Herpesvirus infection of arterial smooth muscle cells can lead to marked accumulation of cholesterol (1) due, in part, to decreased cholesteryl ester (CE) ()hydrolysis(2) . HSV-1 infection has also been linked to decreased intracellular levels of cyclic AMP, a second messenger known to activate cellular CE hydrolases(3) . To date, this second messenger system is the only system known to significantly alter cellular cholesterol metabolism following herpesviral infection. However, HSV-1 infection alters other signal transduction pathways that could potentially affect cellular metabolism of cholesterol. For example, viral activation of tyrosine phosphorylation has been related to viral cytopathologic effects(4) , and, HSV-1 infection can also increase phosphatidylinositol 4,5-bisphosphate levels (5) and inositol phospholipid turnover(6) . Whether these signal transduction pathways, or others, are linked to altered lipid metabolism in the infected cell remains to be defined.

Cholesterol homeostasis in mammalian cells is maintained by the activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol synthesis(7) , and the low density lipoprotein (LDL) receptor, which provides an exogenous source of cholesterol. Sterol-mediated repression of transcription of the genes encoding these proteins inhibits expression of the HMG-CoA reductase and of the LDL receptor genes under conditions in which cholesterol is sufficient for normal cellular maintenance(8, 9, 10, 11) . This prevents excessive accumulation of cholesterol and CE.

The involvement of specific signal transduction pathways and second messengers have been implicated recently in the regulation of cholesterol homeostasis in non-virally infected cellular systems. Activators of protein kinase C (e.g. phorbol ester)- and protein kinase A (e.g. cyclic AMP)-dependent pathways modulate the activities of HMG-CoA reductase (7, 12, 13) and the LDL receptor(14, 15) . Our laboratory has demonstrated that the activities of acidic and neutral cholesteryl ester hydrolases (ACEH and NCEH) are also cyclic AMP-dependent(16) . Cholesterol efflux from cholesterol-loaded cells is also enhanced by activation of protein kinase C and cyclic AMP-dependent protein kinases (17, 18) . In virally infected cells, the link among protein kinase activation, the activation of specific signal transduction systems, and the control of cholesterol trafficking also has not been defined.

HSV-1 DNA encodes, in part, viral structural proteins, envelope glycoproteins, and viral DNA polymerases. It also encodes several proteins that are functionally related to cellular enzymes including a viral protein serine/threonine kinase (VPK)(19, 20, 21, 22) . VPK has distinct substrate specificity but may share some substrates with protein kinases A and C as determined by their ability to phosphorylate synthetic oligopeptides. The cellular substrates for VPK are unknown, but several viral proteins have been identified as substrates(23, 24) . In this report, we provide experimental evidence supporting the hypothesis that a viral kinase (VPK) alters cellular protein kinase-dependent pathways, which regulate the control of cellular cholesterol trafficking and cellular CE accumulation.

EXPERIMENTAL PROCEDURES

Materials

Viruses

Cell Culture

Virus Infection of Cells

Isolation and Labeling of LDL

LDL Binding, Internalization, and Degradation

RNA Isolation and Northern Analysis

LDL Receptor Promoter in Luciferase Reporter Gene Construct

Calcium Phosphate Transfection

Luciferase Assay

Measurement of Cholesterol and Cholesteryl Ester Mass

Cholesteryl Ester Metabolizing Enzymes

Esterification of [H]Oleic Acid into Cellular Cholesteryl Ester

Assay of Virus-induced Protein Kinase (VPK) Activity

Assay of Protein Kinase A and C Activities from Smooth Muscle Cells

Protein Assay

Statistical Analysis

RESULTS

Since no published study to date has addressed the effects of herpesviral infection on cholesterol delivery with its subsequent metabolic fate, we first evaluated the effects of HSV-1 on the binding and uptake of LDL-derived cholesterol and the influence of viral infection on expression of the LDL receptor. The specific binding of I-LDL (at 4 °C) to arterial smooth muscle cells increased 3-9 h post-infection by HSV-1, and decreased to basal levels by 24 h post-infection (Fig. 1A). Furthermore, studies conducted at 37 °C demonstrated that HSV-1 enhanced internalization (Fig. 1B) but decreased degradation of LDL (Fig. 1C).

