Biological Research Laboratories, Syracuse University, Syracuse, New York 13244
Submitted 22 September 2003 ; accepted in final form 16 January 2004
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ABSTRACT |
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sodium-potassium-chloride-cotransporter; human fibroblast cell line; perinuclear accumulation
Human cytomegalovirus (human herpesvirus 5; HCMV) is an opportunistic pathogen infecting 6090% of the population. Although benign in healthy humans, HCMV is capable of causing severe disease in immunocompromised individuals (e.g., acquired immunodeficiency syndrome and organ transplant patients). It is also the leading cause of virally induced birth defects (e.g., Ref. 30). In common with other viral infections, HCMV is known to perturb a number of host cell functions. The majority of these perturbations presumably optimize host cell functions and conditions to support viral persistence and productive viral replication (2, 4, 7, 28, 30). These viral effects are the result of a controlled program of HCMV gene expression executed through virally activated cellular transcription factors. Two features of the HCMV infection process may make host cell ion transporters important optimization targets for this virus. Because the time of HCMV virion production is much longer than for other viruses (72160 h for HCMV vs. 12 h for herpes simplex virus; Ref. 44), host cell survival is very important. This suggests that maintenance of intracellular housekeeping functions in the face of significant viral demands on cellular metabolism will be critical to viral success. A second reason is that host cell optimization for HCMV includes a stereotypical increase of host cell size, volume, and intracellular water content (3, 18, 46). This phenomenon is termed cytomegaly, and, because cell water content increases, it must be accompanied by an increase in intracellular osmolyte content. A nonspecific increase of osmolytes (e.g., via increased plasmalemmal leakiness) will presumably not suffice, given the long infection time of this virus.
Earlier studies demonstrated that cytomegaly and viral replication both strongly depend on the presence of extracellular Na+. Therefore, it is not surprising that HCMV infection is characterized by significant effects on a variety of membrane ion transporters that mediate Na+ transmembrane movements. Examples include the sodium pump (3, 41, 46) and the Na+/H+ exchanger (NHE) (9), as well as the Cl/HCO3 exchanger, which works in concert with the NHE to mediate net uptake of inorganic osmolytes promoting cell volume increases (37). Fons et al. (18) reported that treatment of HCMV-infected cells with amiloride (an inhibitor of NHE) inhibited HCMV replication by almost two orders of magnitude. Similarly, incubation of HCMV-infected cells in low Na+ concentration ([Na+]) growth medium prevented cytomegaly and reduced viral replication (46).
In light of the above, it was surprising to find that HCMV infection nearly abolishes NKCC ion transport (defined as bumetanide-sensitive Cl uptake and loss) and the expression of the NKCC protein in the microsomal subfraction of infected human embryonic lung fibroblast cells (MRC-5 cells; Ref. 41). The bumetanide and Na+ sensitivities of intracellular [Cl], as well as net Cl uptake and loss, were reduced by approximately threefold within 24 h postexposure (PE) to HCMV and abolished by 72 h PE. The drastically decreased NKCC function was paralleled, and could be explained, by a severe reduction (approximately ninefold) of NKCC protein expression in the membrane subfraction (determined 72 h PE; Ref. 41).
NKCC occurs in two isoforms: NKCC1 and NKCC2 (15, 24, 52). NKCC1 is particularly prominent in epithelial tissue, where it is known as the secretory isoform. However, it is also found in most other cell types (including fibroblasts), whereas NKCC2 distribution is limited to specific renal cell types. Posttranslational modifications (glycosylation and phosphorylation) appear to be critical to the ion transport function of both isoforms. NKCC1 is normally heavily glycosylated, and it has been suggested that a decreased level of glycosyslation will eliminate ion transport function of the NKCC protein by preventing its delivery to the plasma membrane (49). Further posttranslational modification occurs when the NKCC is phosphorylated to activate its ion transport function. It is now well established that the degree of phosphorylation of both isoforms of the NKCC protein is directly related to the rate of NKCC-mediated ion transport (23, 25, 34, 35, 52).
