Dual targeting of the human cytomegalovirus UL37 exon 1 protein during permissive infection

Manohara S. Mavinakere1 and Anamaris M. Colberg-Poley1,2

1 Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010, USA
2 Department of Pediatrics, George Washington University, School of Medicine and Health Sciences, 111 Michigan Avenue NW, Washington, DC 20010, USA

Correspondence
Anamaris M. Colberg-Poley
acolberg-poley{at}cnmcresearch.org


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The human cytomegalovirus (HCMV) UL37 immediate-early (IE) gene minimally encodes three protein isoforms that share NH2-terminal sequences. The predominant UL37 isoform detected during HCMV infection was the UL37 exon 1 protein (pUL37x1), which was produced from IE and, more abundantly, through late times of infection. pUL37x1 was localized in both the endoplasmic reticulum (ER) and mitochondria in infected cells. To determine which UL37x1 NH2-terminal residues serve as ER and mitochondrial targeting signals, we examined the subcellular localization of two deletion mutants. pUL37x1{Delta}2–23, which lacks the hydrophobic leader, is neither translocated into the ER nor imported mitochondrially; conversely, pUL37x1{Delta}23–34, lacking the juxtaposed basic residues, was translocated into the ER but only imported weakly into mitochondria. These studies show for the first time the temporal production and localization of pUL37x1 during HCMV infection. The trafficking patterns of mutants suggest that the pUL37x1 targeting signal to ER and mitochondria is bipartite.


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UL36–38 immediate-early (IE) proteins play important roles in human cytomegalovirus (HCMV) DNA replication, anti-apoptosis and growth (Colberg-Poley, 1996; Colberg-Poley et al., 1992, 1998; Goldmacher et al., 1999; Hayajneh et al., 2001a, b; Skaletskaya et al., 2001; Smith & Pari, 1995). The minimal divergence of UL37 exon 1 (UL37x1) residues in primary strains suggests that the UL37x1 protein (pUL37x1) is essential for HCMV growth in humans (Hayajneh et al., 2001a). The UL36 and UL37 exon 3 (UL37x3) open reading frames (ORFs) are non-essential for HCMV growth in culture (Borst et al., 1999; Patterson & Shenk, 1999). Nonetheless, selected UL37x3 domains appear to be important for CMV pathogenesis in vivo (Hayajneh et al., 2001b; Lee et al., 2000).

Post-transcriptional processing of UL37 pre-mRNA is complex and produces at least ten alternatively spliced UL37 RNAs and one unspliced UL37x1 RNA during HCMV infection (Adair et al., 2003; Goldmacher et al., 1999; Kouzarides et al., 1988; Su et al., 2003a, b; Tenney & Colberg-Poley, 1991a, b). All known UL37 ORFs share UL37x1 NH2-terminal but differ in COOH-terminal sequences. The UL37x1 ORF contains two anti-apoptotic domains: the NH2-terminal domain, spanning amino acids (aa) 5–34, targets UL37 proteins to mitochondria, where they interact with the adenine nucleotide translocator and prevent cytochrome c release (Goldmacher et al., 1999; Hayajneh et al., 2001a). This domain includes a prominent hydrophobic leader (aa 1–22) and juxtaposed basic residues (23R, 27RKR).

gpUL37 is produced at low levels and is N-glycosylated in the secretory apparatus of HCMV-infected cells following their release from a protein synthesis block (Al-Barazi & Colberg-Poley, 1996), whereas pUL37x1 is weakly detected by immunofluorescence (IFA) and co-localized with a mitochondrial marker within 24 h post-infection (p.i.) (Goldmacher et al., 1999). Interestingly, pUL37x1 and gpUL37 traffic both to ER and to mitochondria in transfected cells (Al-Barazi & Colberg-Poley, 1996; Colberg-Poley et al., 2000; Goldmacher et al., 1999; Hayajneh et al., 2001a, b; McCormick et al., 2003).

The ER targeting signal of secretory proteins is defined by a hydrophobic segment of the nascent polypeptide, which is often removed by cleavage following ER translocation (Johnson & van Waes, 1999). UL37 proteins have a non-cleavable NH2-terminal strongly hydrophobic leader (Kouzarides et al., 1988; Nielsen et al., 1997). When the signal contains a sufficiently hydrophobic core and is not cleaved by signal peptidase, they function as a membrane anchor (Kanaji et al., 2000).