Figure 1: A, effect of HSV-1 infection on specific I-LDL binding. Smooth muscle cells were grown to 90% confluence in 12-well plates (24 mm) and then placed in serum-free medium (control) or serum-free medium inoculated with HSV-1 (m.o.i. = 0.5) for 3, 6, 9, and 24 h prior to the addition of I-LDL (50 µg/ml) at 4 °C for 2 h. LDL binding was assessed in supernatants following the release of receptor-bound I-LDL by dextran sulfate. Nonspecifically bound I-LDL was determined by incubating smooth muscle cells with a 100-fold excess of unlabeled LDL. Specific LDL binding is determined by subtracting nonspecifically bound counts from total counts released. The data derived from the HSV-1 group are significantly different from the uninfected group at all but the 24-h time point (* = p < 0.05). Binding was normalized to protein content of the cell layer. Points represent the mean of triplicate wells ± S.E. and are representative of two separate experiments. B and C, effect of HSV-1 infection on I-LDL uptake and degradation. Control and HSV-1-infected cells (m.o.i. = 0.5) were incubated for the indicated times prior to the addition of I-LDL (50 µg/ml) at 37 °C for 5 h. Degraded I-LDL was assayed in the supernatant as described under ``Experimental Procedures.'' Surface-bound I-LDL was release by treatment with 4 mg/ml dextran sulfate in PBS, and cell-associated I-LDL was determined after the cells were solubilized in 0.2 N NaOH. Each point represents the mean of quadruplicate wells ± S.E. and is representative of two separate experiments (* = p < 0.05).

To test the hypothesis that HSV-1 infection increased LDL receptor activity by altering the rate of expression of its mRNA, we first examined the influence of HSV-1 on the steady state levels of LDL receptor mRNA. HSV-1 infection enhanced LDL receptor mRNA (normalized to mRNA of glyceraldehyde-3-phosphate dehydrogenase gene) 5-fold after 60 min when compared to control cells (Fig. 2A). Viral-induced degradation of GAPDH mRNA was not observed during the time course of this experiment. Second, since actinomycin D inhibited the HSV-1 induction of the LDL receptor gene (data not shown), we tested the hypothesis that increased mRNA of LDL receptor following HSV-1 infection was due to increased transcriptional activity of the LDL receptor gene. Using a construct composed of the luciferase gene driven by the LDL receptor gene promoter(32) , we observed that by 2 h, HSV-1 infection or PMA stimulation (as a positive control) significantly induced luciferase activity by 5- and 4-fold, respectively, as compared to control cells (Fig. 2B). Luciferase activity in virally infected cells increased 12-fold relative to uninfected cells by 12 h post-infection.

Figure 2: A, HSV-1 infection induces LDL receptor mRNA. Smooth muscle cells were grown in lipoprotein-deficient medium for 24 h prior to infection. Total RNA was isolated from control (uninfected) and HSV-1-infected (m.o.i. = 1.0) smooth muscle cells at the indicated time points (10, 30, 60, and 120 min of HSV-1 infection). Northern blots were hybridized with P-labeled LDL receptor cDNA. Histograms represent densitometric scanning of the LDL receptor mRNA band normalized to mRNA of GAPDH and are expressed as a percentage of control. Insets are the respective autoradiograms with LDL receptor and GAPDH bands labeled and indicated with an arrow. B, HSV-1 infection induces LDL receptor gene promoter activity. Smooth muscle cells were transfected by the calcium phosphate method with LDLRP-Luc, a plasmid luciferase construct consisting of the promoter region from human LDL receptor gene in plasmid Luc containing the luciferase gene. Cells were infected with HSV-1 (m.o.i. = 1.0) or stimulated with PMA (100 nM) as a positive control. The luciferase activities of transfected cells were measured at the indicated times. Data derived from the HSV-1 group and PMA-treated group that are significantly (p < 0.05) different from the control group are indicated by (*). Data are expressed as the mean of quadruplicate wells ± S.E. and are representative of four separate experiments.

Since increased de novo cholesterol biosynthesis can also contribute to the increased mass of cholesterol in HSV-1-infected cells, we tested the hypothesis that HSV-1 infection altered the activity of HMG-CoA reductase. HSV-1 modestly increased HMG-CoA reductase activity relative to uninfected cells (Fig. 3A). This was reflected in a significant increase in HMG-CoA reductase mRNA in infected cells when compared to control cells (Fig. 3B). As expected, HMG-CoA reductase activity and its mRNA level are markedly reduced in both infected and control cells in the presence of 25-hydroxycholesterol and mevalonic acid (Fig. 3, A and B). However, in the presence of 25-hydroxycholesterol and mevalonic acid, there was a significant increase in HMG-CoA reductase activity and its mRNA level in HSV-1-infected cells relative to uninfected cells (Fig. 3, A and B).