The marked but unexpected effect of HCMV to reduce NKCC function and microsomal protein expression raises questions about the fate of this important ion transporter and its role in the productive HCMV infection. The present study follows NKCC gene and protein expression, NKCC plasmalemmal levels, and intracellular localization after HCMV infection. We report that the loss of NKCC function during HCMV infection is not a result of NKCC gene repression. Nevertheless, within 24 h of HCMV infection, plasmalemmal NKCC protein levels are drastically reduced and remain low thereafter. In contrast, plasmalemmal NKCC in mock-infected MRC-5 cells is stable for at least 24 h, with an approximate lifetime of 48 h. Interestingly, >80% of the NKCC protein in mock-infected fibroblasts are intracellular and diffusely distributed, presumably in cytoplasmic vesicles. Despite the near-complete ablation of plasmalemmal NKCC protein, HCMV infection may lead to a slight increase of total cell content of NKCC protein. Confocal images of immunostained cells reveal that, at 72 h PE, NKCC protein accumulates in the perinuclear region of the HCMV-infected fibroblast. These results suggest that HCMV infection prevents NKCC protein from being inserted in the plasma membrane, leading to intracellular retention of NKCC protein, an effect that may be the result of virally induced changes in the intracellular NKCC protein trafficking.
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EXPERIMENTAL PROCEDURES |
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HCMV strain AD169 (American Type Culture Collection) was used in these studies. A continually replenished stock of HCMV was cultured in confluent MRC-5 cells. At the appropriate time, the infected cells were lysed, and the infectivity of the resultant viral stock was assayed by plaque assay (37, 41). For the studies reported here, HCMV infection was carried out by exposing the confluent MRC-5 cells to a virus stock at a multiplicity of infection of approximately three to five plaque-forming units (PFU/cell) for 1 h at 37°C. At such a multiplicity, >95% of the cells were productively infected. To control for nonspecific effects of the cell lysate portion of the viral stock, a parallel set of MRC-5 cells was always mock infected by a 1-h exposure to a lysate of uninfected MRC-5 cells. After the infectious period of exposure, both mock- and virus-treated cells were rinsed with PBS followed by fresh culture medium and returned to cell culture for various PE times.
Gel electrophoresis and Western blotting. Western blot analysis was performed as described in detail in Maglova et al. (41). Protein samples and prestained molecular mass markers were denatured in SDS reducing buffer for 20 min at 65°C. The samples were then electrophoretically separated on 7.5% SDS gel, and the resolved proteins were transferred to a polyvinylidene membrane. The resultant blots were incubated in 5% nonfat milk overnight at 4°C and were subsequently incubated with the monoclonal anti-NKCC antibody T4 (Developmental Studies Hybridoma Bank, Iowa City, IA) for 1 h at room temperature and horseradish peroxidase-conjugated goat-anti-mouse IgG for 1 h at room temperature. The chemiluminescent signal was captured by using X-ray film (Fuji-RX) or directly on a Kodak Digital Science Image Station 440CF (Rochester, NY). Band densities were analyzed by using 1D Image Software (Kodak, Rochester, NY). All data are presented as either representative of several repeats or the means ± SD for multiple repeats.
NKCC cell surface biotinylation and quantification. Cell surface biotinylation was used to label plasmalemmal NKCC protein in MRC-5 cells before and after HCMV infection. MRC-5 cells were grown to confluence. Cells were either mock or HCMV infected and, at appropriate times after infection, washed twice in PBS and once in borate buffer (in mM: 154 NaCl, 10 boric acid, 7.2 KCl, and 1.8 CaCl2; pH 9.0). The surface plasma membrane proteins were then biotinylated by gently shaking the cells for 20 min with 3 ml of a biotinylation solution composed of borate buffer containing 1.5 mg of the membrane-impermeable agent, N-hydroxysulfosuccinimydyl-S,S-biotin (Pierce Biotechnology, Rockford, IL). After 20 min, an additional 3 ml of the same biotinylation solution were added, and the cells were rocked for another 20 min. The cells were washed extensively with quenching buffer (in mM: 120 NaCl and 20 Tris; pH 7.4) to remove excess N-hydroxysulfosuccinimydyl-S,S-biotin and then washed twice with PBS. All labeling and washing manipulations were performed at 4°C. The cell surface biotinylation was verified by using confocal microscopy to visualize the cell membrane-bound biotin labeled with fluorescent-labeled streptavidin.
