Journal of General Virology

The carboxyl terminus of the human cytomegalovirus UL37 immediate-early glycoprotein is conserved in primary strains and is important for transactivation

Wail A. Hayajneh,^1,2 Despina G. Contopoulos-Ioannidis,^1,2† Marci M. Lesperance,^1,3‡ Ana M. Venegas¹ and Anamaris M. Colberg-Poley¹

Center for Virology, Immunology and Infectious Disease Research, Children's Research Institute¹, Department of Infectious Diseases² and Department of Otolaryngology³, Children's National Medical Center, George Washington University School of Medicine and Health Sciences, 111 Michigan Avenue, NW, Washington, DC 20010, USA

The human cytomegalovirus (HCMV) UL37 exon 3 (UL37x3) open reading frame (ORF) encodes the carboxyl termini of two immediate-early glycoproteins (gpUL37 and gpUL37_M). UL37x3 homologous sequences are not required for mouse cytomegalovirus (MCMV) growth in vitro; yet, they are important for MCMV growth and pathogenesis in vivo. Similarly, UL37x3 sequences are dispensable for HCMV growth in culture, but their requirement for HCMV growth in vivo is not known. To determine this requirement, we directly sequenced the complete UL37x3 gene in multiple HCMV primary strains. A total of 63 of the 310 amino acids in the UL37x3 ORF differ non-conservatively in one or more HCMV primary strains. The HCMV UL37x3 genetic diversity is non-random: the N-glycosylation (46/186 aa) and basic (9/15 aa) domains have the highest proportion of non-conservative variant amino acids. Nonetheless, most (15/17 signals) of the N-glycosylation signals are retained in all HCMV primary strains. Moreover, new N-glycosylation signals are encoded by 5/20 primary strains. In sharp contrast, the UL37x3 transmembrane (TM) ORF completely lacks diversity in all 20 HCMV sequenced primary strains, and only 1 of 28 cytosolic tail residues differs non-conservatively. To test the functional significance of the conserved carboxyl terminus, gpUL37 mutants lacking the TM and/or cytosolic tail were tested for transactivating activity. The gpUL37 carboxyl-terminal mutants are partially defective in hsp70 promoter transactivation even though they trafficked similarly to the wild-type protein into the endoplasmic reticulum and to mitochondria. From these results, we conclude that N-glycosylated gpUL37, particularly its TM and cytosolic domains, is important for HCMV growth in humans.

Human cytomegalovirus (HCMV) is a medically significant pathogen that causes congenital infections as well as seriously debilitating or life-threatening infections in immunocompromised patients (Britt & Alford, 1996). The UL36–38 locus appears to be essential for HCMV origin-dependent DNA replication and HCMV growth in vitro (Pari et al., 1995; Smith & Pari, 1995). The UL36–38 locus encodes three UL37 immediate-early (IE) proteins: the UL37 exon 1 (UL37x1) protein (pUL37x1) and two UL37 N-glycoproteins (gpUL37 and gpUL37_M) which differ in the portion of the N-glycosylation domain which they retain (Wilkinson et al., 1984; Kouzarides et al., 1988; Chee et al., 1990; Al-Barazi & Colberg-Poley, 1996; Goldmacher et al., 1999). pUL37x1, gpUL37 and gpUL37_M play significant roles in anti-apoptosis and in the regulation of HCMV DNA replication genes and of cellular genes (Colberg-Poley et al., 1992, 1998; Tenney et al., 1993; Colberg-Poley, 1996; Zhang et al., 1996; Biegalke, 1999; Goldmacher et al., 1999; Hayajneh et al., 2001).

All UL37 IE proteins share their amino-terminal signal sequence (aa 1–22), a strongly charged acidic domain (aa 81–108) and two domains (aa 5–34 and aa 118–147) that are required and sufficient for anti-apoptotic activity (Kouzarides et al., 1988; Goldmacher et al., 1999; Hayajneh et al., 2001). Although UL37x1 homologous sequences are not found in other herpesviruses (Baer et al., 1984; Davison & Scott, 1986; McGeoch, 1989; Gompels et al., 1995; Nicholas, 1996; Rawlinson et al., 1996; Russo et al., 1996; Vink et al., 2000), they are very well conserved in HCMV primary strains, indicating their biological importance for growth in vivo (Hayajneh et al., 2001).

gpUL37, encoded mostly by the downstream UL37 exon 3 (UL37x3) open reading frame (ORF), is an integral membrane protein with a large N-glycosylated domain, a basic domain, a typical hydrophobic transmembrane (TM) domain and a compact cytosolic tail (Kouzarides et al., 1988; Chee et al., 1990; Al-Barazi & Colberg-Poley, 1996). gpUL37 traffics through the endoplasmic reticulum (ER) and Golgi apparatus, to the plasma membrane, and to mitochondria (Al-Barazi & Colberg-Poley, 1996; Colberg-Poley et al., 2000 a). gpUL37 is modified by N-glycosylation in the ER and Golgi apparatus during HCMV infection of permissive cells (Al-Barazi & Colberg-Poley, 1996). gpUL37_M differs from gpUL37 by the absence of aa 178–262, which contain the first six N-glycosylation signals within the N-glycosylation domain (Goldmacher et al., 1999).

In contrast to the UL37x1 gene, UL37x3 homologous sequences are conserved in other betaherpesviruses (Nicholas & Martin, 1994; Gompels et al., 1995; Nicholas, 1996; Rawlinson et al., 1996). UL37x3 homologous sequences are not required for mouse cytomegalovirus (MCMV) growth in culture (Lee et al., 2000). Nonetheless, an MCMV UL37x3 mutant is severely hindered both in growth and in pathogenesis in mice (Lee et al., 2000).

