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Previous Article | Next Article 
Journal of Virology, February 2002, p. 1450-1460, Vol. 76, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.76.3.1450-1460.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, 91054 Erlangen, Germany,1 Departments of Pediatrics and Microbiology, The University of Alabama at Birmingham, Birmingham, Alabama 352332
Received 5 June 2001/ Accepted 25 October 2001
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ABSTRACT |
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INTRODUCTION |
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The envelope glycoproteins of herpesviruses have multiple roles in the viral replication cycle, including essential functions such as attachment, penetration, cell-to-cell spread, envelopment, and maturation of nascent virus particles (29). To understand these processes, it is necessary to have detailed knowledge of the structure and function of envelope glycoproteins. In addition, envelope glycoproteins can elicit a protective virus-neutralizing humoral immune response and thus have been proposed as important components of vaccines.
Among the HCMV structural glycoproteins, three major complexes (designated gCI to -III) have been identified to date (13). The gCI complex is formed by disulfide-linked homodimers of glycoprotein B (gB; also called gpUL55) (2, 3). The gCII complex is composed of gM (IMP; also called gpUL100) and gN (gpUL73), and large parts of this complex are also held together by disulfide bonds (28). Glycoproteins H (gH; also called gpUL75), L (gL; also called gpUL115) and O (gO; also called gpUL74), the constituents of gCIII, form a heterotrimeric, disulfide-linked complex (17, 18, 26). Thus, a hallmark of the structural glycoproteins of HCMV seems to be their formation of disulfide-linked high-molecular-weight complexes. Proteins within the gCI to -III complexes represent a group of structural glycoproteins which are conserved within the herpesvirus family, indicating important or even essential functions for the replication of these viruses (4, 29, 33).
In addition to the glycoproteins, such as gB, gH, gL, and gM, which have identifiable protein counterparts in all herpesviruses (family common), herpesviruses also code for a number of glycoproteins which are expressed only by individual genera of a herpesvirus subfamily (29). These "private" glycoproteins could contribute to the specific properties of individual herpesviruses, such as cell tropism or pathogenicity. For HCMV, very little is known about private structural glycoproteins. To our knowledge, the only reported structural glycoprotein that is not conserved throughout the herpesvirus family is gp48, the product of the UL4 reading frame (7). Interest in this glycoprotein has been limited almost exclusively to the complex transcriptional regulation of its expression (9, 15, 16, 36). Based on the large coding capacity of HCMV for glycoproteins, it can be postulated that HCMV virions will contain a number of glycoproteins which do not have counterparts in other herpesviruses.
We have studied the gene product(s) of reading frame 10, located within the repeat sequences flanking the long unique segment of the viral genome (terminal repeat long [TRL] and internal repeat long [IRL]). In the laboratory-adapted HCMV strain AD169, the reading frame is present twice and termed TRL10 and IRL10, respectively (8). In contrast, low-passage clinical isolates contain only the TRL10 reading frame, since large parts of IRL, including IRL10, are replaced by unique coding sequences (5).
Our data reveal that ORFs IRL10 and TRL10 of HCMV strain AD169 as well as TRL10 of the clinical isolate TB40E are transcribed with early/late kinetics. Using a rabbit antiserum, glycosylated proteins of 22 and 23.5-kDa were detected in cells infected with either virus. The proteins were also detected in purified HCMV virions by immunoblotting as well as immunoelectron microscopy. These results indicated that the product of the TRL10 reading frame was a structural protein. Analysis of the protein in the presence and absence of reducing agents indicated that TRL10, like the other previously described HCMV envelope glycoproteins, forms higher-molecular-weight disulfide-linked complexes.
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MATERIALS AND METHODS |
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Preparation of GST-TRL10 fusion proteins and antisera. The TRL10 carboxyl-terminal region was amplified from AD169 DNA by PCR with the following primers containing either BamHI or SalI sites (underlined): TRL10.124/171s (5'-GGACGGATCCACACGCAAAAAGCTGGAACAA-3' ) and TRL10.124/171as (5'-ATTAGTCGACGACGTTGTCGTCCTCGTCCTC-3'). The PCR product was digested with both endonucleases and inserted into the expression vector pGEX-1P (Pharmacia Biotech, Freiburg, Germany). The resulting plasmid, pGST/TRL10c, contained nucleotides (nt) 370 to 513 of TRL10AD169 fused to the glutathione S-transferase (GST) gene. Correct insertion was confirmed by DNA sequencing.
