected with rabbit polyclonal antibodies and PE-conjugated antirabbit antibodies (red). Slides were examined by fluorescence microscopy at 10 magnification and digital images were captured. The merged images show that cdk4 and cdk2 were present in VZV plaques.
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3. VZV interaction with cyclin-dependent kinases 3.1. Disregulation of the cell cycle When VZV causes primary infection, establishes latency, and reactivates, the virus infects non-dividing and terminally differentiated cells in skin and neurons. When, for example, VZV infects fibroblast cells in the skin, they are in the G0 phase of the cell cycle. In these quiescent cells, regulators such as pRb, p21 and p27 actively suppress biosynthetic pathways for DNA replication and cell division. VZV infection subverts these suppression mechanisms in an unknown manner and results in disregulation of cyclin-dependent kinases. The G0 kinase cdk4 translocates to the nucleus of VZV-infected fibroblasts, and lysates of infected cells contain cdk4 activity while uninfected confluent fibroblasts do not (Fig. 1). The function of cdk4 in normal cells is to phosphorylate pRb, which allows release of the transcription factor E2F that then enters the nucleus to activate many genes involved in cell cycle progression and DNA synthesis. E2F binds to the promoter for cyclin E, the activating partner of cdk2, and cdk2/cycE further phosphorylates pRb, creating a positive feedback signal for E2F [45]. Similar to the induction of cdk4, VZV induces cdk2/cycE expression and activity in infected fibroblasts (Fig. 1). This enzymatic cascade is necessary for passing the restriction point between G1 and S phases. It is during S phase that the cell has abundant nucleotides and enzymes used for DNA synthesis that are necessary for VZV replication. The related herpesvirus, HCMV, also induces cdk2/cycE activity in fibroblasts, yet the infected cells arrest in late G1 phase and do not fully enter S [46 –49]. Cdk activity is critical for altering the intracellular environment of non-dividing cells toward one that favors VZV and HCMV replication; cdk activity is also important for the function of viral proteins, such as the phosphorylation of gI by cdk1.
treated with 25 AM Rosco did not appear to undergo apoptosis, did not lyse during treatment for 48 h, and did not completely arrest in G1 phase, although they grew more slowly than untreated cells. In melanoma cells infected with VZV, 25 AM Rosco and 10 AM Purv caused a 10-fold or greater reduction in VZV yield after 24 h (Fig. 2). Removing the drugs allowed VZV replication to resume, providing further evidence that Rosco and Purv were not cytotoxic during infection. Even after treating with Rosco or Purv for 24 h, VZV proteins were detectable by immunofluorescence microscopy in single cells, indicating that the drugs prevented cell-to-cell spread of the virus. The inability of VZV to spread during Rosco treatment can be explained by the inhibition of VZV mRNA transcription in the presence of Rosco, which would abrogate translation of viral proteins and synthesis of viral DNA (data not shown). Importantly, transcription from the IE62 gene was reduced, and as IE62 is likely essential for transcription of all other VZV genes, this alone could prevent initiation of infection. Rosco inhibits cdks 1, 2, 3, 5, 7, and 9, and Erks 1 and 2 at higher
3.2. Effects of cdk inhibitors The finding that herpesviruses utilize cdks led several groups to test the antiviral effects of cdk inhibitors against HSV, HCMV, VZV and other viruses (Taylor et al. J. Virol., in press) [50,51]. The concept was validated when Olomoucine (Olo), Roscovitine (Rosco) and Purvalanol A (Purv), all purine derivatives that competitively bind to the cdk ATPbinding site, were found to prevent herpesvirus replication [52 –55]. In the case of VZV, Purv was the most potent and Olo the least. The efficacies of Rosco and Purv were measured by the reduction in VZV yield and by quantitation of viral genomes using Real Time PCR (TaqMan), giving EC50 values of 14 AM for Rosco and 1 – 2 AM for Purv (data not shown). Substances that target cell proteins are potentially cytotoxic [56], so the effects of Rosco on the melanoma cells used to cultivate VZV were evaluated. Melanoma cells
Fig. 2. Roscovitine and Purvalanol prevent VZV replication. Melanoma (MeWo) cell monolayers were inoculated for 2 h with VZV-infected MeWo cells that had been briefly sonicated to disrupt the cells, then Rosco or Purv was added, or the cells were mock-treated with tissue culture medium containing the same concentration of drug diluent (DMSO). Virus titers were determined daily in cultures that were mock-treated (black diamonds), had the drug removed after 24 h (green triangles), or were continuously treated with the drugs (blue squares). The effects of Rosco and Purv were reversible, and the yield of VZV from drug-treated cultures was significantly lower than the mock-treated controls (Student’s t test, P < 0.05).