It has been found that HCMV UL37 exons 2 and 3 are not required for HCMV growth in human diploid fibroblasts (HFF) in culture (Borst et al., 1999; Goldmacher et al., 1999). As the UL37x3 ORF is important for MCMV growth and pathogenesis in vivo but not for its growth in culture, we tested whether UL37x3 sequences are important for HCMV growth in vivo. The limited host range of HCMV and the lack of a suitable animal model which fully recapitulates HCMV replication and pathogenesis in vivo makes it difficult to determine whether or not a gene product is essential for HCMV growth. We therefore determined the nucleotide sequences of the complete UL37x3 gene (930 nt) in 20 HCMV primary strains to assess conservation of its ORF in vivo. Sequence diversity of subdomains within ORFs considered to be highly conserved has been found to occur in HCMV primary strains (Chou & Dennison, 1991; Chou, 1992; Meyer-König et al., 1998; Zweygberg Wirgart et al., 1998). Conservation of ORFs or specific subdomains in primary strains at the amino acid level suggests a crucial role of the product for HCMV growth in humans. Conversely, if HCMV is able to grow in vivo despite non-conservative substitutions or deletions in ORFs or subdomains, those ORFs or subdomains are predictably non-essential for HCMV growth in humans. We tested the functional significance of the conserved gpUL37 carboxyl-terminal domains of primary strains by examining mutants in the TM and cytosolic domains for transactivating activity and their ability to traffic like the parental gpUL37 protein.

HCMV primary strains. Clinical specimens used directly for PCR sequencing of HCMV primary strains included a lung biopsy (specimen 1), urine (specimens 6, 11, 13, 15, 32, 52, 53 and 54) and a purified buffy coat preparation from peripheral blood (specimen 72) (Hayajneh et al., 2001). Other HCMV primary strains were PCR-sequenced either from urine cultured only once in HFF cells (specimens 31, 33, 34, 35 and 36) or from supernatant of shell vial assays that were performed on urine specimens (specimens 63, 64, 65, 67 and 68). The specimens had scored HCMV-positive by shell vial assay or culture and were not subcultured further in HFF cells prior to PCR sequencing. HCMV DNA purified from HFF cells infected with HCMV (strain AD169, ATCC) as previously described (Lesperance et al., 1998) served as a positive control for all PCR reactions.

Nested PCR and PCR sequencing. Nested PCR (NPCR) was performed as previously described (Hayajneh et al., 2001). The primers used for PCR and NPCR amplifications are represented in Fig. 1 (a). The sequencing strategy involved NPCR, purification of the NPCR products using QIAquick gel extraction kit (Qiagen) and PCR sequencing using UL37 sequencing primers (Fig. 1 a) as described previously (Hayajneh et al., 2001). Primers 113 (nt 50764–50786), 114 (nt 50156–50176), 115 (nt 50156–50176), 134 (nt 49833–49852), 154 (nt 50765–50786), 173 (nt 50873–50894), 174 (nt 50919–50939) and 175 (nt 49803–49823) allowed for direct PCR sequencing of the complete UL37x3 ORF (nt 49910–50842). UL37x3 sequences from HCMV primary strains were compared to those obtained for HCMV strain AD169, sequenced in parallel. Variant sequences in primary strains were sequenced at least three times to verify their identities independent of PCR amplification.

Fig. 1. (a) The UL37 gene and PCR and sequencing primers. The UL37 exons 1, 2 and 3 are represented at the top of the figure. The approximate nucleotide position of the exons, start (nt 52706) and stop (nt 49910) of translation as well as the splice junctions (nt 52219/50989 and nt 50947/50842) are indicated on the gene. The nucleotide numbering is from the HCMV AD169 sequence (EMBL accession no. X17403, Chee et al., 1990). The arrows on UL37x3 represent PCR and sequencing primers and their polarity. (b) Wild-type and mutant UL37 proteins. gpUL37, gpUL37_M and pUL37x1 are represented at the top. The leader (first cylinder) and the acidic domain (open rectangle) are common to all UL37 proteins and are encoded by UL37x1 sequences (Kouzarides et al., 1988; Goldmacher et al., 1999). The N-glycosylation signals (lollipops), a basic domain (cross-hatched hexagon), TM (second cylinder) and cytosolic tail (line) are encoded by UL37x3 sequences. The dashed line represents the sequences absent from gpUL37_M. The motifs retained by truncation mutants gpUL37 aa 1–461 and gpUL37 aa 1–438 are represented below.

Phylogenetic tree analysis. This was performed on the nucleotide sequences of the HCMV UL37x3 ORF (nt 49910–50842) of the sequenced primary strains and HCMV strain AD169. The analysis was conducted by complete alignment of the nucleotide sequences using the Clustalx program (NCBI) and construction of an unrooted tree using the PHYLIP 3.5 DrawTree program.

gpUL37 carboxyl-terminal mutants. gpUL37 truncation mutants were used to test the functional importance of the conserved carboxyl-terminal TM and cytosolic tail (Fig. 1 b). Mutant gpUL37 aa 1–438 (p605) was created by insertion of a stop linker (5´ CTA GGC CTT AGC GGC CGC TAG 3´) into an NdeI site (nt 50062) within the TM ORF. This mutant gene was then subcloned under the control of the HCMV major IE enhancer/promoter, generating p627. Mutant gpUL37 aa 1–461 (p612), under the control of the major IE promoter, was generated by insertion of the termination linker into the unique NruI site (nt 49992) in the UL37x3 cytosolic ORF (Colberg-Poley et al., 1998). The mutant constructions were verified by nucleotide sequencing (US Biochemical) and the ORFs were independently verified by in vitro transcription and translation (unpublished results).