The biotinylated cells were scraped and solubilized in a small, defined volume of solubilization buffer (in mM: 60 HEPES, 150 NaCl, 3 KCl, 5 tri-sodium EDTA, 3 EGTA, and 1% Triton X-100; pH 7.4), sonicated for 20 s, agitated on a rotating rocker at 4°C for 30 min, and centrifuged at 12,000 g for 30 min to remove insoluble cellular debris. An aliquot of the resulting supernatant was retained to represent the total cell protein fraction (T). The protein content was determined by the Lowry assay by using a Bio-Rad DC protein assay kit (Bio-Rad Life Sciences, Hercules, CA). The remaining supernatant was incubated with avidin-agarose. At least two avidin incubations were performed to assure the extraction of all biotinylated proteins (B). After two consecutive avidin precipitations, the supernatant was retained as the residual fraction (R), and its volume was adjusted to the initial defined volume of solubilized cell protein (defined volume procedure). The avidin-agarose beads were washed five times in solubilization buffer, and bound proteins were eluted in 1/8 of the initial defined volume of sample buffer (110 mM Tris·HCl, 0.9% SDS, 0.8% EDTA, 5% glycerol, 5% 2-mercaptoethanol, and bromphenol blue), yielding a surface biotinylated fraction (B) that is 8x concentrated compared with the T and R fractions.
A semiquantitative Western blot analysis was performed by using the T, R, and B fractions. Equal volumes of all three fractions were loaded on the gel (defined volume gel-loading procedure). The optical density (OD) values (in arbitrary densitometric units) of the NKCC bands for each treatment were normalized to the OD of the NKCC band in parallel mock-infected cells. By normalizing to mock-infected data, one can compare results from different experiments. Results were discarded when the total recovery of protein in the intracellular (R) plus surface fractions (B) was <80% or >98% of the total fraction (T).
Plasmalemmal NKCC lifetime. The cell surface biotinylation protocol described above was used. In this kind of experiment, the cell surface biotinylation was done in sterile conditions by using noninfected MRC-5 cells. Following their biotinylation, cells were returned to culture medium and cultured for an additional 2448 h. Biotinylated fractions were isolated at different times after the biotinylation procedure (1, 24, and 48 h), and semiquantitative Western blots were performed. In all experiments, the level of the biotinylated NKCC immediately after the biotinylation procedure (1 h) was used as the internal, normalizing control to compare data from different experiments.
Immunofluorescence and confocal microscopy. Confluent cells were grown on 12-mm coverslips and were either mock or HCMV infected. At the appropriate time after infection, the cells were fixed in cold methanol or were treated with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100. After preblocking the cells in 20% normal goat serum, they were further incubated with the appropriate primary and fluorochrome-conjugated secondary antibodies. The coverslips were mounted on glass microscope slides, and cells were viewed by using a Zeiss Pascal Confocal microscope (Carl Zeiss, North American, Thornwood, NY). Images were processed by using the Zeiss Pascal software package.
Antibodies.
For Western blot analyses, the anti-NKCC monoclonal antibody T4, designed and developed by Christian Lytle (University of California, Riverside, CA) and Bliss Forbush (Yale University, New Haven, CT), was used. The antibody was obtained from the Developmental Studies Hybridoma Bank (developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA). For immunostaining studies, T4 and the recently available polyclonal anti-NKCC antibodies (TEF-S1 and TEF-S2), specific to the COOH-terminal epitope of NKCC1, were used. These latter antibodies were generous gifts of Dr. John Payne (University of California, Davis, CA) and Christian Lytle. To verify the NKCC colocalization with the perinuclear structure, another polyclonal anti-NKCC antibody (-wNT, specific to the NH2-terminal epitope of NKCC1) was used (16). The
-wNT antibody was kindly provided by Dr. Robert Turner, Membrane Biology Section, Gene Therapy and Therapeutics Branch, National Institutes of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD. In double-labeling immunostaining experiments, anti-immediate early antigen IE1 and 2 monoclonal antibody (Chemicon International, Temecula, CA) was used to stain the nucleus of the HCMV-infected MRC-5 cells.
