gpUL73 (gN) genomic variants of human cytomegalovirus isolates are clustered into four distinct genotypes

S. Pignatelli1, P. Dal Monte1 and M. P. Landini1

Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, St Orsola General Hospital, Via Massarenti 9, 40138 Bologna, Italy1

Author for correspondence: Sara Pignatelli. Fax +39 051 341632. e-mail sarapig{at}med.unibo.it


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Clinical isolates of human cytomegalovirus (HCMV) show differences in tissue tropism, severity of clinical manifestations and ability to establish persistent or latent infections, characteristics that are thought to be related to genomic variation among strains. This work analysed the genomic variants of a new HCMV polymorphic locus, open reading frame (ORF) UL73. This ORF encodes the envelope glycoprotein gpUL73 (gN), which associates in a high molecular mass complex with its counterpart, gM, and induces a neutralizing antibody response in the host. Detailed sequence analysis of ORF UL73 and its gene product from clinical isolates and laboratory-adapted strains shows that this glycoprotein is highly polymorphic, in the N-terminal region in particular. gpUL73 hypervariability is not randomly distributed, but the identified genomic variants are clearly clustered into four distinct genotypes (gN-1, gN-2, gN-3 and gN-4), which are not associated with the gB subtype.


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Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus with a broad spectrum of infectivity, as documented by the several different cell types that can be infected in vitro and by the multiple organ tropism observed during infection in vivo. Differences in tissue tropism, severity of clinical manifestations and ability to establish persistent or latent HCMV infections are thought to be related to genomic variability among strains (Brown et al., 1995 ; Fries et al., 1994 ; Meyer-Konig et al., 1998a , b ). The sequence similaritybetween different isolates varies depending on the region analysed (Bale et al., 1993 ); highly variable regions have been found in the HCMV genome.

HCMV genomic variants have been detected for a variety of genes, such as UL4 (Bar et al., 2001 ), UL144 (Lurain et al., 1999 ), gH (Chou, 1992a ) and gB. The HCMV genes that have been most studied for their polymorphism are those encoding the structural surface glycoproteins gB and gH, mainly because they are the target proteins for neutralizing antibodies, which are usually produced in a strain-specific pattern (Klein et al., 1999 ), and/or because they are involved in virus entry and cell-to-cell virus spread (Navarro et al., 1993 ; Rasmussen et al., 1997 ).

As far as gB is concerned, four genotypes (gB-1, gB-2, gB-3 and gB-4) have been identified (Chou & Dennison, 1991 ; Chou, 1992b ; Fries et al., 1994 ; Meyer-Konig et al., 1998a , b ), although rare non-prototypic variants have been described (Shepp et al., 1998 ; Trincado et al., 2000 ). The relationship between gB types and HCMV pathogenesis or virus cell/tissue tropism is under investigation, but the results are sometimes controversial (Binder et al., 1999 ; Chern et al., 1998 ; Gilbert et al., 1999 ).

Obviously, it is unlikely that one variant gene is the only factor that influences HCMV virulence, tropism and severity of clinical outcome. It seems more realistic that a cooperation of multiple gene variants associates in determining the virulence of each strain.

In this context, our study performed a detailed analysis of genomic variants of a new HCMV polymorphic locus, open reading frame (ORF) UL73, which is highly conserved among members of the family Herpesviridae. The protein homologues of ORF UL73 are Epstein–Barr virus (EBV) BLRF1, pseudorabies virus UL49.5, herpes simplex virus (HSV) Saimiri ORF 53, human herpesvirus-6 (HHV-6) U46 and murine CMV UL73. ORF UL73 encodes the envelope glycoprotein gpUL73 (gN) (Dal Monte et al., 2001 ). This glycoprotein associates in a high molecular mass complex with gM and is able to induce neutralizing antibodies in the host (Dal Monte et al., 2001 ; Mach et al., 2000 ).

The entire UL73 ORF was amplified by PCR from 38 HCMV clinical isolates and 4 laboratory-adapted strains (AD169, Towne, Davis and Toledo) and sequenced directly to determine the extent of sequence variability. Strain source, clinical data, outcome of HCMV disease (when available) and genotyping results are summarized in Table 1.