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concentrations, so numerous reactions could be the basis of its antiviral effects, and different stages of infection may require different cdks [55]. Understanding the antiviral mechanism of cdk inhibitors is presently an area of intense interest. 3.3. Roscovitine inhibits cdk2 Closer examination of the effects of cdk inhibitors on VZV replication revealed two important findings: first, that the cdk2 activity induced by VZV was inhibited by Rosco, and second, that no VZV mutants resistant to Rosco or Purv arose. These results support the concept that the antiviral effects of cdk inhibitors are mediated through interference with cellular functions, not by targeting virus proteins. Increasing amounts of Rosco inhibited cdk2 activity from VZV-infected fibroblasts, whereas mock-infected confluent
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(non-dividing) fibroblasts had no detectable cdk2 activity (Fig. 3). Thus, the cdk2 in VZV-infected cells is likely complexed with cyclin E or cyclin A and is sensitive to Rosco. Experiments are in progress to determine which cyclin is bound to cdk2 during VZV infection. It remains a possibility that cdk inhibitors bind to and inactivate viral proteins, although this has been studied in detail and no HSV proteins bound to immobilized Purvalanol [57]. A second line of evidence that cdk inhibitors do not target VZV proteins was that resistance to these compounds did not develop. VZV was cultivated for 10 weeks in the presence of sub-inhibitory concentrations of Rosco, Purv, and acyclovir. As expected, resistance to acyclovir arose within 1 week and the EC50 increased to >40 AM. In contrast, no VZV resistant to >5 AM Rosco or >0.5 AM Purv was detected. Efforts to select HSV mutants resistant to Rosco were also unsuccessful [51]. Nucleoside analogs such as acyclovir, which are highly effective and well-tolerated treatments for herpesvirus infections, are dependent on viral thymidine kinases (TK) and ultimately target the viral DNA polymerase, causing chain termination [58]. Mutations in the VZV TK or polymerase genes result in resistance to acyclovir, and this has occasionally been reported in patients who are given longterm acyclovir therapy for chronic zoster [59 –61]. A potentially great advantage of cdk inhibitors as antiviral agents is that resistance is extremely unlikely to arise by mutations in the virus.
4. Conclusions and perspectives
Fig. 3. Roscovitine inhibits cdk2 activity induced by VZV infection. Confluent HFF cells were infected with VZV or mock infected for 7 days. Subconfluent melanoma cells are rapidly dividing and so were used as a positive control for cdk2 activity. Cells were harvested in RIPA buffer, cdk2 was immunoprecipitated, and phosphorylation of histone H1 was performed in a kinase assay that contained 0, 1, 5, or 20 AM Rosco. Arrowheads indicate the position of 32P-histone. Cdk2 activity was detected only in VZV-infected HFF’s and the positive control, and Rosco inhibited the kinase activity.
Viral enzymes have been the targets of choice in developing chemotherapeutic drugs because they have advantages of selectivity and low toxicity. However, viruses also depend on cellular kinases that can be attractive targets. The tremendous success of anti-herpesvirus drugs such as acyclovir and ganciclovir attests to using viral kinases, thymidine kinase in the case of HSV and UL97 kinase in the case of HCMV, to activate these nucleoside analogs in infected cells. The concept that viral protein kinases are useful antiviral targets had been overlooked until Shugar [62] outlined this strategy in 1999, and described the enzymatic characteristics of multiple viral protein kinase targets. At that time, inhibitors of cellular kinases were just beginning to receive attention as antiviral agents [50,51]. As more basic research into this area is accomplished, interest may grow in targeting antiviral agents toward cell proteins. Coen and Schaffer [63] brought this idea forward in a recent review. Progress may be rapid since there are many kinase inhibitors and other compounds that have been developed as anticancer agents, and some of these may have antiviral activity against a broad range of viruses. VZV utilizes viral and cellular kinases to achieve its replication, and these kinases are essential for pathogenesis. The role of kinases in VZV replication was obscured when the virus was cultivated in rapidly dividing tumor cell lines,
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which express abundant cell kinases and biosynthetic machinery, but became evident in systems using non-dividing cells and whole human tissues. Primary cells such as human foreskin fibroblasts (HFFs) and human tissue systems such as the SCID-hu model are far less convenient than cultured tumor cells, but they will be necessary for future studies on the function of kinases in VZV infections. In a recently developed human skin explant model, Rosco inhibited the growth of VZV at the same levels used in cultured cells (Taylor and Moffat, unpublished data). The skin explant model will be used to determine the optimal doses of cdk inhibitors on VZV replication in whole skin, which will lead directly to in vivo experiments in SCID-hu mice. Studies are planned to test the antiviral activity of Rosco and Purv in the SCID-hu model, using implanted mini diffusion pumps to continuously deliver the drugs. Cdk inhibitors have already been useful tools to clarify the complex interactions between virus and cell proteins, and if successfully developed, they show great promise for new treatments for VZV, especially as topical agents.
Acknowledgements Robert Morton provided excellent technical assistance. This work was supported by the Hendricks Research Fund, SUNY Upstate Medical University and Public Health Service grant R01 AI052168 (J.F.M.).
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