Plasmid DNA was used to transform Escherichia coli BL21 for expression of GST fusion proteins. The fusion protein was induced and purified from E. coli lysates according to the manufacturer’s instructions. One half of the purified glutathione-Sepharose 4B (Pharmacia Biotech)-coupled fusion protein was treated with PreScission protease (Pharmacia Biotech) to release the viral peptide. A mixture of pGST/TRL10c, the 34-kDa fusion, and pTRL10cut, the 6-kDa cleaved viral peptide, was used to raise an antiserum in rabbits. The resulting serum was affinity purified on the GST/TRL10c fusion protein by standard methods.
Preparation of eukaryotic expression constructs and DNA transfection. The entire TRL10 reading frame was amplified from AD169 cosmid pCM1052 (11) with the following primers that contain XhoI and HindIII restriction sites (underlined): TRL10 forward (5'-GGATCTCGAGATGTATCCGCGTGTAATG-3') and TRL10 reverse (5'-ATACAAGCTTGACGTTGTCGTCCTCGTC-3'). The PCR product was cleaved and inserted into the expression vector pcDNA3.1myc/His (Invitrogen, Carlsbad, Calif.). The resulting plasmid, pcTRL10myc/his, encoded full-length pTRL10 fused to a C-terminal Myc/His tag. The integrity of pcTRL10myc/his was confirmed by DNA sequencing.
293T cells were transfected with the respective DNA using Lipofectamine Plus reagent (Gibco-BRL) according to the manufacturer’s suggestion except that the transfection mixture consisted of 1 µg of DNA, 95 µl of DMEM and 6 µl of Lipofectamine Plus reagent. The mixture was added to a cell culture dish (3.5-cm diameter, seeded with 2 x 105 cells 1 day before). After 48 h, cells were harvested, washed three times with phosphate-buffered saline (PBS), and stored at -20°C until used.
Image analysis. Cos7 cells grown on glass cover slips in 24-well plates were transfected with 3 to 5 µg of plasmid pcTRL10myc/his using calcium chloride as described (35). Two days later, the cover slips were washed and fixed in 2.0% paraformaldehyde in PBS. The fixed cells were permeabilized with NP-40-containing buffer and then blocked with 20% normal goat serum (28). Primary antibodies, including an anti-Myc monoclonal antibody, rabbit anticalreticulin (endoplasmic reticulum [ER] marker), and rabbit anti-gm130 (Golgi marker) were then added. Following a wash, antibody binding was detected with a Texas red-conjugated anti-mouse immunoglobulin G (IgG) or fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Southern Biotechnology Associates, Birmingham, Ala). The anticalreticulin serum was purchased from Affinity Bioreagents, Boulder, Colo. and the gm130 antiserum was kindly provided by Elizabeth Sztul (University of Alabama, Birmingham, Ala.). Images were collected using a Leitz Dialux fluorescence microscope fitted with a Photometrics charge-coupled device camera. Images were processed using Image Pro software and Adobe Photoshop as previously described (28).
DNA sequence analysis. To establish the coding sequences for TRL10 in clinical isolates, DNA was extracted from lysates of infected fibroblasts and amplified by PCR using primers TRL10forward and TRL10SEQAS (5'-GACGGCGTTCGATGAACTTCC-3'). Nucleotide sequences were determined on an ABI 377 using the dye terminator sequencing kit according to the manufacturer’s instructions (PE Applied Biosystems, Foster City, Calif.). DNA and amino acid sequence evaluation was performed by use of the University of Wisconsin Genetics Computer Group software.
SDS-PAGE and immunoblotting. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10 to 15% polyacrylamide gels under standard conditions. Proteins were transferred to nitrocellulose membranes, and membranes were blocked with PBS containing 0.1% Tween 20 and 5% powdered milk. Antibodies and sera were diluted in PBS containing 0.1% Tween 20. For detection of primary antibody binding, horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody and the enhanced chemiluminescence detection system (Pharmacia Biotech) were used according to the manufacturer’s instructions. Removal of N-linked oligosaccharides was carried out using recombinant peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) (New England Biolabs, Beverly, Mass.) according to the manufacturer’s specification.