Enzymatic assays. Transactivation of the human hsp70 promoter–chloramphenicol acetyltransferase (pHB-CAT) construction by gpUL37 and the gpUL37 mutant was measured using a modification of the previously published procedure (Colberg-Poley et al., 1992). Briefly, HeLa cells were transfected with pHB-CAT and the expression vectors for gpUL37, mutant gpUL37 or negative control glycoprotein B (gB) using calcium phosphate coprecipitation. Transfection efficiency was normalized by co-transfection of pCH110 (Pharmacia), carrying E. coli lacZ under the control of the SV40 early promoter (Colberg-Poley et al., 1992). Cells were harvested at 48 h after transfection and assayed for both CAT and -galactosidase (-gal) activities as previously described (Colberg-Poley et al., 1992). Briefly, CAT reactions contained 80 µg of protein extract, 0.25 M Tris–HCl pH 8.0, 1 mM acetyl-coenzyme A (Pharmacia) and [¹⁴C]chloramphenicol. The acetylated forms of [¹⁴C]chloramphenicol were resolved from the unacetylated [¹⁴C]chloramphenicol by thin layer chromatography and each was quantified by scintillation counting. Transfected cells were assayed for -gal activity by conversion of chlorophenol red -galactopyranoside (Boehringer Mannheim) (Eustice et al., 1991). Protein concentrations of the extracts were determined using Bio-Rad Protein Determination Reagent as previously described (Colberg-Poley et al., 2000 b). Fold inductions were calculated by dividing the normalized CAT/-gal activity of each group by the normalized CAT/-gal activity of the negative control group (gB) (Colberg-Poley et al., 1992).

Confocal laser scanning microscopy. HFF cells were transiently transfected with expression plasmids for mutant gpUL37 aa 1–461 (p612), mutant gpUL37 aa 1–438 (p627), wild-type gpUL37 (p414) or control gB (p370) using LipofectAmine as previously described (Colberg-Poley et al., 2000 a). Cells were harvested 48 h after transfection, fixed in methanol and stained simultaneously with rabbit polyvalent Ab1064 (1:200, against gpUL37 aa 27–40), mouse anti-protein disulphide isomerase (PDI, 1:25, StressGen) and human autoimmune serum against mitochondria (1:25, ImmunoVision) at 37 °C for 1 h. Secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:50, Southern Biotechnology), Texas red (TR)-conjugated goat anti-mouse IgG (1:50, Kirkegaard and Perry Laboratories) and cyanine 5 (Cy5)-conjugated goat anti-human IgG (1:100, Jackson ImmunoResearch). Analyses were performed with a Bio-Rad MRC1024 confocal laser scanning microscope (Center for Microscopy and Image Analysis, George Washington University, USA) as previously described (Colberg-Poley et al., 2000 a). Emission signals were measured at 520 (FITC), 615 (TR) and 670 (Cy5) nm. Individual signals were captured sequentially to avoid spurious overlap of the emission signals. Individual optical sections were examined to determine co-localization of gpU37 mutants with cellular markers. Images were generated using Adobe Photoshop (version 4.0), Bio-Rad confocal microscopy plug-ins and Microsoft Publisher 98.

Fig. 1 represents the HCMV UL37 genomic sequences, the UL37x3 primers and the wild-type UL37 proteins and truncation mutants. The HCMV strain AD169 UL37x3 sequence was as previously published (Kouzarides et al., 1988). The UL37x3 ORF is maintained in all sequenced HCMV primary strains. Although substitutions, in-frame insertions and in-frame deletions were found within the HCMV UL37x3 ORF of primary strains, none resulted in termination codons or frame shifts of the UL37x3 ORF. The nucleotide and amino acid variations are not randomly scattered throughout the UL37x3 ORF; rather, they are located predominantly in the extracellular domains of gpUL37/gpUL37_M (Fig. 2). The UL37x3 genetic diversity spanned the first three-quarters of the ORF. A total of 88 of the 310 aa in the UL37x3 ORF differ from AD169, 63 of them non-conservatively in one or more strains (Table 1).

Fig. 2. Genetic diversity of the UL37x3 ORF in HCMV primary strains. The number of primary strains whose sequence differs from that of AD169 is indicated for each residue of the UL37x3 ORF in the histogram. The map above the histogram indicates the corresponding portion of gpUL37.

Table 1. Proportion of UL37x3 variant amino acids in primary strains

N-glycosylation subdomain (aa 206–391)

As viral glycoproteins are potentially subject to the host immune response, we anticipated that the gpUL37/gpUL37_M N-glycosylated subdomain (aa 206–391) might contain the largest number of variant amino acids. A total of 67 residues of the 186 aa differed: of these, 46 residues differed non-conservatively in one or more primary strains (Table 1). Thus, we found that the N-glycosylation ORF differs from AD169 more frequently than does the upstream UL37x1 ORF (Hayajneh et al., 2001) but comparably with the HCMV UL144 ORF (Lurain et al., 1999).

gpUL37 (including signals 1–17) and gpUL37_M (including signals 7–17) have large N-glycosylation domains (Fig. 1 b). As the UL37x1 hydrophobic leader lacks diversity in all HCMV primary strains sequenced (Hayajneh et al., 2001), targeting of gpUL37/gpUL37_M encoded by HCMV primary strains to the ER is predicted. To test this prediction and the importance of post-translational modification of gpUL37/gpUL37_M in vivo, we examined the conservation of N-glycosylation signals. Two N-glycosylation signals, signals 6 and 15, are deleted in-frame in 10/20 and 9/20 primary strains, respectively (Table 2). Nonetheless, the majority of the N-glycosylation signals (NXT/S) either lack diversity (signals 1–5, 7–12 and 17) or are conserved (signals 13, 14 and 16) in all sequenced primary strains because of second (X) amino acid variants, which do not alter the recognition signal. Moreover, five strains contain an in-frame insertion of consensus N-glycosylation signals either between signals 11 and 12 (signal 11*, NAT, 1/20) or between signals 13 and 14 (signal 13*, NVT, 4/20). Thus, while overall genetic diversity within the UL37x3 N-glycosylation domain is notable, the N-glycosylated character of gpUL37/gpUL37_M is retained by virtue of the conserved UL37x1 hydrophobic leader and multiple N-glycosylation signals.