Figure 3: A, effects of HSV-1 Infection on activity of HMG-CoA reductase in smooth muscle cells. Smooth muscle cells were grown to 95% confluence in Medium 199 containing 10% fetal calf serum. Prior to HSV-1 infection (m.o.i. = 0.5), half of the flasks were replaced with serum-free medium containing lipoprotein-deficient serum (LPDS, 5 mg/ml) plus 25-hydroxycholesterol (5 µg/ml) and mevalonic acid (20 mM), and incubated 6 h at 37 °C. After 6 h, the cells were washed, harvested, homogenized, and assayed for HMG-CoA reductase activity as described in Experimental Procedures. The data are expressed as nmol/h/mg protein. Data derived from the HSV-1-infected group are significantly different (p < 0.05) from the uninfected group in both the presence and absence of 25-hydroxycholesterol and mevalonic acid. Sample1 (Control), control cells grown in Medium 199 containing LPDS (5 mg/ml); sample2 (HSV), HSV-1-infected cells grown in Medium 199 containing LPDS (5 mg/ml); sample3 (Control/CHOL+MEV), control cells grown in Medium 199 containing LPDS plus 25-hydroxycholesterol (CHOL) and mevalonic acid (MEV); sample4 (HSV/CHOL+MEV), HSV-1-infected cells grown in Medium 199 containing LPDS plus 25-hydroxycholesterol and mevalonic acid. Data represent the mean of quadruplicate wells ± S.E. This figure is representative of two such experiments. B, Northern blot analysis of mRNA of HMG-CoA reductase in HSV-1-infected smooth muscle cells in the presence or absence of 25-hydroxycholesterol and mevalonic acid. Smooth muscle cells were grown to 95% confluence in Medium 199 containing 10% fetal calf serum. Prior to HSV-1 infection (m.o.i. = 0.5), the medium was replaced with Medium 199 containing LPDS (5 mg/ml) or Medium 199 containing LPDS (5 mg/ml) plus 25-hydroxycholesterol (5 µg/ml) and mevalonic acid (20 mM). Total RNA was isolated following incubation at 37 °C for 6 h. Northern blots were hybridized with P-labeled cDNAs of HMG-CoA reductase and GAPDH. The position of the 4.7-kilobase mRNA of HMG-CoA reductase is marked. Histograms represent densitometric scanning of HMG-CoA reductase mRNA normalized to mRNA of GAPDH. Uninfected cells without 25-hydroxycholesterol and mevalonic acid are assigned (normalized to) a value of 1, and the other treatment groups are expressed relative to control cells. Inset is an audioradiogram of the Northern blot probed with HMG-CoA reductase as labeled. Sample1 (Control), control cells grown in Medium 199 containing LPDS (5 mg/ml); sample2 (HSV), HSV-1-infected cells grown in Medium 199 containing LPDS (5 mg/ml); sample3 (Control/CHOL+MEV), control cells grown in Medium 199 containing LPDS plus 25-hydroxycholesterol (CHOL) and mevalonic acid (MEV); sample4 (HSV/CHOL+MEV), HSV-1-infected cells grown in Medium 199 containing LPDS plus 25-hydroxycholesterol and mevalonic acid.

We next tested the hypothesis that HSV-1 infection altered CE-hydrolytic enzymes, namely ACEH and NCEH, since these enzymes have been documented to participate in the regulation of cholesterol/CE levels in the cell. Both ACEH and NCEH activities were significantly decreased (by 45% and 50%, respectively) in HSV-1-infected cells relative to control cells at 24 h post-HSV-1 infection (Fig. 4). These data are consistent with the decrease in LDL degradation observed in HSV-1-infected cells (Fig. 1C) and provide another mechanism by which HSV-1 promotes cellular CE accumulation.