In contrast, most cellular mitochondrial proteins are synthesized as cytosolic precursors, are post-translationally targeted to mitochondria by NH2-terminal amphipathic alpha helices containing basic residues, and are imported into mitochondria by translocases in the outer and inner mitochondrial membranes (Ni et al., 1999; Rapaport, 2002; von Heijne, 1986). Mitochondrial type I membrane proteins can contain weakly hydrophobic leaders and adjacent basic residues (Kanaji et al., 2000). The number of basic residues and the hydrophobicity of the leader can determine whether the secretory or mitochondrial pathway is targeted (Kanaji et al., 2000).

Given the large number of possible UL37 products, we wished to determine which UL37 protein is produced most abundantly at different times of HCMV infection. Human diploid fibroblasts (HFFs) were uninfected or HCMV-infected (strain AD169) at an m.o.i. of 3 p.f.u. per cell. Cells were harvested at various times of infection and fractionated as described below. HeLa cells were lipofected as described (Colberg-Poley et al., 2000). Briefly, cells (4–8x106) were transfected with plasmid DNA (20 µg) expressing pUL37x1{Delta}2–23-myc (p856), pUL37x1{Delta}23–34-myc (p857), pUL37x1 (p327) or an empty vector (p790, pFLAG-CMV5a, Sigma) using Lipofectamine 2000 (Invitrogen). To increase the copy number of transfected DNA, an SV40 T antigen expression vector (pCMV-T1-708, 5 µg) was co-transfected. Subcellular compartments were fractionated at 24 h after transfection as described below.

Mitochondria were purified on discontinuous sucrose gradients as described (Colberg-Poley et al., 2000) with the following modifications. Briefly, cells in MTE buffer (0·27 M mannitol, 10 mM Tris/HCl, 0·1 mM EDTA, pH 7·4) supplemented with protease inhibitor cocktail (Sigma) were lysed by sonication. Nuclei and cellular debris were removed by centrifugation (700 g, 10 min). Mitochondria were pelleted (15 000 g, 10 min) and the post-mitochondrial supernatant was used for ER purification. Pelleted crude mitochondria were purified in discontinuous sucrose gradients, diluted in MTE buffer and pelleted (15 000 g, 10 min). Purified ER was isolated as described (Paulik et al., 1988). Briefly, the post-mitochondrial supernatant was layered onto a sucrose step gradient (1·3, 1·5 and 2·0 M sucrose in 10 mM Tris/HCl, pH 7·6) and banded by centrifugation (87 000 g, 90 min). The ER fraction was collected, diluted in MTE buffer and pelleted by centrifugation (87 000 g, 45 min). Purified mitochondria and ER were resuspended in PBS and stored at -80 °C until use. Protein concentrations were determined using the BCA reagent kit (Pierce).

Fractionated proteins were examined by Western blot analyses using rabbit anti-UL37x1 aa 27–40 (Ab1064), mouse anti-Myc (MAb9E10, BAbCO), goat anti-dolichyl phosphate mannose synthase 1 (DPM1) (I-20, Santa Cruz Biotechnology), or mouse anti-glucose regulated protein 75 (GRP75) (SPA-825, StressGen Biotechnologies) and the corresponding horseradish peroxidase-conjugated secondary antibodies (Ab) (1 : 2500–1 : 4000, Bio-Rad). Goat anti-DPM1 is an affinity purified polyclonal Ab raised against an internal peptide of human DPM1, a subunit of the ER protein, DPM synthase. Mouse anti-GRP75 is a monoclonal (IgG1) Ab against the human mitochondrial matrix heat-shock protein, GRP75. Western blot analyses were carried out by the chemiluminescent method using the ECL Detection System (Amersham Pharmacia Biotech) and were digitized using ScanWizard Pro version 1.21 and imported into Adobe Photoshop 5.0 LE and Microsoft PowerPoint 2000.

This is the first study of the temporal production of UL37 proteins during permissive HCMV infection. As UL37x1 RNA is polysome-associated within 8 h p.i. (Tenney & Colberg-Poley, 1991a), we examined the production of UL37 proteins in HCMV-infected cells at IE, early and late times. pUL37x1 (~20 kDa) was detected at low abundance within 8 h p.i. (Fig. 1). Its abundance increased by early times and remained abundant through late times of HCMV infection (Fig. 1A). At the early and late times of HCMV infection, a broad band (~20–23 kDa) was detected. The kinetics of pUL37x1 production paralleled closely those of UL37x1 unspliced RNA during HCMV infection (Kouzarides et al., 1988; Tenney & Colberg-Poley, 1991a, b) but not of the much lower abundance UL37 spliced transcripts (Adair et al., 2003). Moreover, the mass corresponds well with the predicted (19 kDa) and observed (24 kDa) mass of pUL37x1 (Chee et al., 1990; Tenney & Colberg-Poley, 1991b). These findings argue compellingly that the Ab1064 reactive species is pUL37x1.



