Department of Physiology and Immunology, Medical Faculty, University of Rijeka, Brae Branchetta 20, 51000 Rijeka, Croatia
Correspondence
Pero Luin
perol{at}medri.hr
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ABSTRACT |
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INTRODUCTION |
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Murine cytomegalovirus (MCMV) is a betaherpesvirus that establishes productive cytolytic or non-productive latent infection in cells. The MCMV virion consists of an icosahedral protein capsid surrounding the double-stranded DNA genome, which has to be released into the cytoplasm (reviewed by Luin & Jonji
, 1995
). The entry route of MCMV and the very early events of its transport into the nucleus are still poorly understood. Various pathways are used by herpesviruses to penetrate host cells. Membrane fusion is the most common process, occurring either at the cell plasma membrane or within endocytic vesicles (reviewed by Smith & Helenius, 2004
). Among herpesviruses, differences in entry are observed, depending not only on the type of virus, but also on the targeted cell type (Miller & Hutt-Fletcher, 1992
). Human cytomegalovirus (HCMV) enters the cell by pH-independent fusion of the viral envelope with the plasma membrane, which is initiated by a signal generated inside the glycoprotein B cytosolic domain after binding to the cellular receptor (Tugizov et al., 1999
). Other events after the entry of betaherpesviruses, prior to the initiation of gene expression, require triggering of a signalling cascade that allows local reorganization of the cytoskeleton and propagation of viral components through the cytosol. It has been reported that the cellular protein kinases C (PKCs) are required in the early phase of influenza virus entry into the cell nucleus (Root et al., 2000
) and, in later phases, for herpesvirus nucleocapsid assembly and egress from the nucleus (Mettenleiter, 2002
; Muranyi et al., 2002
).
Protein kinases are key intracellular elements of signal-transduction pathways that control gene expression, modulate metabolic processes and allow the cell to respond to constantly changing extracellular conditions or to intracellular modulations induced by intracellular microbial agents (Krajcsi & Wold, 1998). The PKCs comprise a large superfamily of related proteins that carry out diverse regulatory roles in many cellular processes (Newton, 1997
). Based on structural variations and biochemical properties, the PKC family of proteins can be categorized into three groups: classical (cPKC), novel (nPKC) and atypical (aPKC). cPKCs are calcium- and phospholipid-dependent, whereas nPKCs and aPKCs do not require any calcium for their activities (Newton, 1997
). Activation of the cPKC and nPKC isoforms typically involves recruitment to membranes and interaction with both phosphatidylserine and the second messenger diacylglycerol (DAG). Tumour-promoting phorbol esters can activate both cPKCs and nPKCs, whereas aPKCs are insensitive to phorbol esters and DAG (Way et al., 2000
).
Small molecules that act by inhibition of novel molecular targets, such as the cellular and viral protein kinases, are currently being considered as therapeutic candidates against herpesviruses (Wathen, 2002). Among the small-molecule kinase-inhibitor candidates, the H-series isoquinolinesulfonamide inhibitors (H-7, H-8 and H-89) inhibit protein kinase activity by competitively inhibiting ATP interactions with eukaryotic kinases (Hidaka et al., 1984
). Within the group of H-series inhibitors, H-7 is known to be non-selective in its action and inhibits a wide range of different protein kinases (Quick et al., 1992
). In addition to the H-series of PKC inhibitors, staurosporine (Ward & O'Brian, 1992
) is a non-specific kinase inhibitor that inhibits both Ca2+-dependent and -independent PKCs at the conserved site in the catalytic domain. Sangivamycin, a potent inhibitor of PKCs, competes with the binding of ATP to the catalytic fragment (Loomis & Bell, 1988
). Calphostin C (CC) (Kobayashi et al., 1989
; Tamaoki & Nakano, 1990
), an inhibitor of the classical and novel PKC isoforms, and bisindolylmaleimide II (BIM II) (Toullec et al., 1991
), an inhibitor of different PKC isozymes, are highly potent and specific inhibitors of PKCs. The inhibitory potential of BIM II is dependent on concentration, showing more specificity and selectivity for members of the cPKC family at the nanomolar range and to the nPKCs at the micromolar range (Basu, 1998
; Wilkinson et al., 1993
).
In the present study, we used the specified protein kinase inhibitors to explore the role of protein phosphorylation mediated by PKCs in the very early events of MCMV infection, prior to initiation of the replication cycle and viral gene expression. We have found that virus replication was inhibited by these inhibitors in a dose-dependent and reversible manner, and that the block occurred at very early stages of infection.
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METHODS |
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Reagents and antibodies.