Table 2. Conservation of gpUL37 N-glycosylation signals in HCMV primary strains.

Three cysteines (aa 291, 366, 453) encoded by the HCMV UL37x3 gene are conserved in the human herpesvirus 6 UL37x3 homologous ORF (Nicholas & Martin, 1994). These residues are conserved in all the HCMV primary strains sequenced (data not shown).

The divergent basic subdomain (aa 368–382)

The UL37x3 basic subdomain partially overlaps with the end of the N-glycosylation subdomain. The basic residues are the most diverse within the UL37x3 ORF, 13 of the 15 residues differ, nine non-conservatively (Table 1). The conservative variants include ³⁷¹K R, ³⁷³T S or A, ³⁷⁴V L or I, ³⁷⁶L I, ³⁷⁷T A, ³⁷⁸R K, ³⁸⁰K R and ³⁸²K R (Table 3). The non-conservative variants include ³⁶⁹F L, ³⁷²R G, ³⁷⁵K S, ³⁷⁶L F, ³⁷⁷T M or deletion, ³⁷⁹N R or K, ³⁸⁰K N, Q or deletion, ³⁸¹T Q or deletion and ³⁸²K I. Thus, four basic subdomain residues (³⁷¹K, ³⁷³T, ³⁷⁴V and ³⁷⁸R) varied only conservatively while nine residues (³⁶⁹F, ³⁷²R, ³⁷⁵K, ³⁷⁶L, ³⁷⁷T, ³⁷⁹N, ³⁸⁰K, ³⁸¹T and ³⁸²K) varied both conservatively and non-conservatively.

Table 3. Amino acid diversity in the UL37x3 basic domain ORF of HCMV primary strains.

The TM ORF (aa 433–459) lacks diversity in HCMV primary strains

In strong contrast to the N-glycosylation and basic subdomains, no amino acid differences were observed in the UL37x3 TM domain of any of the 20 sequenced HCMV primary strains (Fig. 2 and Table 1). Because the 27 TM residues of gpUL37/gpUL37_M serve as an anchor for this type I integral membrane protein (Al-Barazi & Colberg-Poley, 1996), we expected that all residues encoded by primary strains would be hydrophobic. However, the complete lack of diversity in the primary strain UL37x3 TM ORF was surprising and suggests an important role beyond that of a hydrophobic anchor, likely involving direct interactions with other membrane proteins.

The conserved cytosolic tail (aa 460–487)

The compact gpUL37/gpUL37_M cytosolic tail has consensus PKC (⁴⁷⁸STK), CKII (⁴⁷⁹TKND) and Tyr (⁴⁶¹RDLLEDFRY) phosphorylation sites. The residues contained within these consensus phosphorylation sites lack diversity in all the sequenced primary strains (Fig. 2 and Table 1). Two cytosolic tail residues (⁴⁷⁶S G and ⁴⁸⁵R W), which are not contained within consensus phosphorylation sites, were found to differ infrequently (2/20 and 3/20, respectively). The ⁴⁷⁶S G variation is conservative while the other is not.

Phylogenetic analysis of HCMV UL37x3 DNA sequences

Visual inspection of the HCMV UL37x3 ORF indicated that some strains were closely related in nucleotide sequence. To test this suggestion, we performed a phylogenetic analysis of the HCMV UL37x3 nucleotide sequences to generate an unrooted tree (Fig. 3). Based upon this analysis, five primary branches of HCMV primary strains were observed. These are named I–V starting with the cluster containing strain AD169. Group I viruses (strains 34, 52, 53, 64, 65 and AD169) differ by no more than seven of the 930 nucleotides in the UL37x3 ORF from strain AD169. Group II (strains 1, 6, 11, 13, 32, 33, 63 and 68) differ by 93–120/930 nt from AD169 UL37x3 ORF. Group III (strains 15 and 36), group IV (strains 31, 54, 67 and 72) and group V (strain 35) differ by 100–143/930 nt from the strain AD169 UL37x3 ORF. We also generated an unrooted tree using the amino acid sequences of the UL37x3 ORF from the HCMV primary strains. All the strains remained in the same groups, as seen in the nucleotide phylogenetic analysis, indicating that translationally silent mutations had not resulted in any significant convergence of the UL37x3 amino acid sequence (data not shown).