Figure 4: Effects of HSV-1 infection on the cholesteryl ester hydrolytic (ACEH and NCEH) and synthetic (ACAT) enzyme activities in smooth muscle cells. Confluent smooth muscle cells were infected with HSV-1 (m.o.i. = 0.5). After a 24-h incubation, medium was removed. Cells were scraped from the flasks after two washes with ice-cold PBS, harvested, and homogenized, and samples were assayed for ACEH, NCEH, and ACAT as described under ``Experimental Procedures.'' ACEH, NCEH, and ACAT activities are expressed as nmol/h/mg protein. ACEH and NCEH activity in HSV-1-infected groups are significantly different (p < 0.05) from control groups. ACAT activity is not significantly different (p > 0.05) between HSV-1-infected and control groups. Data represent the mean of quadruplicate wells ± S.E. This figure is representative of two such experiments.

We also determined if HSV-1 infection altered the rate of CE synthesis as measured by the incorporation of isotopic fatty acid into nascent CE. At the initiation of the experiments, [H]oleic acid-albumin mixture in the absence of FCS was added to infected and uninfected cells. CE synthesis was increased in HSV-1-infected cells relative to uninfected cells (Fig. 5). However, HSV-1 infection did not alter intrinsic ACAT activity (Fig. 4). These data support the concept that HSV-1 infection increases cholesterol esterification by increasing substrate (cholesterol) availability.

Figure 5: Esterification of [H]oleic acid into cellular CE pool in HSV-1-infected and uninfected smooth muscle cells. Smooth muscle cells were grown to 95% confluence, and half the groups were infected with HSV-1 (m.o.i. = 0.5). Esterification of free cholesterol with [H]oleic acid into cellular CE was assessed in cells incubated with a [H]oleic acid-albumin mixture (final concentration of 100 µM oleate, 20 µM albumin) and LDL (25 µg/ml/well). Cell lipids were extracted, and radioactivity in cellular CE was measured after separation by thin layer chromatography. Data derived from the HSV-1-infected group (after 6 h post-infection) are significantly different (p < 0.05) from the uninfected group. Data represent the mean of quadruplicate wells ± S.E. This figure is representative of two experiments.

Next, since inhibition of efflux could contribute to cholesterol and CE retention within the virally infected cell (as it does in uninfected cells), we evaluated the effect of HSV-1 on cholesterol efflux in the presence of HDL as a plasma cholesterol acceptor particle. During a 30-h experimental period, HSV-1 infection significantly reduced cholesterol efflux compared to uninfected cells (Fig. 6).

Figure 6: Efflux of cholesterol from cellular CE in HSV-1-infected and uninfected smooth muscle cells exposed to HDL. Smooth muscle cells were grown to 95% confluence and then incubated for 24 h at 37 °C with a [H]oleic acid-albumin mixture (final concentration of 100 µM oleate, 20 µM albumin) and LDL (50 µg/ml). After 24 h, cells were washed and infected with HSV-1 (m.o.i. = 0.5) or mock infection. After 2 h, the cells were washed with medium and replaced with medium containing HDL (400 µg/ml). At the designated times, medium was removed and the cells were washed (three times with ice-cold PBS containing BSA, and three times with ice-cold PBS). Cell lipids were extracted, and radioactivity in the remaining cellular CE (cholesteryl [H]oleate) was measured over time after separation by thin layer chromatography. The data derived from the HSV-1 group are significantly (p < 0.05) different from the uninfected control group at each time point. Data represent the mean of quadruplicate wells ± S.E. This figure is representative of two similar experiments.

As noted earlier, protein kinases have been implicated in regulation of cellular cholesterol trafficking in vascular cells(13, 16, 32, 44, 45, 46, 47) and viral-induced protein kinase (VPK) has been identified in HSV-1-infected cells(19, 20, 21, 22) . To test the hypothesis that VPK activity mediates HSV-1 induced alterations in CE content, we tested a VPK mutant in which the portion of the HSV-1 genome encoding VPK had been deleted(25) . Arterial smooth muscle cells were inoculated (m.o.i. of 0.1) with wild-type HSV-1, HSV-1 VPK mutant, or an HSV-1 VPK ``repair'' mutant (in which the sequence encoding VPK is reinserted in the HSV-1 VPK mutant). CE accumulation was significantly and reproducibly increased (>50-fold) in HSV-1-infected cells compared to mock-infected smooth muscle cells. Infection by the HSV-1 VPK mutant did not effect CE accumulation, whereas smooth muscle cells infected with the HSV-1 VPK repair mutant had CE accumulation equivalent to wild-type HSV-1. Viral infection (HSV-1, VPK mutant, or VPK repair mutant) did not significantly alter free cholesterol content from levels observed in uninfected cells (Fig. 7). To rule out the possibility that failure of the VPK mutant to induce CE accumulation resulted from reduced infectivity of smooth muscle cells, infectivity and replication of the VPK mutant were determined by plaque assay. There were no significant differences in the number of plaques produced in smooth muscle cells by wild-type HSV-1 or the HSV-1 VPK mutant (data not shown).