H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride] (1050 µM), phorbol 12-myristate 13-acetate (PMA) (10 nM), CC (50 nM to 0·5 µM), BIM II (50 nM to 10 µM), sangivamycin (1050 µM), EDTA (1·5 mM), [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] (W7) (10 µM) and a mAb against -tubulin (clone DM 1A) were provided by Sigma-Aldrich. mAbs croma-101 (anti-IE1) and croma-103 (anti-E1), produced in our laboratory, were used as tissue-culture supernatant or semipurified ascites.
Immunofluorescence.
MEFs, grown in six-well tissue-culture plates on glass coverslips, were used for immunofluorescence experiments. Before staining, they were washed with PBS, permeabilized with ice-cold methanol, blocked with PBS containing 2 % FCS and incubated with mAbs diluted in PBS containing 2 % FCS and 0·3 % NaN3. Bound antibodies were visualized by fluoresceinated goat anti-mouse IgG (Becton Dickinson). After immunofluorescence, cells were stained by Evans blue (red fluorescence of the cytoplasm) and analysed by fluorescent microscopy.
Metabolic labelling and immunoprecipitation.
Cells were incubated for 30 min at 37 °C in methionine-free MEM (Gibco) and labelled with [35S]methionine (Amersham Biosciences) at 200 µCi (7·4 MBq) ml1. After labelling, cell monolayers were washed three times with cold PBS and lysed in a buffer containing 10 mM Tris/HCl (pH 7·6), 150 mM NaCl, 1 % SDS, 1 % sodium deoxycholate and 1 mM PMSF. After centrifugation at 14 000 r.p.m. for 20 min, the cellular extracts were used for immunoprecipitation. Cellular lysates were precleared with 50 % protein ASepharose slurry (Amersham Biosciences) at 4 °C for 30 min. Immunoprecipitation of the immediate-early 1 (IE1) and early 1 (E1) viral proteins was performed with ascitic fluid of mAbs croma-101 and croma-103 (3 µl each). Immune complexes were retrieved with protein ASepharose (50 µl of 50 % slurry) at 4 °C for 1 h, eluted at 96 °C by incubation with SDS sample buffer [0·125 M Tris/HCl (pH 6·8), 20 % glycerol, 3 % SDS, 2 % -mercaptoethanol, 0·05 % bromphenol blue] and analysed by SDS-PAGE (10 % gel) under reducing conditions. Polyacrylamide gels with metabolically labelled proteins were exposed to autoradiography film containing scintillating emulsion (Biomax MR; Kodak).
Western blot analysis.
Cellular extracts for Western blot analysis were prepared in SDS sample buffer, separated by reducing SDS-PAGE and blotted onto a PVDF Western blotting membrane (Roche Diagnostics) at 6070 V for 2 h. PVDF membranes were washed in Tris/HCl-buffered saline (TBS) (50 mM, pH 7·5) and incubated in 1 % blocking reagent (Roche Diagnostics) for 2 h, followed by 1 h incubation with mAbs (1 : 500 dilution of ascitic fluid), three cycles of washing (TBS with 0·1 % Tween 20; TBS-T buffer) and 45 min incubation with peroxidase-conjugated goat anti-mouse Ig antibody (1 : 1000 dilution) in TBS buffer containing 0·5 % blocking reagent. After washing with TBS-T buffer, immunocomplexes were visualized by using a substrate solution containing 0·013 mM diaminobenzidine, 0·02 % H2O2 and 0·03 % NiCl/CoCl2 in PBS. For a negative control, Western blots of MCMV-infected cells were probed with isotype-mached irrelevant mAbs (IgG1 and IgG2a) (Jackson ImmunoResearch Laboratories). Signals of bands on Western blot membranes were quantified with a calibrated imaging densitometer (GS-710; Bio-Rad).
Virus-titre assays.
MEFs were treated for 1 h with inhibitors, dissolved in MEM containing 5 % FCS or appropriate vehicles (PBS, DMSO, ethanol) used for preparation of the inhibitor, and infected with MCMV at an m.o.i. of 10 by using the centrifugal enhancement of infectivity technique at 800 g for 30 min. Thirty hours after infection, cell cultures were subjected to three cycles of freezing and thawing to release infectious virus particles from cells. Number of infectious particles in tissue-culture homogenates was determined by titration on monolayers of subconfluent MEF cultures in 48-well plates by the standard viral-plaque assay, as described previously (Luin et al., 1994
). Titration was carried out in four replicates and results were presented as number of infectious virions (p.f.u.) and compared by using the MannWhitney U test.