Fig. 3. Phylogenetic analysis of HCMV UL37x3 nucleotide sequences in 20 independent primary strains. The analysis was conducted using Clustalx and PHYLIP DrawTree programs on the UL37x3 ORF (nt 50842–49910). Numbers on the tree correspond to the strain numbers used in the manuscript or to HCMV strain AD169. The scale bar indicates % nucleotide diversity. The primary branches are labelled sequentially (I–V) starting with the cluster containing AD169.

gpUL37 carboxyl-terminal mutants are partially defective for hsp70 promoter transactivation in transfected cells

The low variability of gpUL37 carboxyl-terminal TM and cytosolic sequences in most primary strains suggested their functional importance for HCMV growth in vivo. Therefore, we set out to determine the contribution of the TM and cytoplasmic subdomains to the known regulatory activity of gpUL37 for hsp70 promoter transactivation (Colberg-Poley et al., 1992, 1998; Tenney et al., 1993; Zhang et al., 1996). We examined two gpUL37 truncation mutants, gpUL37 aa 1–461 and gpUL37 aa 1–438 (Fig. 1 b), for their ability to transactivate hsp70 promoter activity (Fig. 4). The CAT activities of triplicate wild-type gpUL37 extracts converted about 90–94 % (29238, 31131 and 33952 c.p.m.) of total [¹⁴C]chloramphenicol (31133, 34578 and 37000 c.p.m.) to its acetylated forms. Mutant gpUL37 aa 1–461, which retains the TM domain and 2 aa of the cytosolic tail, transactivated hsp70 expression (10.7±1.1-fold) to levels about 30 % of wild-type gpUL37 (38.5±0.48-fold). Mutant gpUL37 aa 1–438, which lacks an intact TM and the cytosolic domain, transactivated hsp70 promoter-driven expression (3.75±0.6-fold) to levels about 10 % of wild-type gpUL37. gB, an HCMV structural glycoprotein, served as a negative control for these experiments (1.0±0.0-fold). These results suggest that the gpUL37 carboxyl-terminal cytosolic tail and TM domain, encoded by the UL37x3 sequences which lack diversity, play key functional roles in transactivating activity.

Fig. 4. The gpUL37 carboxyl-terminal TM domain and cytosolic tail are important for hsp70 promoter transactivation. HeLa cells were transfected with pHB-CAT, pCH110 and expression vectors encoding wild-type gpUL37, mutant gpUL37 aa 1–461, mutant gpUL37 aa 1–438 or gB. Cells were harvested at 48 h post-transfection and the extracts were analysed for CAT and -gal activities. The normalized CAT activities of each group were compared to the normalized mean CAT activity of cells transfected with the negative control, gB. Shown is a representative experiment with the average of triplicate samples for each group. The error bars indicate the standard error of the means.

gpUL37 carboxyl-terminal mutants traffic to the ER and to mitochondria in HFF cells

As mutants gpUL37 aa 1–461 and gpUL37 aa 1–438 are partially defective in hsp70 promoter transactivation, we examined their trafficking in HFF cells to determine if it was similar to that of the wild-type gpUL37 (Fig. 5). Wild-type gpUL37 is known to traffic to the ER and to mitochondria (Colberg-Poley et al., 2000 a). For these studies, HFF cells were transfected with expression vectors encoding gpUL37 aa 1–461 (Fig. 5 a–f) or gpUL37 aa 1–438 (Fig. 5 g–l) and stained simultaneously with anti-gpUL37 (Ab1064), anti-PDI and anti-mitochondrial antibodies. gpUL37 aa 1–461 (Fig. 5 a) and gpUL37 aa 1–438 (Fig. 5 g) were detected using Ab1064. The ER (Fig. 5 b, h) and mitochondria (Fig. 5 d, j) were clearly stained by their respective antibodies. In the optical sections shown, gpUL37 aa 1–461 (Fig. 5 c, e) and gpUL37 aa 1–438 (Fig. 5 i, k) co-localized with the ER and mitochondrial markers as indicated by the yellow and aquamarine overlaps, respectively. Overlap between the two compartments containing the mutant glycoproteins (white overlap) was observed (Fig. 5 f, l). Taken together, these results suggest that gpUL37 aa 1–461 and gpUL37 aa 1–438 traffic through the secretory apparatus and to mitochondria, as wild-type gpUL37 does.

Fig. 5. Mutant gpUL37 aa 1–461 and gpUL37 aa 1–438 traffic to the ER and to mitochondria in HFF cells. HFF cells transiently expressing gpUL37 aa 1–461 (a–f) or gpUL37 aa 1–438 (g–l) were stained with rabbit Ab1064, mouse anti-PDI (ER) and human autoimmune anti-mitochondrial antibodies. The cells were then stained with FITC–anti-rabbit, TR–anti-mouse and Cy5–anti-human IgG antibodies. Confocal optical sections (z=0.5 µm) show gpUL37 aa 1–461 alone (a, FITC) or merged with ER (c, FITC/TR), with mitochondria (e, FITC/Cy5) or with ER and mitochondria (f, FITC/TR/Cy5). Control staining of ER alone (b, TR) and of mitochondria alone (d, Cy5) are shown. Confocal optical sections show gpUL37 aa 1–438 alone (g, FITC) or merged with ER (i, FITC/TR), with mitochondria (k, FITC/Cy5) or with ER and mitochondria (l, FITC/TR/Cy5). Control staining of ER alone (h, TR) and of mitochondria alone (j, Cy5) are shown. Bars represent 30 µm.

The UL37x3 gene has been deleted in an HCMV mutant with no apparent effect on the viability of HCMV in cultured fibroblasts (Borst et al., 1999). Although the dispensability of the UL37x3 gene has been tested in cell culture, it has not been determined for HCMV growth in humans. This is particularly pertinent to HCMV, as the MCMV UL37x3 homologous sequences were also found not to be essential for MCMV growth in mouse fibroblasts in culture; yet, the MCMV UL37x3 mutant was severely attenuated for its growth and pathogenicity in mice.