Figure 7: Cholesterol and CE contents of arterial smooth muscle cells infected with wild-type HSV-1, HSV-1 VPK mutant, and HSV-1 VPK repair mutant and of uninfected smooth muscle cells. Arterial smooth muscle cells were cultured with or without HSV-1 or HSV-1 mutants for 24 h (m.o.i. = 0.1). Medium was then removed, cells were washed (three times with ice-cold PBS containing BSA, and three times with ice-cold PBS) and lipids extracted in hexane/isopropanol (3:2). Cholesterol (CH) and CE were quantified by GLC as described under ``Experimental Procedures.'' Data represent the mean of 5 T25 flasks ± S.E. The mean values derived from the HSV-1 and HSV-1 VPK repair mutant are significantly (* = p < 0.05) different from the mock-infected and HSV-1 VPK mutant.

Finally, we quantified VPK activity in HSV-1-infected smooth muscle cells. Relative to mock-infected cells, wild-type HSV-1 infection increased VPK activity approximately 4-fold (mock-infected; 375 ± 8.8 cpm/µg protein, HSV; 1410 ± 7 cpm/µg protein) at the 24-h time point (Fig. 8A). Kinase activity in VPK mutant-infected cells was similar to that in mock-infected cells (VPK mutant; 377 ± 6 cpm/µg protein). Kinase activity in VPK repair mutant-infected cells was increased 2.5-fold relative to mock-infected cells (VPK repair mutant; 948 ± 43 cpm/µg protein). VPK activity was not reduced by an anti-PKC monoclonal antibody, which does not recognize VPK, but was reduced by the protein kinase inhibitors staurosporine and calphostin C (Fig. 8B). Neither staurosporine nor calphostin C affected HSV-1 infectivity or replication as determined by plaque assay (data not shown). In contrast to the effects of HSV-1 on VPK activity, HSV-1 infection transiently increased PKA activity at 1-3 h post-infection but was reduced by 60% relative to uninfected cells after 24 h (Fig. 8C). This effect appears to parallel the trend in cyclic AMP reduction following HSV-1 infection(3) .

Figure 8: HSV-1 VPK activity, inhibition of VPK activity by protein kinase inhibitors, and PKA activity in HSV-1-infected smooth muscle cells. Smooth muscle cells grown to confluence in T-75 flasks were infected with HSV-1 (m.o.i. = 0.5) for 0.2, 1, 3, 6, 12, and 24 h. At the indicated times, medium was removed, cells were washed and assayed for activity of VPK and PKA as described under ``Experimental Procedures.'' Panel A, kinase activity in uninfected cells was assayed and assigned a relative value of 1. Relative VPK activity (mean ± S.E.) in HSV-1-infected cell lysates is not significantly (p < 0.05) different from HSV-1-infected cell lysates incubated with an anti-PKC monoclonal antibody at any time point as determined by analysis of variance. Panel B, protein kinase inhibitors staurosporine (0.5 nM) or calphostin C (100 nM) were added to HSV-1-infected cells. VPK activity was assayed at the indicated times. A relative value of 1 was assigned to VPK activity at the 6-h time point. The data (mean ± S.E.) derived from the HSV-1 group are significantly (* = p < 0.05) different from the HSV-1 group incubated with inhibitors. Panel C, PKA activity in uninfected (control) cells was assayed and assigned a relative value of 1. Relative PKA activities (mean ± S.E.) derived from the HSV-1-infected group are significantly (p < 0.05) different from the uninfected group at all time points except 0.2 h. These data are representative of three experiments.