RNA isolation and DNA microarray analysis. RNA was isolated from confluent MRC-5 cells that had been either mock or HCMV infected (30 and 72 h PE). Cells were rinsed with prewarmed PBS and then lysed by using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA). Residual genomic DNA was digested with RNA-free DNase I. DNase I and other protein contaminants were removed by two extractions with buffer-saturated phenol-chloroform. Total RNA was precipitated in propanol, washed in ethanol, and resuspended in diethylpyrocarbonate-treated water. An Oligotex mRNA Midi Kit (Qiagen, Valencia, CA) was used for isolation of mRNAs by following the manufacturer's instructions. The purity and integrity of the isolated mRNA were confirmed spectroscopically and electrophoretically by separation of mRNAs on a 1.2% agarose gel containing formaldehyde. Samples (4 µg) of high-quality mRNAs were stored at 80°C and sent to Incyte Genome Systems (St. Louis, MO) for further RT-PCR, DNA amplification, Cy3/Cy5 labeling, and DNA microarray hybridization. Results were obtained from Incyte Human UniGEM version 2.0 (30 h PE) and Incyte Human UniGEM version 1.0 (72 h PE) DNA microarrays (30-h PE sample URL: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM9974; 72-h PE sample URL: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM9973). The limit of statistically significant differential expression, as recommended by Incyte Genome Systems, was 1.74-fold.
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RESULTS |
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More evidence that NKCC is predominantly localized intracellularly was provided by means of a different, more quantitative protocol in which both the biotinylated and the nonbiotinylated (presumably intracellular) protein fractions were isolated and directly compared. After the intact cells were biotinylated, total cell proteins were fractionated into biotinylated and residual or nonbiotinylated subfractions by using the defined volume technique (see EXPERIMENTAL PROCEDURES for details). This permits a direct comparison between the NKCC OD signals of the three fractions (total, T; biotinylated, B; and residual, R). Figure 3A is a representative Western blot analysis (n = 3) of the three protein fractions by using the defined volume gel-loading procedure. We compared the OD of the NKCC bands from the three subfractions, taking into account that the biotinylated fraction is eightfold concentrated relative to the original cell lysate. In Fig. 3B, the OD data from the three experiments were normalized to the OD of the total NKCC fraction. This plot shows the fractions of biotinylated and nonbiotinylated (intracellular) NKCC to be 12 and
85%, respectively, of the total NKCC level detected in quiescent MRC-5 cells (Fig. 3B). These data are in good agreement with the estimated level of biotinylated NKCC obtained from Fig. 2, A and B. Both types of studies indicated that <20% of total cellular NKCC protein resides in the plasmalemma of the MRC-5 cells. These results imply that the nonbiotin-labeled NKCC protein resides intracellularly, presumably in endosomal vesicles.
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HCMV induces accumulation of NKCC in a perinuclear structure.
Because HCMV infection results in decreased plasmalemmal, but not total cellular, content of NKCC protein, the most likely mechanism of the reduction of NKCC function is by altered NKCC protein delivery to the plasmalemma. Our observation that late in the infection NKCC can be detected only in a detergent-solubilized (1% Triton X-100) cell lysate suggests that NKCC should be located within the infected MRC-5 in a virus-inducible, detergent-soluble structure. These observations initiated additional confocal microscopy studies to learn whether any changes in the NKCC intracellular distribution can be observed late in the HCMV infection of MRC-5 cells. By 72 h PE (viral potency of 35 PFU/cell), almost all MRC-5 nuclei show the presence of NKCC clustering (red) very close to the enlarged, kidney bean-shaped nuclei (green; Fig. 7, A and B). Cell nuclei of HCMV-infected MRC-5 cells were visualized by using an antibody to immediate early HCMV proteins expressed very early in the infection that are mainly limited to the cell nuclei (53). A control experiment using fluorophore-conjugated IgG did not detect any clustering, confirming the specificity of the NKCC antibody (Fig. 7A, top right corner). The aggregated NKCC protein distribution observed in the HCMV-infected cells (Fig. 7) differs significantly from the diffuse distribution of this protein observed in uninfected cells (Fig. 4, A and B). We have confirmed that the perinuclear structure contains NKCC protein by using several anti-NKCC antibodies made to the COOH and NH2 termini of the protein (TEF-S1/S2, Fig. 7A, and -wNT, Fig. 7B, respectively) for immunolocalization (see EXPERIMENTAL PROCEDURES for details).
