Center for Virology, Immunology and Infectious Disease Research, Childrens Research Institute1, Department of Infectious Diseases2 and Department of Otolaryngology3, Childrens National Medical Center, George Washington University School of Medicine and Health Sciences, 111 Michigan Avenue, NW, Washington, DC 20010, USA
Author for correspondence: Anamaris Colberg-Poley. Fax +1 202 884 3985. e-mail acolberg{at}cnmc.org
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Abstract |
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Introduction |
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All UL37 IE proteins share their amino-terminal signal sequence (aa 122), a strongly charged acidic domain (aa 81108) and two domains (aa 534 and aa 118147) 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., 2000a
). gpUL37 is modified by N-glycosylation in the ER and Golgi apparatus during HCMV infection of permissive cells (Al-Barazi & Colberg-Poley, 1996
). gpUL37M differs from gpUL37 by the absence of aa 178262, 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.
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Methods |
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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. 1a
) as described previously (Hayajneh et al., 2001
). Primers 113 (nt 5076450786), 114 (nt 5015650176), 115 (nt 5015650176), 134 (nt 4983349852), 154 (nt 5076550786), 173 (nt 5087350894), 174 (nt 5091950939) and 175 (nt 4980349823) allowed for direct PCR sequencing of the complete UL37x3 ORF (nt 4991050842). 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.
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gpUL37 carboxyl-terminal mutants.
gpUL37 truncation mutants were used to test the functional importance of the conserved carboxyl-terminal TM and cytosolic tail (Fig. 1b). Mutant gpUL37 aa 1438 (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 1461 (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 promoterchloramphenicol 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 TrisHCl 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., 2000b
). 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 1461 (p612), mutant gpUL37 aa 1438 (p627), wild-type gpUL37 (p414) or control gB (p370) using LipofectAmine as previously described (Colberg-Poley et al., 2000a ). Cells were harvested 48 h after transfection, fixed in methanol and stained simultaneously with rabbit polyvalent Ab1064 (1:200, against gpUL37 aa 2740), 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., 2000a
). 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 gpUL37 mutants with cellular markers. Images were generated using Adobe Photoshop (version 4.0), Bio-Rad confocal microscopy plug-ins and Microsoft Publisher 98.
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Results and Discussion |
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gpUL37 (including signals 117) and gpUL37M (including signals 717) have large N-glycosylation domains (Fig. 1b). As the UL37x1 hydrophobic leader lacks diversity in all HCMV primary strains sequenced (Hayajneh et al., 2001
), targeting of gpUL37/gpUL37M encoded by HCMV primary strains to the ER is predicted. To test this prediction and the importance of post-translational modification of gpUL37/gpUL37M 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 15, 712 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/gpUL37M is retained by virtue of the conserved UL37x1 hydrophobic leader and multiple N-glycosylation signals.
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The divergent basic subdomain (aa 368382)
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.
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The conserved cytosolic tail (aa 460487)
The compact gpUL37/gpUL37M 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 IV 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 93120/930 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 100143/930 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).
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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/gpUL37M. 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 206391), 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., 2000a
). 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/gpUL37M 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 368382) to be consensus for HCMV because these residues appear in50% 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/gpUL37M function in vivo. It is notable that gpUL37/gpUL37M 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/gpUL37M 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/gpUL37M 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 1461 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/gpUL37M 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.
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Acknowledgments |
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Footnotes |
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b Present address: Department of Hygiene and Epidemiology, University of Ioannina Medical School, Ioannina 45110, Greece.
c Present address: Department of Otolaryngology, Head and Neck Surgery, University of Michigan Medical Center, F6905 C. S. Mott Hospital, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0241, USA.
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References |
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Received 5 February 2001;
accepted 30 March 2001.