Identification of HCMV essential genes has been hindered by the limited host range of the virus and the lack of a suitable animal model for studying HCMV infection in vivo. To determine the requirement for UL37x3 sequences for HCMV growth in vivo, we therefore directly sequenced the complete UL37x3 gene in HCMV primary strains and compared it to that of HCMV strain AD169, sequenced in parallel. HCMV (AD169) is a laboratory strain, which has been extensively passaged for more than 40 years in laboratories. This prolonged passage of strain AD169 in culture has resulted in its loss of multiple genes encoding other HCMV glycoproteins (Cha et al., 1996) but not of UL37x3 sequences (Kouzarides et al., 1988). Sequence diversity of subdomains within ORFs considered to be highly conserved has been found to occur in HCMV primary strains (Chou & Dennison, 1991; Chou, 1992; Meyer-König et al., 1998; Zweygberg Wirgart et al., 1998). Thus, when ORFs or specific subdomains are conserved in primary strains at the amino acid level, a crucial role of the product for HCMV growth is implied. Conversely, non-conservative substitutions or deletions of ORFs or subdomains in primary strains suggest a non-essential role of the ORF or its subdomain for HCMV growth in humans.

Identification of regions of the HCMV genome with low levels of nucleotide variation is also important for designing primers for PCR detection of HCMV DNA in clinical specimens. We discovered significant strain-specific sequence diversity in the 5´ three-quarters of the UL37x3 gene which encode the N-glycosylation and basic subdomains of gpUL37/gpUL37_M. These sequences differ comparably with the HCMV UL144 ORF (Lurain et al., 1999). In contrast, the low diversity of the UL37x3 nucleotide sequences encoding the TM and cytosolic tail makes these sequences suitable for diagnostic primer design. Our TM and cytosolic tail primers were found to be unique to HCMV DNA by searching the combined sequence databases (W. A. Hayajneh & A. M. Colberg-Poley, unpublished results).

Although the overall UL37x3 nucleotide diversity is notable, construction of an unrooted tree was possible. The tree indicated that there are at least five primary branches, some of which have multiple members (groups I, II and IV). The length of the branches indicates that UL37x3 sequences have diverged more than UL37x1 sequences (Hayajneh et al., 2001). Notably, the position of strain AD169 in the tree indicates that this laboratory strain has not significantly diverged its UL37x3 sequences during its in vitro culture over the last 40 years from those present in other group I primary strains isolated within the last 5 years.

At the amino acid level, the N-glycosylation subdomain was diverse and comparable to the UL144 ORF (Lurain et al., 1999) in its diversity. In the N-glycosylation subdomain (aa 206–391), we consider ²⁰⁷V/I, ²²²C, ²⁵¹Q, ²⁹³T, ³²⁸H, ³³⁸V, ³⁹²V and ³⁹⁴S to be consensus because these residues are encoded in 50 % of the HCMV strains. We note, however, that within the N-glycosylation subdomain, the vast majority of the N-glycosylation signals are retained. Although we know that gpUL37 is N-glycosylated (Al-Barazi & Colberg-Poley, 1996), we do not know which N-glycosylation sites are post-translationally modified. The functional significance of N-glycosylation for gpUL37 is unclear, but these N-glycosylation sites are known to be required for modification and processing of gpUL37 in the ER and Golgi apparatus. N-glycosylation of gpUL37 may be needed to ensure proper folding and transport of the protein through the ER and the Golgi apparatus to the cell surface (Colberg-Poley et al., 2000 a). A defect in transactivating activity was documented in a gpUL37 mutant lacking the first two-thirds of the N-glycosylation domain (H. Zhang & A. M. Colberg-Poley, unpublished results). Taken together with the previously observed lack of diversity of the gpUL37 hydrophobic leader sequence (Hayajneh et al., 2001), these results suggest that post-translational processing of gpUL37/gpUL37_M by N-glycosylation, observed during productive HCMV infection of HFF cells in culture (Al-Barazi & Colberg-Poley, 1996), is important for its function during HCMV growth in vivo.

Multiple residues within the basic domain ORF differ in most primary strains when compared to AD169. We consider ³⁷¹R, ³⁷⁴L and ³⁷⁸K of the basic subdomain (aa 368–382) to be consensus for HCMV because these residues appear in 50 % of the primary strains. As the basic domain has the highest proportion of non-conservative variant residues and the overall basic charge of the subdomain is reduced in some strains (e.g. strain 72), we conclude that the basic subdomain is not essential for gpUL37/gpUL37_M function in vivo. It is notable that gpUL37/gpUL37_M has another basic subdomain, which is encoded by the UL37x1 ORF. The overall basic charge within that subdomain is conserved in primary strains (Hayajneh et al., 2001). Thus, the basic subdomain encoded by UL37x3 may be redundant for gpUL37/gpUL37_M function and thus non-essential for HCMV growth in vivo. Alternatively, UL37x3 function may require the presence of only a small number of basic residues which are retained in all HCMV primary strains, although this seems unlikely.

High rates of substitution of UL37x3 basic subdomain sequences are suggested by genealogical analysis showing that certain substitutions have been adopted more than once by strains in different groups. For example, the consensus sequence at residue ³⁷⁷T (ACG) is replaced by ³⁷⁷M (AUG) in strains 15, 35 and 36 from two different groups. Moreover, consensus ³⁷¹K (AAG) is replaced by ³⁷¹R in group II strains by two different codons [(AGA) in strains 1, 11 and 32 and (AGG) in strains 6, 13, 33, 63 and 68] while group IV [(AGA) in strain 72] and group V [(AGG) in strain 35] have also acquired ³⁷¹R. The new consensus residue ³⁷⁴L is encoded by CUU in group II and V strains and by UUA in group IV strain 72. The use of different codons to encode the same residues by divergent strains and by strains within the same group suggests high rates of substitution, particularly within the UL37x3 basic subdomain.