DISCUSSION

Herpesviruses have been implicated as potential etiologic agents in the pathogenesis of human atherosclerosis(48, 49) . In animal models of this disease, there is a significant accumulation of cholesterol and CE within the arterial wall(2) ; this is also mimicked in vitro(2) . Viral infection results in reduced CE hydrolytic activities, which could explain, in part, one mechanism for the retention of lipid in these cells. Previously, we have shown that the cytoplasmic (neutral) CE hydrolase can be activated by a cyclic AMP-dependent protein kinase, and that a reduction in its activity is accompanied by reduced cellular cyclic AMP activity in response to viral infection(3) . However, cholesterol trafficking can also be regulated by other second messenger systems, especially those systems that activate cellular protein kinases. This study was undertaken in order to identify potential mechanisms by which herpesviral infection and replication can alter influx, intracellular metabolism, and efflux of cellular lipid.

We have shown for the first time that HSV-1 infection can increase binding and internalization of labeled I-LDL to arterial smooth muscle cells (Fig. 1, A and B). Increased surface expression of the LDL receptor by HSV-1 infection resulted from increased transcription of the LDL receptor gene (Fig. 2B) and, subsequently, an increase in steady state levels of LDL receptor mRNA (Fig. 2A). Transcription assays using an LDL receptor promoter/luciferase construct demonstrated that increased transcription occurred through activation of the promoter of the LDL receptor gene (Fig. 2B). Some cellular genes can be directly activated by HSV-1 infection(50, 51, 52) . Activation of these genes may result directly from viral entry or may require viral protein synthesis (53) . It has been speculated that the binding of HSV-1 to the cell surface could act as a ``mitogen-like stimulation'' (6) and that this mitogenic effect is mediated by virion-associated protein(s), which may be sufficient to induce expression of specific cellular genes. In this context, HSV-1 may be activating signal transduction pathways similar to those activated by growth factors and cytokines, which we (32, 54, 55) and others (56, 57, 58, 59, 60) have shown to induce surface and mRNA expression for the LDL receptor. HSV-1 infection may increase LDL receptor gene transcription through a mechanism involving phosphorylation of cellular proteins. This could involve phosphorylation of a protein that activates or contributes to the activation of LDL receptor gene transcription; alternatively, phosphorylation may inactivate an inhibitor of LDL receptor gene transcription. The mechanism by which LDL receptor activity is induced by other protein kinases (13, 46) is unknown.

Protein kinase A (PKA) constitutes an important cellular signal transduction pathway following the activation of adenylate cyclase. We determined if HSV-1 infection would alter PKA activity in the arterial cells and relate this to changes to cytoplasmic CE hydrolysis. HSV-1 infection transiently increased in PKA activity during early periods of infection, then decreased to 40% of the control samples at 24 h post-infection (Fig. 8C). A similar transient induction of adenylate cyclase activity, cyclic AMP levels, and PKA activity have also been reported after infection of various cells with other human herpesvirus, followed by an eventual decrease in PKA activity(61) . We also observed a 45% decrease in ACEH activity and a 50% decrease in NCEH at this 24-h time point (Fig. 4). This demonstrates a parallel and correlative reduction in PKA and CE hydrolase activities. The reduction in ACEH activity is reflected in a decreased degradation of I-LDL in HSV-1-infected cells (Fig. 1C), while the reduction of NCEH activity is paralleled by the results of our cholesterol efflux experiments, which show a reduced capacity of HSV-1-infected cells to release cholesterol (Fig. 6). A reduction in cytoplasmic hydrolysis of CE leads to less free cholesterol available for efflux and more CE remaining within the cell. The regulatory process involved in cytoplasmic CE hydrolysis is related, in part, to intracellular levels of cyclic AMP and the subsequent activation of cyclic AMP-dependent protein kinases. Cyclic AMP can activate CE hydrolase activities in a variety of cell types(2, 36, 44, 45) , and AMP-activated protein kinases can also regulate several key enzymes of lipid metabolism in normal cells(47) . Thus, such a metabolic sequence of events, in turn, may activate cytoplasmic CE hydrolase by covalent phosphorylation(62) .