Membrane topology was investigated via protease digestion. Gradient-purified virions were resuspended in MSB buffer containing 150 mM potassium acetate, 5 mM magnesium acetate, 50 mM HEPES (pH 7.6), and 200 mM sucrose. For solubilization of virions, Triton X-100 was added to a final concentration of 1% (wt/vol). Protease digestion was performed with 20 µg of proteinase K per ml for 2 h at 37°C. All samples were subjected to PAGE and immunoblot analysis immediately after incubation.
RNA methods. For Northern blot analysis, whole-cell RNA was isolated from noninfected or infected cells at 7, 24, 48, and 72 h postinfection using an RNA isolation kit (Qiagen, Hilden, Germany). For gel electrophoresis, 20 µg of each RNA preparation was denatured with glyoxal and separated in 1% agarose gels containing 40% formaldehyde. Northern blot analysis was carried out according to standard procedures. The TRL10-specific antisense probe was 32P labeled in a single-strand PCR using full-length TRL10 as the template. As an internal control, the filter was hybridized with a glyceraldehyde-3-phosphate dehydrogenase-specific probe.
For reverse transcription (RT)-PCR, infection was blocked with either 150 µg/ml cycloheximide or phosphonoformic acid at a concentration of 185 µg/ml. Total RNA was isolated using the High Pure RNA isolation kit (Roche Diagnostics, Indianapolis, Ind.), and RT-PCR was carried out with the Titan One Tube RT-PCR System (Roche Diagnostics) following the manufacturer‘s specifications.
Immunoelectron microscopy. For immunoelectron microscopy, purified virions were labeled with an antibody directed against pTRL10 as well as with preimmune sera and a control antibody against gB, monoclonal antibody 27-287. Detection of specific antibody binding was achieved by using protein A-gold. Stability tests at high ionic strength (14) ensured that the gold particles had been quantitatively covered by protein A, thus minimizing the possibility of nonspecific binding. The samples were subjected to labeling prior to fixation with 1% (vol/vol) glutaraldehyde and negative staining with 4% (wt/vol) uranyl acetate following published procedures (14). The specimens, which were mounted on 400-mesh, carbon-coated copper grids, were observed at an accelerating voltage of 80 kV.
Nucleotide sequence accession numbers. The nucleotide sequences for the TRL10 coding sequences from clinical isolates have been assigned the following accession numbers from GenBank: AF432083, AF432084, AF432085, AF432086, AF432087, AF432088, AF432089, AF432090, AF432091, and AF432092.
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RESULTS |
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N exchange at position 38 (8). Low-passage HCMV isolates, in contrast to the laboratory-adapted strains, have a different genetic organization with respect to the TRL and IRL regions. In these isolates, reading frames IRL14 to IRL1 of the IRL sequence are replaced by a unique sequence containing at least 19 genes not found in the laboratory-adapted strains (5). Thus, in contrast to the laboratory-adapted strains, recent clinical isolates contain only a single TRL10 allele (see Fig. 1). The presence of two ORFs which are part of repeat sequences in strain AD169 as opposed to a single unique reading frame in clinical isolates poses a problem in nomenclature, since the coding sequence in clinical isolates corresponds to IRL10AD169 rather than TRL10AD169 (see below). Nevertheless, to avoid confusion and to facilitate consistency throughout the paper, we will designate the gene and its product TRL10 and gpTRL10, respectively, being aware that in low-passage isolates TRL10 is not part of repeat sequences and that in AD169 transcripts and protein products cannot be unequivocally assigned to TRL10 or IRL10.
Since we wanted to study the protein product of the TRL10/IRL10 reading frames, we included both the laboratory-adapted strain AD169 and the clinical isolate TB40E (37) in our studies to account for the different number of alleles. To verify that TB40E carried only a single TRL10 coding sequence, the genome was cloned into overlapping cosmids. Partial DNA sequencing revealed that the genome arrangement of TB40E was homologous to that of HCMV strain Toledo. Sequence analysis also confirmed the presence of an ORF in strain TB40E with close homology to TRL10 from strain AD169 (see Fig. 7). It should be noted that TRL10TB40E contained the DN exchange at position 38 which is characteristic of the IRL10AD169 sequence (see Fig. 7).