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RESULTS |
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Expression of the IE1 protein could be detected by immunoprecipitation after 30 min and reached a maximum between the first and third hours after infection (Fig. 1a). At 2 h p.i., IE1 could be detected by Western blotting (not shown) and, at 3 h p.i., it could be detected by immunofluorescence, showing a diffuse nuclear staining except in distinct areas of the nucleus (Fig. 1b
). The E1 protein is expressed later, as a product of the first set of early genes (Bühler et al., 1990
). It is expressed throughout the early phase (Fig. 1a
) and it can be visualized in the nucleus at 3 h p.i. by immunofluorescence, with a characteristic speckled distribution (Fig. 1b
).
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H-7 reversibly blocks entry of MCMV into the nucleus
Our previous data indicated that the inhibitory effects of H-7 on MCMV replication occur at very early times of infection. To test the inhibitory effects on virus entry into the cell, we treated fibroblasts with H-7 and infected them with MCMV. Two hours after infection, H-7 was washed out and, 3 h later, the cells were examined by immunofluorescence. IE1 and E1 expression after washout was similar to that in cells that were infected in the absence of inhibitor, whereas it was almost completely absent in the presence of H-7 (Fig. 3). These data indicate that H-7 reversibly blocks entry of MCMV into the cell or unpacking of the nucleocapsid and its transport into the nucleus.
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MEFs were treated with selected inhibitors and the expression of IE1 and E1 was determined 3 h after infection by immunofluorescence and Western blotting. BIM II, tested in nanomolar and micromolar concentrations, showed little inhibitory effect on the expression of IE1 (Fig. 4a) and E1 (not shown). At the 10 µM concentration of BIM II, a large proportion of cells expressed IE1 and E1, whilst increasing its concentration enhanced its inhibitory action, but also its toxic effect on cells (data not shown). In contrast, sangivamycin (Fig. 4a
) and CC (Fig. 4b
) blocked IE1 and E1 expression completely at micromolar concentrations.
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DISCUSSION |
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H-7, a competitive inhibitor of the ATP-binding site at the catalytic domain of a protein kinase (Hidaka et al., 1984), prevented MCMV infection in a dose-dependent and reversible manner. H-7 and other isoquinolinesulfonamide inhibitors, such as H-8 and H-89, are inhibitors of a wide range of protein kinases, including PKC, PKA and PKG (Engh et al., 1996
). H-89 has a similar inhibitory effect to H-7 (N. Ku
i
, unpublished data), although H-89 shows the highest specificity for PKA (Hidaka & Kobayashi, 1992
). Besides H-7, sangivamycin, a wide-range protein kinase inhibitor (Loomis & Bell, 1988
), showed almost-complete inhibition of MCMV infection, starting at very early stages, which resulted in almost-complete inhibition of virion assembly and release of infectious virions. Sangivamycin competes with binding of ATP to the catalytic fragment of PKC and thus inhibits the PKC family (Loomis & Bell, 1988
). Distinct from H-7, an additional inhibitory effect of sangivamycin is attributed to competition with the phosphatidylserine-binding site, and it does not exert its action through the lipid-binding/regulatory domain (Loomis & Bell, 1988
).
Furthermore, we compared the effects of two more specific PKC inhibitors that exhibit differential specificity toward PKC isozymes: CC, a highly potent and specific inhibitor of DAG/phorbol ester-sensitive PKCs (Kobayashi et al., 1989; Tamaoki & Nakano, 1990
) that acts by inhibition of phorbol dibutyrate and other phorbol esters binding to PKC by competition with DAG (Redman et al., 1995
), and BIM II, an inhibitor with isozyme specificity for cPKCs and nPKCs (Toullec et al., 1991
). CC, which inhibits the classical and novel PKC isoforms (Tamaoki & Nakano, 1990
), completely prevented IE1/E1 expression and formation of infectious virions at submicromolar concentrations. In contrast, BIM II had a partial inhibitory effect on MCMV replication only at micromolar concentrations, whilst in a nanomolar range, it had no effect at all. It has been reported that the specificity of BIM II on types of PKCs is concentration-dependent (Basu, 1998
). cPKCs are the most sensitive to BIM II, for which IC50 values are in the nanomolar range, whilst nPKC can be inhibited by micromolar concentrations (Basu, 1998
; Wilkinson et al., 1993
). Thus, our experiments with BIM II indicate the involvement of nPKCs, rather than cPKCs, in early events of MCMV infection. In addition, within the PKC-activation cascade at the plasma membrane, BIM II acts at the end, whereas CC acts at the beginning of recruitment and phosphorylation reactions of PKC that are required to generate the mature, phosphorylated form (Parekh et al., 2000
), where a BIM II-sensitive molecule amplifies the signal from the CC-sensitive molecule. This can be an explanation for the stronger inhibitory activity of CC, especially if it is known that the effects of PKC phosphorylation are concerned primarily with its intrinsic catalytic activity and ability to phosphorylate and modify the actions of its own downstream targets. Enhancing effects of PMA support the evidence for involvement of the classical and novel subclasses of PKC, which have a phorbol ester-binding site and could be stimulated by PMA. In addition, the absence of inhibitory effect after cytoplasmic Ca2+ depletion by the calcium chelator EDTA and inhibition of calmodulin by W7 (O'Brian & Ward, 1989
) indicates the involvement of PKCs that are not stimulated by or dependent on Ca2+ ions. cPKCs are calcium- and phospholipid-dependent, whereas nPKCs and aPKCs do not require any calcium for their activities (Newton, 1997
). Therefore, our results suggest the role of DAG/phorbol ester-dependent, but calcium-independent, PKC during the early stage of MCMV infection.