The activity of HMG-CoA reductase, like that of the LDL receptor, is down-regulated by cholesterol, 25-hydroxycholesterol, and mevalonic acid(7, 9, 10, 11) . We found that there was a modest increase in HMG-CoA reductase activity in infected cells compared to uninfected cells cultured in serum-containing medium (Fig. 3A). As expected, 25-hydroxycholesterol and mevalonic acid in serum-free medium down-regulated HMG-CoA reductase activity (Fig. 3A) and the mRNA of HMG-CoA reductase in both infected and uninfected cells (Fig. 3B). However, in the presence 25-hydroxycholesterol and mevalonic acid, there was a significant increase in both HMG-CoA reductase activity and HMG-CoA reductase mRNA in HSV-1-infected cells compared to uninfected cells (Fig. 3, A and B). This suggests that viral infection may abrogate the repressive effect of sterols on HMG-CoA reductase activity and HMG-CoA reductase mRNA synthesis. HSV-1 infection increased the esterification of [H]oleic acid into the synthesis of nascent CE as compared to uninfected cells (Fig. 5). It is likely that this is the result of increased substrate (cholesterol) availability either from exogenous (LDL receptor) or from endogenous (HMG-CoA reductase) sources, since we observed no increase in ACAT activity in response to viral infection (Fig. 4).

HSV-1 infection also led to a significant accumulation of CE (Fig. 7). These results are consistent with our previous findings(1) . We hypothesized that VPK could be directly or indirectly involved in the modulation of cholesterol metabolism in infected cells. This viral protein kinase has been isolated(19, 20, 21, 22) , and a correlation between induction of VPK activity and herpesviral infection has been established(19) . To identify the biochemical mechanisms responsible for this large increase in CE, particularly the involvement of VPK, we tested the ability of HSV-1 VPK mutant to alter CE mass. Infection by both wild-type HSV-1 and the VPK repair mutant increased CE accumulation in the cell by >50-fold. However, the CE content of smooth muscle cells was equivalent to uninfected cells following infection with an HSV-1 VPK mutant (Fig. 7). This VPK activity could not be inhibited with an anti-PKC antibody, but could be inhibited with the kinase inhibitors staurosporine and calphostin C (Fig. 8, A and B).

In summary, we have identified altered signal transduction pathways (increased VPK and decreased cellular PKA activity) that may predispose to changes in cholesterol/CE binding, intracellular metabolism, and efflux following HSV-1 infection. We show that HSV-1 infection: 1) increases LDL binding and uptake, LDL receptor mRNA steady state levels, and transcription of the LDL receptor gene; 2) increases CE synthesis and HMG-CoA reductase activity but reduced CE hydrolysis and cholesterol efflux; 3) decreases both lysosomal and cytoplasmic CE hydrolytic (ACEH and NCEH) activities, where the latter enzyme is PKA-sensitive; and 4) reduces PKA activity by 6 h post-infection, which we believe explains the decreased ACEH and NCEH activity in these cells. Each one of these cellular mechanisms, working alone or in concert, may lead to CE accumulation.

Finally, we have demonstrated an association between VPK activity and CE accumulation utilizing HSV-1 VPK mutants. The mechanism by which VPK alters cellular host cholesterol trafficking is unknown. Although VPK possesses similarities to eukaryotic protein kinases A and C, the cellular substrates for VPK are unknown. Interestingly, it has been shown that VPK can phosphorylate viral substrates, but it has also been speculated that VPK may phosphorylate some host proteins(25) . This is the first demonstration that increased VPK activity is associated with altered activities of kinase-dependent proteins involved in cholesterol trafficking. However, we have not conclusively demonstrated that VPK specifically phosphorylates serine/threonine residues on those proteins involved in cholesterol metabolism. It is noteworthy, however, that CE accumulation following HSV-1 infection in vascular smooth muscle cells is abrogated in cells infected with an HSV-1 VPK mutant, which clearly implicates this kinase in altered lipid metabolism following herpesviral infection.

FOOTNOTES

*

This work was supported in part by National Institutes of Health Grants HL-07423, HL-46403, and HL-45343 (to D. P. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom reprint requests and correspondence should be addressed: Depts. of Biochemistry and Pathology, Cornell University Medical College, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6470; Fax: 212-746-8789.

()

The abbreviations used are: CE, cholesteryl ester; HSV-1, herpes simplex virus type-1; m.o.i., multiplicity of infection; ACEH, acid (lysosomal) cholesteryl ester hydrolase; NCEH, neutral (cytoplasmic) cholesteryl ester hydrolase; ACAT, acyl-CoA:cholesterol acyltransferase; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; VPK, virus-induced protein kinase; PKC, protein kinase C; PKA, protein kinase A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDL, low density lipoprotein; LPDS, lipoprotein-deficient serum; HDL, high density lipoprotein; FCS, fetal calf serum; PBS, phosphate-buffered saline.

ACKNOWLEDGEMENTS

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