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RT-PCR analysis of TRL10 transcription following treatment of infected cells with cycloheximide or phosphonoformic acid were consistent with classification of the TRL10 transcripts in the early/late kinetic class (data not shown). Thus, although we cannot definitively identify transcripts encoding the TRL10 RNA in either strain, the data are consistent with the interpretation that TRL10 RNA was expressed with early/late kinetics.
In order to characterize the protein product of ORF TRL10, fibroblasts were infected with AD169 or TB40E, cell lysates from virus-infected cells were prepared at different time points after infection and subjected to immunoblot analysis. The protein was detected using a polyclonal rabbit antiserum raised against a bacterial fusion protein comprising amino acids 124 to 171 of TRL10AD169. Two proteins of 22-kDa and 23.5-kDa molecular mass were detected with the antiserum beginning 48 h after infection (Fig. 2A). These proteins were not detected in uninfected cells or by the preimmune serum. The signal intensity increased considerably between 48 h and 72 h.
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The predicted primary amino acid sequence of both TRL10AD169 and TRL10TB40E contains two motifs for N-linked glycosylation in the extraluminal portion of the molecule. To determine whether the protein was in fact glycosylated, lysates from infected cells 72 h postinfection were treated with endoglycosidases and subsequently analyzed by immunoblotting. Treatment of lysates from AD169-infected cells with 1PNGase F, which removes complex as well as high-mannose N-linked sugars, resulted in the loss of both the 23.5-kDa and the 22-kDa proteins. Instead a single protein of 20 kDa was detected in these lysates (Fig. 2B).
Treatment with Endo H, which removes only high-mannose carbohydrates, resulted in the nearly complete loss of signal from the 23.5-kDa protein and the disappearance of the signal from the 22-kDa protein. Again, a new species of 20 kDa was detected in these samples. Similar results were obtained with lysates from TB40E-infected cells except that the 22-kDa and 20-kDa signals appeared as doublets (Fig. 2B). The proteins of 32 kDa and 34 kDa that were also detected in this analysis represent nonspecific staining, since they were also detected with the preimmune serum (Fig. 2A). To control for possible proteolytic activity which may have occurred during the enzymatic digestion, blots were stripped and redeveloped using antibody 27-287, which is specific for the gp58 part of glycoprotein B (Fig. 2B). In agreement with published data, we observed an 8-kDa reduction with PNGase F and a 3-kDa reduction with Endo H (3). Partial digests were not seen.
These findings were most consistent with the interpretation that the 22-kDa protein contained exclusively high-mannose N-linked carbohydrates, whereas the signal at 23.5 kDa was produced by two proteins, of which the vast majority contained high-mannose forms, whereas a small fraction was modified by complex sugars. In any case, our results clearly show that the protein product of the TRL10 reading frame was a glycoprotein, and therefore we have designated it gpTRL10.
gpTRL10 is a structural glycoprotein. To analyze whether gpTRL10 represents a structural glycoprotein, HCMV virions were purified in glycerol-tartrate gradients and analyzed for the presence of gpTRL10 in immunoblots. A diffusely migrating band at 22 to 34 kDa was detected using the TRL10-specific rabbit antiserum (Fig. 3A). Upon treatment of the viral lysates with PNGase F, the diffusely migrating proteins disappeared and a protein of 20 kDa was generated. Digestion of viral lysates with Endo H did not alter the migration of the virion 22- to 34-kDa protein (Fig. 3A). Identical results were obtained using dense bodies as the antigen (data not shown). The 25-kDa protein detected in the virion lysate after Endo H digestion was also demonstrated in a control lane which contained only Endo H, indicating nonspecific reactivity of the rabbit serum with Endo H (Fig. 3A) These data indicated that gpTRL10 was a virion structural protein which, in contrast to the intracellular forms of this protein, contained only complex N-linked sugars.