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Table 1. HCMV clinical samples

 
All virus strains were propagated in human embryonic lung fibroblasts (HELF). Virus infection and DNA extractions were performed as described previously (Dal Monte et al., 2001 ). Genomic DNA was used to amplify the UL73-coding sequence by PCR using the primers forward-U73, 5' TTCGGTCGGTCAACATCGTAAG 3', and reverse-L73, 5' CACCCACGTATGTAAACCTTAC 3'. The conditions for PCR were 35 cycles of 1 min at 96 °C, 1 min at 50 °C and 1 min at 72 °C. Both UL73 strands were sequenced by the dideoxynucleotide chain-termination method using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems), according to the manufacturer’s protocol. Purified sequencing ladders were analysed using the ABI PRISM 377 sequencer (Applied Biosystems).

Using the AD169 strain as the arbitrary reference sequence, we provide evidence that the 5' half of the ORF (nt 1–261) is highly polymorphic, although some changes occur throughout the remainder of the coding sequence. Furthermore, the identified variants are not randomly distributed, but are clearly clustered into four distinct groups. These four groups have been subjected to phylogenetic analysis to generate a tree (Fig. 1a) and a distance matrix. Phylogenetic analysis was performed using the method described by Hein & Stovlbaek (1996) , which is included in the Lasergene 1999 System package (DNASTAR). A detailed summary of the results is presented in Table 2. The four main groups demonstrate sequence identities in the range of 80–100% among the 42 strains sequenced. The reference group comprising the AD169 strain was referred to as group 1 and includes the six strains DB, GR, HDs, LN, SB and HR, whose UL73 shares {cong}100% sequence similarity to AD169. Group 2 (Can2, Can4, Can7, Can8 and DL) differs from group 1 because of three nucleotide deletions and 60–62 nucleotide substitutions, of which 45% are non-synonymous. Strains B-AL, B-AS, BD, ML, TS, FL and PS belong to group 3, with 49–51 nucleotide substitutions, of which 53% are non-synonymous with respect to group 1. As shown in Fig. 1(a), the branch identifying group 4 can be divided into three phylogenetically linked sub-groups. Sub-group 4a contains eight strains (BC, Can10, BO, LC, ZV, SR, HDu and MS), which diverge from AD169 by six nucleotide deletions and 51–55 nucleotide substitutions, of which 69% are non-synonymous. The Towne and Davis laboratory strains plus the five clinical isolates BR, LV, RL, SE and CR belong to sub-group 4b, which, when compared to group 1, show six nucleotide deletions and 76–79 nucleotide substitutions, of which 56% are non-synonymous. Strains RC, VL, PM, PN, GJ, MN, BN and Toledo form group 4c and show nine nucleotide deletions and 78–79 nucleotide substitutions, of which 58% are non-synonymous with respect to AD169.



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Fig. 1. (a) Results of phylogenetic analysis of the 42 UL73 DNA sequences. The tree was generated using the MegAlign software. Scale measures distance among sequences. The reliability of the branching orders was estimated by bootstrapping (1000 re-iterations). Bootstrap values (% after 1000 iterations) for major branches are shown. (b) Alignment of UL73 amino acid sequences of the clinical and laboratory-adapted strains and comparison using the AD169 sequence as the arbitrary reference sequence. Dots indicate identity and dashes indicate deletions. Strains are listed according to the sequence groups determined by phylogenetic analysis of the DNA sequences. The amino acid sequences were aligned using the MultiAlign software (http://dot.imgen.bcm.tmc.edu.9331/multi-align/multi-align.html), option MAP (Huang, 1994 ), and displayed as a printable output by the BOXSHADE server (http://www.ch.embnet.org/software/BOX_form.html).