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DISCUSSION |
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Reduced microsomal NKCC is not the result of NKCC message repression. The possibility that a virally directed repression of host cell protein messages causes the reduction of microsomal NKCC protein content was tested. The results of our DNA microarray analysis showed that, 30 h after HCMV infection (a time at which plasmalemmal NKCC is already greatly reduced; see Fig. 5), there was no significant effect on NKCC1 message levels (Fig. 1). Similarly, the microarray results show no significant HCMV effects on the message levels of several other membrane ion transporters for which we have reported HCMV-induced functional changes (NHE: Ref. 9; Cl/HCO3 exchanger, anion exchanger: Ref. 37; Na-Cl-cotransporter: Ref. 39). It is of interest that, in the case of the NHE, we have preliminary evidence that the increased function may be mediated by an increase in the protein levels of the NHE2 isoform relative to the NHE1 (38).
Reduced microsomal NKCC cannot be solely the result of reduced net NKCC protein synthesis.
We next tested whether the decrease of microsomal NKCC protein reflected the net decrease of total cellular NKCC protein content brought about by virally mediated effects on NKCC protein synthesis and/or degradation. A generalized reduction of host cell protein expression seems unlikely, as we have demonstrated increased function for several ion transport processes (i.e., the Na-K-ATPase, the NHE, and Cl/HCO3 exchange; Refs. 3, 9, 37) and increased protein expression of the Na-K-ATPase (41). In contrast to the approach of Maglova et al. (41) in which the NKCC protein content of a microsomal preparation was determined, for the present study, we solubilized total cell membrane proteins using Triton X-100 (see EXPERIMENTAL PROCEDURES). As Fig. 6 clearly shows, HCMV infection does not result in a decrease of total, detergent-solubilized NKCC protein when one corrects for the cell loss of 25% that occurs during the first 24 h PE (41). In fact, our data suggest that, by 96 h PE, the content of NKCC protein per remaining HCMV-infected cell may have increased by as much as 3040% over that in the mock-infected control cells. This implies that net synthesis of the NKCC protein continues during the infection. The relative contributions of synthesis and degradation to the observed net increase of NKCC protein remain to be determined.
Despite the results just described, HCMV infection clearly caused functional inhibition of the NKCC (41). An independent approach was taken to test whether the loss of plasmalemmal NKCC protein could account for the functional NKCC inhibition. The plasmalemmal subfraction of cellular NKCC protein was biotinylated and separated, and the amount was determined by using semiquantitative Western blotting. As seen in Fig. 5, HCMV infection reduced the plasmalemmal component of NKCC protein by 75% between 6 and 24 h PE. Plasmalemmal NKCC content continued to decrease as the infection progressed so that, by 96 h PE, plasmalemmal NKCC content was reduced
90% relative to mock-infected cells.
To put this pathological, HCMV-induced rate of net loss of plasmalemmal NKCC protein into a physiological perspective, we estimated the turnover rate of plasmalemmal NKCC in uninfected cells. The amount of biotinylated NKCC protein remaining 24 and 48 h after the biotinylation procedure was used for this estimate. The changes were quantitatively assessed by comparing the linear regression slopes of the biotinylated fractions 24 and 48 h after biotinylation to the slope obtained immediately after the biotinylation procedure (1 h). Interestingly, there was no detectable change during the first 24 h after surface membrane biotinylation. However, by 48 h following biotinylation, 75% of the biotinylated NKCC protein was degraded (compare Fig. 2, B and D). It is possible that, during the lag period between biotinylation and degradation of the biotinylated NKCC protein, recycling of NKCC protein in and out of the plasmalemma occurs before its ultimate destruction. As discussed below, other workers have provided some evidence that NKCC protein may cycle in and out to the plasmalemma, perhaps depending on its phosphorylation state (11, 12, 23). In summary, in uninfected MRC-5 cells, biotinylated NKCC protein is not degraded for at least 24 h but then disappears over the next 24 h, whereas, after HCMV infection, there is a net removal of plasma membrane (biotinylated) NKCC protein that is near completion 24 h PE. This suggests that the virus causes a significant change in trafficking of this integral membrane protein to the plasmalemma. At least part of the viral effect must involve enhanced endocytosis of the plasmalemmal NKCC. In fact, HCMV infection is known to cause an increased rate of endocytosis (20, 57), and HCMV infection has been reported to reduce cell surface expression of EGF receptors (17).