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To further define the virion association of gpTRL10, we treated extracellular virions with detergent to disrupt the virion envelope and then digested solubilized virions with proteinase K. Neither a known tegument protein, pp28 (UL99), nor gpTRL10 could be detected in virions which were initially solubilized with detergent and then digested with proteinase K (Fig. 4). In the absence of detergent, the virion pp28 in the tegument remained intact in the face of proteinase K digestion. In contrast, when untreated virions were digested with proteinase K, the forms of gpTRL10 migrating between 22 and 34 kDa were no longer present, but an immunoreactive form of gpTRL10 of approximately 16 kDa was detected (Fig. 4). Because the gpTRL10 antiserum is directed at the carboxyl terminus, this finding provided evidence that gpTRL10 was exposed on the envelope of HCMV and its topology was most consistent with that of a type I glycoprotein.
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3 kDa).
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Complex formation of gpTRL10. All but one of the structural glycoproteins of HCMV which have been characterized thus far have been shown to form disulfide-linked complexes (2, 3, 26, 28). It was therefore of interest to investigate potential complex formation of gpTRL10. Lysates of cells transfected with the plasmid encoding TRL10myc or gradient-purified virions were solubilized in the presence and absence of the reducing agent 2-mercaptoethanol and analyzed by immunoblotting.
In the presence of 2-mercaptoethanol, protein bands of the expected sizes between 22 and 34 kDa were detected (Fig. 6). In the absence of 2-mercaptoethanol, higher-molecular-weight proteins were seen. In lysates from TRL10myc-transfected cells, proteins of 50 to 60 kDa and >90 kDa were detected by the Myc-specific antibody in the nonreduced lysates. However, it should be noted that a significant fraction of gpTRL10 remained in monomeric form (Fig. 6). In contrast, the 22- and 34-kDa forms of gpTRL10 present in viral lysates completely disappeared in the samples which were solubilized in the absence of 2-mercaptoethanol. Instead, a broadly migrating band between 40 and 90 kDa was detected by the TRL10 antiserum, demonstrating formation of a disuldfide-bonded complex which contained gpTRL10 as at least one protein component and likely a second and unidentified virion protein.
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On the nucleotide level, the gpTRL10 coding sequences showed overall homology of from 93.7 to 99.7%. On the amino acid level, the putative membrane anchor and carboxy-terminal regions between residues 86 and 171 were highly conserved among the isolates. The only differences in the predicted sequence were a KN exchange at position 150 and an ND exchange at position 170. The predicted extraluminal part of the protein showed a somewhat higher degree of variation (1.2 to 15.5% amino acid dissimilarities). However, the position and number of cysteine residues as well as potential N-linked glycosylation sites were perfectly conserved among the isolates. Note that the aspartic acid (D) in position 38 of TRL10AD169 seems to be the exception, since all other isolates have an asparagine (N) at the same position and thus resemble IRL10AD169-like sequences.
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DISCUSSION |
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N exchange at position 38 (8). Whether similar divergence occurs in other laboratory strains is unknown. The genomic area from both strains gave rise to numerous RNAs, making an assignment of the TRL10-specific transcripts via Northern blot analysis not possible even by using strand-specific probes. However, our results suggested that the TRL10 ORF was transcribed with early/late kinetics, which is in agreement with results obtained by microarray analysis (6).
Computer algorithms predict that gpTRL10 is a type I glycoprotein with a theoretical molecular mass of 20.5 kDa. The protein exhibits no amino acid homology to other herpesvirus proteins, including the closely related betaherpesviruses, murine cytomegalovirus (34), and human herpesvirus types 6 and 7 (12). In agreement with the predicted mass of the TRL10 protein, a polypeptide of 20 kDa was observed in infected cells after removal of N-linked carbohydrates.
Interestingly, in infected cells gpTRL10 existed in two predominant forms having molecular masses of 22 and 23.5 kDa. These molecules were modified by the addition of high-mannose sugars, as indicated by their sensitivity to Endo H. The most likely explanation for the expression of the two forms of gpTRL10 in infected cells was the presence of differentially glycosylated forms of gpTRL10. Potential sites for the addition of N-linked carbohydrates were present at amino acid positions 48/49 and 56 in the extraluminal domain of the gpTRL10. The presence of high-mannose carbohydrates on the vast majority of gpTRL10 present in virus-infected fibroblasts suggested that these molecules were likely localized to the ER. In addition to the predominant Endo H-sensitive forms, we also identified immunoreactive gpTRL10 which contained complex N-linked sugars, as reflected by their resistance to Endo H digestion and sensitivity to Endo F. Forms of TRL10 modified with complex carbohydrates were far less abundant in infected cells. In contrast, the gpTRL10 present in extracellular viral particles contained only complex N-linked sugars.