 

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Table 2. UL73 genotype frequency and nucleotide/amino acid substitutions

 
The non-synonymous nucleotide substitutions lead to a wide spectrum of amino acid changes. A sequence alignment compiled using the MultiAlign software of the 42 gpUL73 amino acid sequences obtained from clinical and laboratory-adapted strains is shown in Fig. 1(b). Phylogenetic analysis of the proteins is consistent with results described for DNA, thus maintaining the division of HCMV strains into four main groups (tree not shown). As summarized in Table 2, a large number of amino acid substitutions was detected using group 1 as the arbitrary reference sequence. In particular, group 2 shows a single amino acid deletion and 21–22 amino acid substitutions, of which 59% are non-conservative substitutions. Group 3 displays 20–21 amino acid substitutions, of which 43% are non-conservative. Sub-group 4a shows two amino acid deletions and 21 amino acid substitutions, of which 52% are non-conservative, while sub-group 4b shows two amino acid deletions and 26–28 amino acid substitutions, of which 64–65% are non-conservative. Sub-group 4c presents three amino acid deletions and 26–28 amino acid substitutions, of which 56–64% are non-conservative.

The alignment also shows a high level of amino acid identity (96–100%) among strains within each group, with occasional substitutions being maintained across the clusters. Genotypic frequencies of gpUL73 variants demonstrate that gN-4 is the most representative variant in our population (Table 2).

The clustered variants demonstrated polymorphism in the N-terminal region of the protein, with most of the DNA mutations in the C-terminal region resulting in silent mutations (Fig. 1b). This finding, together with data discovered recently on the biochemical properties of gpUL73 (Dal Monte et al., 2001 ; data not shown), the predicted features of this protein and its ability to induce neutralizing antibody (Britt & Auger, 1985 ), suggest that the polymorphic N-terminal domain is exposed at the external surface of the virus particle. This kind of polymorphic region is considered to be an important domain for the immune response during natural infection and usually represents a target for immunological selective pressure.

Furthermore, previous reports (Mach et al., 2000 ) demonstrated that gN associates with its glycosylated counterpart, gM, forming a highly immunogenic envelope complex, gCII (Kari et al., 1986 ). Considering that gM seems to be highly conserved (Lehner et al., 1991 ) and that our prototype strains AD169, Can4, PS, ZV, BR and RC (Table 2) have an almost identical gM sequence (data not shown), we can speculate that the polymorphic gpUL73 (gN) could be the main protein responsible for the immunogenicity of gCII. Thus, a detailed analysis of the in vivo immune response to gN and gM and to the gN–gM complex is required to confirm this hypothesis and elucidate the role of gN in HCMV immunopathogenesis.

On the contrary, the high degree of conservation observed in the putative transmembrane region and in the C-terminal domain strongly suggest that these regions are not under immunological pressure and their functions have to be maintained for virus growth and/or virus integrity.

However, sequence variability does not seem to influence the expression of gN. In fact, an immunofluorescence assay was used to test the expression of gpUL73 in HELF infected with the prototype stains and stained using the rabbit polyclonal antibody PAbUL73, as described previously (Dal Monte et al., 2001 ); the protein was always detected.

UL73 encodes a structural glycoprotein, predicted to be a type I transmembrane protein, with 33 O-glycosylation sites and 18 phosphorylation sites, according to Meta-Predict-Protein server. A comparison of gpUL73 putative post-translational modifications from group-specific prototypes showed that, despite the observed hypervariability, the basic structural features, such as glycosylation/phosphorylation sites and transmembrane domains, are essentially shared by strains from all of the clusters. In particular, the putative transmembrane domain (aa 104–122 relative to AD169) and two phosphorylation sites (Y86–T134) are conserved in the prototype strains. However, O-glycosylation/phosphorylation sites can vary in number, although they do involve the same region (aa 17–71), where non-synonymous nucleotide changes often convert one potential glycosylation site to another. Thus, sequence variability seems to affect only partially the conserved structural features of the putative gpUL73.

Comparison of gpUL73 with the counterpart proteins from other herpesviruses demonstrated that homologues encode for shorter polypeptides, with markedly lower putative post-translational modifications and, in some cases (HHV-6, HSV-Saimiri and EBV), an additional transmembrane domain. These data are in agreement with the finding that fully processed HCMV gpUL73 (gN) has a molecular mass of 39–53 kDa (putative molecular mass of 15 kDa), while its homologues are lighter (8–14 kDa) and present a limited number of O- and N-linked sugars (Jons et al., 1996 ; Lake et al., 1998 ).