HCMV infection causes redistribution of NKCC protein within the cell. We have confirmed that HCMV infection causes the near-complete removal of plasmalemmal NKCC, while at the same time resulting in a slight increase in total cellular NKCC protein. To better understand this, we needed information on the normal subcellular distribution of the NKCC in our human fibroblast cell line. Two approaches were used to address the question of the cellular distribution of NKCC protein in uninfected fibroblasts.
By means of biotinylation and Western blotting protocols, we demonstrated that, in uninfected, quiescent MRC-5 cells, the large majority (80%) of total cellular NKCC protein is intracellular, not plasmalemmal (
20%; Figs. 2 and 3). Confocal imaging of immunostained, uninfected MRC-5 cells qualitatively confirms this conclusion. Furthermore, the confocal images show in uninfected cells that the intracellular NKCC protein is diffusely distributed throughout the intracellular space in very small, punctate structures, which we assume are vesicles (see Fig. 4, A and B). By contrast, we confirm that, in an epithelial cell line, T84, the NKCC protein accumulates in, and very near to, the plasmalemma.
To date, studies on the normal subcellular distribution of NKCC1 have been limited to polarized epithelial cells where immunostaining techniques have detected NKCC1 predominantly on the plasma membrane (e.g., Ref. 43). In contrast to the present finding in fibroblasts, D'Andrea et al. (11) showed that, for T84 cells (a human intestinal cell line), 80% of cellular NKCC protein were located in the basolateral membrane. Our results (Fig. 4, C and D) are consistent with this conclusion. About 20% were localized to vesicle-like structures in the cytosolic region immediately subjacent to the basolateral membrane. These findings led to the suggestion that the cytoplasmic vesicles represent a "reserve" source of NKCC1 ion-transporting units that can be inserted into the plasmalemma in response to the appropriate signal (11, 12). Similar vesicles have been reported for the NKCC2 isoform by using immunofluorescent techniques in epithelial cells isolated from the loop of Henle (45). Forbush's group (23) used electron microscopy to demonstrate a significant pool of NKCC2 in cytoplasmic vesicles located just under the plasmalemma in cells of the thick ascending limb of the loop of Henle. They further showed that the phosphorylated transporter appears to be restricted to the apical cell membrane of the cells and suggested that NKCC phosphorylation may be necessary for NKCC2 protein translocation to the cell surface.
HCMV infection of MRC-5 cells leads to a perinuclear accumulation of NKCC protein.
Our results clearly show that, following HCMV infection in MRC-5 cells, there is a remarkable change in the subcellular distribution of NKCC protein. By 72 h PE, the plasmalemmal NKCC content was reduced by 8085% (see Fig. 5B), but the amount of intracellular NKCC protein was unchanged or slightly increased. In addition, intracellular NKCC protein was no longer diffusely distributed throughout the cytoplasm. Rather, it had aggregated into a relatively large perinuclear accumulation (see Fig. 7). Thus this perinuclear structure must represent the source of the detergent-soluble NKCC protein detected by Western blot analysis of HCMV-infected cells (e.g., Fig. 6). The detergent solubility suggests that the NKCC protein remains in a lipid bilayer membrane. Therefore, we assume the reason that we earlier reported NKCC to be depleted from the microsomal fraction (41) is because, in the absence of detergent, the perinuclear structure sediments at a low centrifugation rate.
Perinuclear accumulations of membrane and/or cytosolic proteins, termed "aggresomes" (e.g., Refs. 21, 22, 27), have been reported in a variety of experimental or pathological situations. The term "conformational diseases" has been coined for diseases exhibiting such structures (e.g., Ref. 22). Aggresomes may represent cellular responses to misfolded or unassembled proteins (21, 29). Aggresomes are envisaged to be holding stations that recruit chaperones and proteasomes for the purpose of degrading the defective proteins residing therein. Thus it is possible that HCMV infection results in the misfolding of the NKCC protein that, in turn, causes the protein to accumulate in an aggresome. This would explain the loss of plasmalemmal NKCC protein. However, we have three pieces of evidence that argue against the perinuclear structure being an aggresome. First, aggresomes are said to be detergent insoluble (27), including being insoluble in Triton X-100 (21), and, as we have shown in this report, the NKCC perinuclear structure is detergent soluble (see Figs. 5 and 6). Second, the protein in aggresomes is believed to be deglycosylated (27). The NKCC protein before and after HCMV infection runs at 159 kDa (see Figs. 2, 5, and 6), whereas the unglycosylated NKCC protein has a predicted mass of
130 kDa. Third, aggresomes are enclosed in a characteristic vimentin cage (21, 27). We have been unable to demonstrate the presence of such a structure in HCMV-infected cells (LM Maglova, WE Crowe, and JM Russell, unpublished observations).