There are at least two possible explanations for the differences between the sugar modifications on the gpTRL10 in virus and that found in infected cells. The first is that the rabbit antiserum recognized predominantly forms of gpTRL10 modified with simple N-linked sugars. This possibility seems unlikely, since it was raised against an amino acid sequence at the C terminus of the molecule (amino acids 124 to 171) which is distal to the proposed N-linked glycosylation sites. Furthermore, transiently expressed gpTRL10 containing a C-terminal Myc epitope tag was modified nearly exclusively with simple N-linked sugars. The second and more likely possibility was that the gpTRL10 containing simple, high-mannose modifications was produced in excess during virus infection and its transport from proximal compartments of the secretory pathway was rate limiting. This resulted in overabundance of the gpTRL10 containing only simple, high-mannose sugar modifications within infected cells. Similar findings have been noted for other HCMV glycoproteins, including gB. Within infected cells, a significant amount of gB can be detected as an uncleaved polyprotein modified with simple, high-mannose sugars (3).
The predominance of forms of gpTRL10 containing only simple sugars could result from either an inefficient folding program, as has been proposed for gB, or perhaps the lack of sufficient expression of associated transporter molecules required for its transport to more distal sites of the secretory pathway. Consistent with this latter explanation was the finding that gpTRL10 in extracellular virions was approximately 8 to 10 kDa larger than intracellular forms of gpTLR10. Because this difference in size was believed to be a result of the presence of heterogeneous complex carbohydrate modification on virion gpTRL10, we would argue that gpTRL10 must associate with a second viral protein to traffic to distal compartments of the secretory pathway for terminal carbohydrate modifications and entry into a cytoplasmic assembly compartment.
Our findings indicated that gpTRL10 was present in a higher-molecular-weight complex. Expression of the TRL10 ORF in transfected cells indicated that in the absence of a reducing agent, the gpTRL10 formed higher-molecular-weight complexes or aggregates; however, a significant amount of the protein remained in monomeric form. In contrast, when extracellular virions were disrupted in the absence of a reducing agent, only higher-molecular-weight immunoreactive forms between 40 and 90 kDa were detected and none of the lower-molecular-weight forms. Together, these findings indicated that in the absence of other viral proteins, gpTRL10 could form higher-molecular-weight species, perhaps as a result of aggregation or nonnative disulfide bond formation between unpaired cysteines. In contrast, the gpTRL10 found in extracellular virions was present almost entirely as a higher-molecular-weight complexes. This result provided additional support for the argument that gpTRL10 was complexed with a second viral protein.
Consistent with this argument was the lack of intracellular transport and carbohydrate processing of gpTRL10 when it was expressed in the absence of other viral proteins. Thus, it appeared that similar to better-described HCMV envelope glycoproteins, gpTRL10 was complexed with other viral proteins, most likely through disulfide bonds, and that intracellular transport and incorporation of the protein into the virion progeny required complex formation. We have not identified the viral protein(s) in the complex and therefore cannot definitively exclude the possibility that the larger, complex forms of gpTRL10 were homodimers or dimers of differentially processed forms of gpTRL10. However, the ER retention and the lack of terminally glycosylated forms of gpTRL10 in cells transfected with a plasmid encoding the TRL10 ORF suggested that a virus function was required for its correct intracellular localization. In this respect, gpTRL10 might be similar to other HCMV glycoproteins such as gH, gL, gO, gM, and gN, which also require complex formation for proper folding and transport to the cell surface (19, 28, 38).
Structural glycoproteins of HCMV have been studied in a number of previous investigations. Solubilization of envelope constituents and subsequent analysis by various chromatographic methods have led to the characterization of three glycoprotein complexes (gCI to -III) within the envelope of HCMV (13). The glycoprotein composition of these complexes has been defined by investigators in several laboratories. The gCI complex is formed by homodimeric molecules of gB (2, 3). Glycoprotein complex II is composed of gM and gN, whereas glycoproteins H, L, and O together constitute the gCIII complex (17, 18, 26, 28). A protein corresponding to the size of gpTRL10 was not detected during the course of these studies, suggesting that this polypeptide was not an additional component of one of these complexes. However, other studies have noted the existence of envelope constituents with molecular weights similar to that of gpTRL10, although at a low abundance (1, 10). Thus, it remains to be determined if gpTRL10 is associated with one of the previously defined gCI to -III complexes or with an as yet unidentified envelope protein.