Residues in the UL37x3 TM ORF lack diversity in all HCMV primary strains sequenced. This retention of identity, which is not required to maintain its role as a hydrophobic anchor, suggests that specific residues in the TM subdomain are required for gpUL37/gpUL37_M to interact with other proteins within the membrane. Indeed, a gpUL37 mutant lacking the TM and cytosolic tails is more defective in transactivating activity than a mutant lacking the cytosolic tail alone. This residual transactivating activity of gpUL37 aa 1–461 as measured by hsp70 promoter transactivation in HeLa cells suggests that the TM likely contributes to the transactivating activity of gpUL37 measured by this assay.

Most residues of the UL37x3 cytosolic tail ORF are conserved in primary strains, with only two amino acids (⁴⁷⁶S and ⁴⁸⁵R) found to differ, one non-conservatively. Furthermore, substitution of these residues occurred infrequently in the sequenced primary strains. These residues are not part of the cytosolic tail consensus phosphorylation sites, which lack diversity in all primary strains sequenced. These results suggest that phosphorylation of the cytosolic tail is important for gpUL37/gpUL37_M function. Defective transactivation of the hsp70 promoter construction by the gpUL37 mutant lacking the carboxyl-terminal cytosolic tail supports this hypothesis.

The carboxyl-terminal domain of the MCMV UL37x3 protein is believed to play an important role in the optimal growth and pathogenesis of MCMV in vivo (Lee et al., 2000). Consistent with that finding, we propose that the strong conservation of the TM and cytosolic tail subdomains of gpUL37 implies that these UL37x3 subdomains have an essential role for HCMV growth in humans not detected during its growth in culture.

The authors wish to thank Drs Holland, Rodríguez and Jantausch for constant encouragement and support, Dr Joe Felsenstein for advice concerning the PHYLIP analysis, and Drs Ted Schutzbank and Joseph Campos for providing clinical specimens. This work was supported by Grant-in-Aid 9750012N from the American Heart Association (to A.M.C-P), Children's Research Institute (CRI) Discovery Funds, and an CRI Fellowship Award (to W.A.H.).

† Present address: Department of Hygiene and Epidemiology, University of Ioannina Medical School, Ioannina 45110, Greece.

Al-Barazi, H. O. & Colberg-Poley, A. M. (1996). The human cytomegalovirus UL37 immediate-early regulatory protein is an integral membrane N-glycoprotein which traffics through the endoplasmic reticulum and Golgi apparatus. Journal of Virology 70, 7198–7208.

Baer, R., Bankier, A. T., Biggin, M. D., Deininger, P. L., Farrell, P. J., Gibson, T. J., Hatfull, G., Hudson, G. S., Satchwell, S. C., Seguin, C., Tuffnell, P. S. & Barrell, B. G. (1984). DNA sequence and expression of the B95-8 Epstein–Barr virus genome. Nature 310, 207–211.

Biegalke, B. J. (1999). Human cytomegalovirus US3 gene expression is regulated by a complex network of positive and negative regulators. Virology 261, 155–164.

Borst, E.-M., Hahn, G., Koszinowski, U. H. & Messerle, M. (1999). Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants. Journal of Virology 73, 8320–8329.

Britt, W. J. & Alford, C. A. (1996). Cytomegalovirus. In Fields Virology, 3rd edn, pp. 2493–2523. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.

Cha, T.-A., Tom, E., Kemble, G. W., Duke, G. M., Mocarski, E. S. & Spaete, R. R. (1996). Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. Journal of Virology 70, 78–83.

Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M., Cerny, R., Horsnell, T., Hutchison, C. A., III, Kouzarides, T., Martignetti, J. A., Preddie, E., Satchwell, S. C., Tomlinson, P., Weston, K. M. & Barrell, B. G. (1990). Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Current Topics in Microbiology and Immunology 154, 125–169.

Chou, S. (1992). Effect of interstrain variation on diagnostic DNA amplification of the cytomegalovirus major immediate-early gene region. Journal of Clinical Microbiology 30, 2307–2310.

Chou, S. & Dennison, K. M. (1991). Analysis of interstrain variation in cytomegalovirus glycoprotein B sequences encoding neutralization-related epitopes. Journal of Infectious Diseases 163, 1229–1234.

Colberg-Poley, A. M. (1996). Functional roles of immediate early proteins encoded by the human cytomegalovirus UL36–38, UL115–119, TRS1/IRS1 and US3 loci. Intervirology 39, 350–360.

Colberg-Poley, A. M., Santomenna, L. D., Harlow, P. P., Benfield, P. A. & Tenney, D. J. (1992). The HCMV US3 and UL36–38 immediate early proteins regulate gene expression. Journal of Virology 66, 95–105.

Colberg-Poley, A. M., Huang, L., Soltero, V. E., Iskenderian, A., Schumacher, R.-F. & Anders, D. G. (1998). The acidic domain of pUL37x1 and gpUL37 plays a key role in transactivation of HCMV DNA replication gene promoter constructions. Virology 246, 400–408.

Colberg-Poley, A. M., Patel, M. B., Erezo, D. P. P. & Slater, J. E. (2000 a). Human cytomegalovirus UL37 immediate-early regulatory proteins traffic through the secretory apparatus and to mitochondria. Journal of General Virology 81, 1779–1789.

Colberg-Poley, A. M., Soltero, V. E. & Schumacher, R. F. (2000 b). Analysis of cytomegalovirus gene expression by transfection. In Methods in Molecular Medicine, vol. 33, pp. 67–77. Edited by J. Sinclair. Totowa, New Jersey: Humana Press.

Davison, A. J. & Scott, J. E. (1986). The complete DNA sequence of varicella-zoster virus. Journal of General Virology 67, 1759–1816.