All PKC inhibitors used in this study have different mechanisms of action and their inhibitory effects are concentration-dependent. Reduction of their concentration did not result in uniform increase of IE1 and E1 expression in all cells, but rather with an increase in the proportion of cells that expressed IE1 and E1 proteins to the level achieved in the absence of inhibitor. Thus, it seems that cells that escape the effect of an inhibitor express IE1 and E1 almost to normal levels. As the cells cannot escape exposure to the solubilized inhibitor, the outcome for an individual cell must be determined by the number of viral particles that interact successfully with that cell. This may indicate that the inhibitory activity is an all or nothing effect (Root et al., 2000), suggesting a specific early block: those virions that escape drug inhibition go on to give normal levels of protein expression.
It is known that preceding activation of PKC induces a considerable increase in HCMV infection of endothelial cells and that activity of PKC is modulating factor for HCMV infectivity (Slobbe-van Drunen et al., 1997). The mechanism by which HCMV and MCMV enter the cell is unclear, but it is known that the early events in response to HCMV, including phosphorylation of the cell-surface glycoprotein H receptor, is mediated by tyrosine kinases rather than PKCs (Keay & Baldwin, 1996
). This may indicate that cell-phosphorylation processes mediated by PKCs are involved in steps of viral transport to the nucleus and viral protein expression after virion internalization. Several groups have suggested a role for PKC in endocytosis, as well as for virus entry into the cell (Constantinescu et al., 1991
; Root et al., 2000
; Siecskarski & Whittaker, 2002
). The entry of several enveloped viruses, including rhabdoviruses, alphaviruses, poxviruses and herpesviruses, has been proposed to require PKC for the processes of viral movement through the cell. These results have been based on the action of protein kinase inhibitors, such as H-7 and staurosporine (Constantinescu et al., 1991
). More recently, it has been shown that the successful entry of influenza virus requires PKC (Root et al., 2000
). In the presence of BIM I, influenza virus is prevented from entering the cell or from escaping endosomes and accumulates in cytoplasmic vesicles near the cell periphery. Furthermore, the role of PKC has been confirmed in herpesvirus nucleocapsid egress from the cell nucleus during the enveloping process (Muranyi et al., 2002
).
Cellular protein kinases could be required for remodelling of the cellular cytoskeletal network. The size and structural organization of the cytoskeleton itself would provide a serious obstacle to the diffusion of virus nucleocapsid, viral protein complexes and even individual proteins in the absence of regulated and facilitated transport. The composition of the cytoskeleton makes free diffusion minimal. Thus, by utilizing the trafficking network of the cytoskeleton, virus and its components would traverse quickly through the cytoplasm to direct their perinuclear accumulation (reviewed by Campbell & Hope, 2003). The efficient transport system of the eukaryotic cell is mainly composed of actin microfilaments, which are used for short-range transport, and microtubules, which are used for long-range transport that is partially regulated by phosphorylation mediated by PKC (Keenan & Kelleher, 1998
). The actin network is required primarily for virion entry, and microtubules for active transport of viral proteins to the nuclear membrane (Campbell & Hope, 2003
). Disruption of actin and microtubular networks by cytochalasin D and nocodazole synergistically prevented expression of the IE1 and E1 proteins in our model (N. Ku
i
, unpublished data). Similarly, it has been demonstrated that intact microtubules are required for the infection of epithelial cells with adenovirus and herpes simplex virus (Sodeik et al., 1997
).
In the past, the search for antiviral drugs was focused mainly on replicases and other viral enzymes. One of the most effective ways of preventing virus infection and disease is to prevent the initial entry of virions into their target cells or very early events during infection. Thus, further studies on very early events of virus replication will add to our knowledge of virus entry and transport to the cell nucleus, as well as understanding the contribution of cellular mechanisms and signalling pathways.
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ACKNOWLEDGEMENTS |
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Received 28 October 2004;
accepted 15 April 2005.
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