Since strains used in this study were passaged in cell culture, we verified that in vitro propagation did not modify or select variants present in the clinical specimen inoculum, as in vitro passaging of the virus is considered to be partly responsible for strain selection (Sinzger et al., 1999 ). The gpUL73 genotypes obtained from eight pairs of clinical isolates at low (3–7) and high (30–33) passage number in cell culture were compared. No difference between gN variants passaged at low and high numbers was detected, indicating either that gN is stable during HCMV culture or that more in vitro passages are necessary for strain selection and/or adaptation.

We also determined whether different genotypes can be found in the same patient. For that purpose, the UL73 sequence was obtained from five patients over a follow-up period of 6 months or from the urine and saliva of two patients (ZV and HD). In the first case, identical gN sequences were obtained from all patients throughout follow-up, indicating either that these genotypes are stable during in vivo infection, as shown for HHV-8 latent nuclear antigen and ORF-K1 (Gao et al., 1999 ; Zong et al., 1999 ) and HCMV UL144 (Lurain et al., 1999 ), or that a longer period of time is needed for genotype change. In the second case, the same gN genotype was detected in both clinical specimens of ZV, while the other one (HD) displayed two different variants, gN-1 in saliva and gN-4 in urine, although the specimens showed the same gB-2 type (Table 1). The presence of multiple genotypes within the same patient will make difficult the determination of whether a correlation exists between a particular gN genomic variant and the degree of HCMV pathogenicity, as drawn previously for gH and gB (Vogelberg et al., 1996 ).

To determine whether gpUL73 variants correlate with gB groups, a 410 bp fragment encompassing the proteolytic cleavage site of gB was sequenced, as described previously (Lurain et al., 1999 ). As shown in Table 1, most of our strains showed a gB-1 (Towne prototype, 69% frequency) or a gB-2 (AD169 prototype, 28·5% frequency) genotype. Toledo and Davis laboratory-adapted strains, uncharacterized previously for gB hypervariability, showed a gB-3 and gB-1 genotype, respectively. No mixture of gB types was found. Neither was a correlation between gB type and gN type observed in our population.

The comparison between gpUL73 (gN) and gB variability shown in this study suggests two main points: (a) gB and gpUL73 (gN) sequence clusters are not phylogenetically linked, as reported recently for the UL144 and UL4 genotypes (Bar et al., 2001 ; Lurain et al., 1999 ) and (b) gpUL73 (gN) seems to be more polymorphic than gB. In fact, two predominant gB types (gB-1 and gB-2) were found in our population, while four main gN variants were detected in the same samples.

The hypothesis that gpUL73 is more variable than other surface glycoproteins stresses the need for an in-depth analysis of this protein, as it may play an important role as a stimulator of the host immune response or as a modulator of cell-specific virus tropism and could represent a useful marker to distinguish HCMV strains.

One gene variation may contribute to differences in HCMV pathogenicity depending on the function of its product. Envelope glycoproteins are highly conserved among herpesviruses because of their role in virus–cell interaction and host immunological responses; thus, their variations could have strong effects on virus ‘behaviour’ (Britt & Mach, 1996 ; Rajcani & Vojvodova, 1998 ). In the case of HSV-1, gB variation has been shown to influence pathogenicity (Goodman & Engel, 1991 ). Also, HCMV gB polymorphism has been related to cell tropism and virulence (McCarthy et al., 1991 ; Pulliam, 1991 ).

In conclusion, the precise definition of the genotypes of single viral glycoproteins present in clinical strains, their immunological properties and their role in virus cell tropism might be extremely useful to understand virus pathogenicity.


   Acknowledgments
 
We thank Dr Alessandro Ripalti for reviewing the manuscript. This work was partially supported by the University of Bologna ‘Young Researcher Award’ (1999), the AIDS projects of the Italian Ministry of Public Health (60%) and the Italian Ministry of University and Scientific Research (40%).


   Footnotes
 
The UL73 sequences of the 38 clinical strains have been assigned GenBank accession nos AF309969AF310006.


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Received 30 May 2001; accepted 25 July 2001.