Alternatively, the perinuclear accumulation of NKCC protein may be associated with the perinuclear accumulation of viral proteins. Large DNA viruses cause perinuclear accumulations of viral proteins, and it has been suggested that these structures play a role in viral assembly (26). In the specific case of HCMV, Britt's group (54, 55) has described a perinuclear structure containing at least six HCMV proteins that are ultimately found in the tegument and envelope of formed virions. Although these accumulations of viral proteins share some common features with aggresomes, they also have some very different properties. For instance, in common with aggresomes, the HCMV-induced structures have a characteristic perinuclear location and form around the microtubule organizing center. On the other hand, they are not surrounded by vimentin (see above), they are disrupted by nocodazole, and there is no evidence of colocalization with lysosomes (54, 55). Thus it is of interest that we have reported preliminary evidence that the NKCC protein in the perinuclear structure is colocalized with several HCMV tegument proteins (40). The possibility that host cell NKCC protein shares a structure with HCMV tegument and envelope proteins raises interesting possibilities as viral factories are said to tend to exclude host cell proteins (26).
Is NKCC functional inhibition related to host cell K+ requirements and viral effects on the cell cycle? A number of investigators have noted that increases of uptake of K+ occur during the early portion of the mammalian cell cycle (e.g., Refs. 32, 42, 58). A rise in intracellular [K+] ([K+]i) may be important for mammalian cell growth and protein synthesis (31). There are reports that mitogens stimulate functional NKCC activity (e.g., Refs. 8, 47) and that overexpression of the NKCC protein will stimulate cell proliferation (48). In light of these findings, it is very interesting to note that HCMV infection stimulates the host cells to enter the cell cycle, but causes the cycle to halt at the G1/S interface (e.g., Refs. 6, 33; reviewed in Ref. 19). Presumably, this viral tactic serves the purpose of optimizing cellular conditions for viral DNA synthesis (56) while preventing host cell DNA synthesis. Halting the host cell cycle appears to be critical as cells that are able to continue through their normal cell cycle following HCMV infection undergo an abortive or nonreplicating viral infection (1). We suggest that the downregulation of NKCC ion transport function may subserve the need of the virus to halt the cell cycle at the G1/S interface. This would help explain why the NKCC, a prime candidate to cause cell volume increase, is downregulated in HCMV-infected, cytomegalic cells. We (JM Russell, LM Maglova, and WE Crowe) have unpublished data showing that HCMV infection results in slight but significant decrease in [K+]i. Thus it is possible that NKCC inhibition occurs to prevent the increase of [K+]i that may normally occur during the early phase of the mammalian cell cycle.
NKCC protein downregulation: a possible mechanism for HCMV-induced neurological birth defects. Congenital HCMV infection is now the leading cause of virally mediated neurological birth defects afflicting between 0.5 and 3% of all live births in the world (e.g., Ref. 10). In addition to mental retardation and learning disabilities, deafness often occurs in infants born to mothers infected with HCMV during their pregnancy. Although the central nervous system is the major target organ for tissue damage by HCMV infection in the developing fetus, little is known about the molecular mechanisms responsible for the pathogenesis of tissue damage of congenital cytomegalovirus infection. The results reported here may begin to offer an explanation for at least some of these pathological effects. Thus we have demonstrated that HCMV infection blocks NKCC function by causing its removal from the plasmalemma. It is becoming increasingly clear that this cation-chloride-coupled cotransport mechanism may play important roles in normal neuronal development and function (e.g., Ref. 13, 51). For example, developmental studies have shown that various regions of the developing brain have carefully timed changes in the relative levels of expression of NKCC1 and the K-Cl cotransporter isoform 2. Thus it is possible that a congenital HCMV infection may result in neurological deficits as a result of disruption of this sequential change in dominance of the two cation-coupled cotransporters. In addition, it has been shown that the inner ear contains NKCC1 that is likely involved with the secretion of endolymph fluid. Mice whose NKCC1 gene has been knocked out demonstrate inner ear structural changes and deafness (14).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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