Because of its location in the virion envelope, gpTRL10 is potentially capable of inducing neutralizing antibodies and thus could represent a target of the humoral immune response during natural infection. Several studies have investigated antibody specificities against structural proteins of HCMV in detail (21, 22, 23, 24). Proteins which could correspond to gpTRL10 were not well described. However, in SDS-PAGE, gpTRL10 migrated as a very diffuse band and could have escaped detection. Other envelope glycoproteins such as gp116 (the amino-terminal component of gB) and gN, which have been shown to be highly immunogenic are also not routinely detected during immunoblot analysis with human sera.
Induction of strain-specific antibodies is a well-known phenomenon in HCMV biology, and antigenic variation could have limited antibody recognition of gpTRL10 by human sera (20, 44, 46). A number of glycoproteins have been described as polymorphic and thus able to induce strain-specific antibodies (30, 42). Our analysis of gpTRL10 coding sequences from clinical isolates revealed some sequence polymorphism in the amino-terminal part of the molecule. Since the extraluminal part is rather small (62 amino acids after removal of the signal peptide) and therefore could contain only a limited number of antibody binding sites, even a limited degree of variability would be sufficient to result in induction of strain-specific antibodies. Interestingly, computer algorithms indicate that the most variable domain between amino acids 40 and 60 is also highly antigenic.
Several other interesting findings were revealed by the sequence analysis. All low-passage isolates contained the DN substitution at position 38 of TRL10AD169 and thus corresponded to the IRL10AD169 sequence. This result suggested that the terminal repeats of AD169 may have originated as a duplication of the internal repeat sequences. Moreover, eight of the isolates that were analyzed contained an ND substitution at position 170. This substitution resulted in the extension of the acidic cluster at the C terminus of gpTRL10. Acidic clusters of membrane-bound proteins are known to play important roles in protein trafficking to the trans-Golgi network and are a common motif in the carboxyl terminus of herpesvirus glycoproteins (43). Whether this additional acidic residue in the C terminus of TRL10 resulted in a change in the intracellular trafficking of gpTRL10 from these clinical isolates of HCMV is unknown; however, it was of some interest that the TRL10 nucleotide sequence from most clinical isolates had this amino acid substitution.
Structural glycoproteins of herpesviruses can be grouped into two general classes, those that are conserved in members of the same subgroup of viruses or even throughout the entire family of herpesviruses and those glycoproteins that are unique to members of a specific subgroup of viruses. The conserved glycoproteins which have homologs in most members of the herpesviruses have been studied extensively and have been shown in many cases to be required for herpesvirus replication, including essential roles in particle assembly and early events in virus infection (4, 27, 33). Much less is known about structural glycoproteins which are unique to members of subgroups of herpesviruses. These proteins likely play very critical roles in the in vivo replication and pathogenic behavior of the respective viruses. An obvious example is the gp350/220 of Epstein-Barr virus, which binds to the cellular receptor CD21 on B cells (40, 41). Homologs of this viral protein have not been described in other alpha-, beta-, and gammaherpesviruses and point to a unique role of this protein in the in vivo replication of Epstein-Barr virus. Other examples include gD of HSV, a glycoprotein which has been shown to have an important role in adsorption (45).
A gD homolog has not been identified in betaherpesviruses such as HCMV, a finding which underscores the requirements of different viral glycoproteins for the unique and often characteristic biological behavior of herpesviruses. Whether gpTRL10 plays an essential role in the biology of HCMV remains to be determined; however, the maintenance of this ORF in the genome of clinical isolates strongly argues for an essential role in the replication of this virus in the natural host. A more complete study of the function of envelope glycoproteins whose expression is restricted to individual members of a virus group could provide insight into the role of these virion envelope components in specific aspects of infection, such as cell tropism and disease induction, which in turn define the unique phenotypes of individual herpesviruses.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung, and the National Institutes of Health.
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FOOTNOTES |
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