Eustice, D. C., Feldman, P. A., Colberg-Poley, A. M., Buckery, R. M. & Neubauer, R. H. (1991). A sensitive method for detection of -galactosidase in transfected mammalian cells. BioTechniques 11, 739–743.

Goldmacher, V. S., Bartle, L. M., Skaletskaya, A., Dionne, C. A., Kedersha, N. L., Vater, C. A., Han, J.-W., Lutz, R. J., Watanabe, S., McFarland, E. D., Kieff, E. D., Mocarski, E. S. & Chittenden, T. (1999). A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to bcl-2. Proceedings of the National Academy of Sciences, USA 96, 12536–12541.

Gompels, U. A., Nicholas, J., Lawrence, G., Jones, M., Thomson, B. J., Martin, M. E. D., Efstathiou, S., Craxton, M. & Macaulay, H. A. (1995). The DNA sequence of human herpesvirus 6: structure, coding content, and genome evolution. Virology 209, 29–51.

Hayajneh, W. A., Colberg-Poley, A. M., Skaletskaya, A., Bartle, L. M., Lesperance, M. M., Contopoulos-Ioannidis, D. G., Kedersha, N. L. & Goldmacher, V. S. (2001). Sequence and antiapoptotic functional domains of the human cytomegalovirus UL37 exon 1 immediate early protein are conserved in multiple primary strains. Virology 279, 233–240.

Kouzarides, T., Bankier, A. T., Satchwell, S. C., Preddy, E. & Barrell, B. G. (1988). An immediate early gene of human cytomegalovirus encodes a potential membrane glycoprotein. Virology 165, 151–164.

Lee, M., Xiao, J., Haghjoo, E., Zhan, X., Abenes, G., Tuong, T., Dunn, W. & Liu, F. (2000). Murine cytomegalovirus containing a mutation at open reading frame M37 is severely attenuated in growth and virulence in vivo. Journal of Virology 74, 11099–11107.

Lesperance, M. M., Contopoulos-Ioannidis, D. G., Gutiérrez, M. P. & Colberg-Poley, A. M. (1998). PCR detection of human cytomegalovirus DNA in clinical specimens using novel UL37 exon 3 and US3 primers. Clinical and Diagnostic Laboratory Immunology 5, 256–258.

Lurain, N. S., Kapell, K. S., Huang, D. D., Short, J. A., Paintsil, J., Winkfield, E., Benedict, C. A., Ware, C. F. & Bremer, J. W. (1999). Human cytomegalovirus UL144 open reading frame: sequence hypervariability in low-passage clinical strains. Journal of Virology 73, 10040–10050.

McGeoch, D. J. (1989). The genomes of the human herpesviruses: contents, relationships, and evolution. Annual Review of Microbiology 43, 235–265.

Meyer-König, U., Haberland, M., von Laer, D., Haller, O. & Hufert, F. T. (1998). Intragenic variability of human cytomegalovirus glycoprotein B in clinical strains. Journal of Infectious Diseases 177, 1162–1169.

Nicholas, J. (1996). Determination and analysis of the complete nucleotide sequence of human herpesvirus 7. Journal of Virology 70, 5975–5989.

Nicholas, J. & Martin, M. E. D. (1994). Nucleotide sequence analysis of a 38.5-kilobase-pair region of the genome of human herpesvirus 6 encoding human cytomegalovirus immediate-early gene homologs and transactivating functions. Journal of Virology 68, 597–610.

Pari, G. S., Field, A. K. & Smith, J. A. (1995). Potent antiviral activity of an antisense oligonucleotide complementary to the intron–exon boundary of human cytomegalovirus genes UL36 and UL37. Antimicrobial Agents and Chemotherapy 39, 1157–1161.

Rawlinson, W. D., Farrell, H. E. & Barrell, B. G. (1996). Analysis of the complete DNA sequence of murine cytomegalovirus. Journal of Virology 70, 8833–8849.

Russo, J. J., Bohenzky, R. A., Chien, M.-C., Chen, J., Yan, M., Maddalena, S., Parry, J. P., Peruzzi, D., Edelman, I. S., Chang, Y. & Moore, P. S. (1996). Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8). Proceedings of the National Academy of Sciences, USA 93, 14862–14867.

Smith, J. A. & Pari, G. S. (1995). Expression of human cytomegalovirus UL36 and UL37 genes is required for viral DNA replication. Journal of Virology 69, 1925–1931.

Tenney, D. J., Santomenna, L. D., Goudie, K. B. & Colberg-Poley, A. M. (1993). The human cytomegalovirus US3 immediate early protein lacking the putative transmembrane domain regulates gene expression. Nucleic Acids Research 21, 2931–2937.

Vink, C., Beuken, E. & Bruggeman, C. A. (2000). Complete DNA sequence of the rat cytomegalovirus genome. Journal of Virology 74, 7656–7665.

Wilkinson, G. W. G., Akrigg, A. & Greenaway, P. J. (1984). Transcription of the immediate early genes of human cytomegalovirus AD169. Virus Research 1, 101–116.

Zhang, H., Al-Barazi, H. O. & Colberg-Poley, A. M. (1996). The acidic domain of the human cytomegalovirus UL37 immediate early protein is dispensable for its transactivating activity and localization but is not for its synergism. Virology 223, 292–302.

Zweygberg Wirgart, B., Brytting, M., Linde, A., Wahren, B. & Grillner, L. (1998). Sequence variation within three important cytomegalovirus gene regions in isolates from four different patient populations. Journal of Clinical Microbiology 36, 3662–3669.

JGV Direct table of contents

© 2001 SGM

This article is now available in the July 2001 print issue of JGV (vol. 82, 1569–1579). The complete issue of the journal may be seen in electronic form on JGV Online.