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Fig. 1. Temporal expression and ER translocation of pUL37x1 in HCMV-infected HFFs. HFFs were infected with HCMV at an m.o.i. of 3 p.f.u. per cell. Uninfected control cells and HCMV-infected cells were harvested at 8, 32, 56 and 80 h p.i. Proteins (20 µg) from total (A) or purified endoplasmic reticulum (ER) (B) were resolved by SDS-PAGE and blotted onto PVDF membranes. Molecular mass markers were resolved in parallel. The membranes were treated with Ab1064 (1 : 1000) (top), stripped and then reprobed with anti-DPM1 Ab (1 : 500) (bottom). The arrowheads indicate the migration of pUL37x1 species. (C) Wild-type pUL37x1 and mutants. The NH2-terminal hydrophobic signal (cylinder), basic (+) and acidic (-, rectangle) residues of pUL37x1 are represented. The sequences retained in pUL37x1{Delta}2–23 and pUL37x1{Delta}23–34 are indicated below the wild-type pUL37x1. The UL37x1 aa 27–40 epitope and the COOH-terminal Myc tag (hexagon) reactive with UL37 antisera (Ab1064) or anti-Myc Ab, respectively, are shown.

 
To determine whether pUL37x1, similar to gpUL37, is co-translationally ER-translocated in infected cells, purified ER from HCMV-infected cells was examined using Ab1064 (Fig. 1B top). As predicted by its hydrophobic signal, pUL37x1, detected as a doublet, was translocated into the ER of HCMV-infected cells. The ER integral transmembrane protein DPM1 was readily detected in purified ER fractions from uninfected and HCMV-infected HFFs (Fig. 1B bottom). This doublet contains differentially phosphorylated pUL37x1, as treatment with calf intestinal phosphatase or with bacterial alkaline phosphatase reduced the molecular mass of the upper band to that of the lower band (unpublished results).

To determine whether, following its production, pUL37x1 is dually targeted to both ER and mitochondria in infected cells, we examined these purified subcellular fractions from HCMV-infected HFFs (Fig. 2). Dual localization of pUL37x1 in the ER and mitochondria of infected HFFs was detected prior to (16 h p.i.) and during (24 h p.i.) HCMV oriLyt DNA synthesis. In addition, pUL37x1 was detected in mitochondria at late times of HCMV infection (80 h p.i.) (unpublished results). As in Fig. 1(B), pUL37x1 was detected as a doublet. Mitochondrial pUL37x1 migrated at a lower molecular mass than the ER species, suggesting the possibility that the two species might be differentially phosphorylated. Consistent with this possibility, the stable presence of HCMV glycoprotein B in the plasma membrane is determined by its phosphorylation state at Ser900 (Fish et al., 1998). Anti-DPM1 and anti-GRP75 Ab showed the purity of the fractionated ER and mitochondria, respectively. Thus, pUL37x1 is dually targeted into the secretory apparatus and mitochondria of HCMV-infected cells.



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Fig. 2. HCMV pUL37x1 traffics both into the ER and into mitochondria of permissively infected cells. HFFs were infected with HCMV and harvested at 16 and 24 h p.i. Cells were fractionated into endoplasmic reticulum (ER) and mitochondria (mito). Fractionated proteins (20 µg) were then resolved by SDS-PAGE and blotted onto a PVDF membrane. Molecular mass markers were resolved in parallel. The arrowheads indicate the migration of pUL37x1 species. The membrane was reacted with Ab1064 (1 : 1000) (top), stripped and then reprobed with Ab for a mitochondrial marker (anti-GRP75, 1 : 2000) (middle), and then for an ER marker (anti-DPM1, 1 : 500) (bottom).

 
We next tested which NH2-terminal UL37x1 sequences target pUL37x1 to the ER and to mitochondria. As the UL37x1 domain which targets pUL37x1 to mitochondria (Hayajneh et al., 2001a) includes its hydrophobic signal, we tested the ER translocation and mitochondrial importation of pUL37x1{Delta}2–23-myc in transfected cells. pUL37x1{Delta}2–23 (about 23 kDa) was not detectable in either ER or mitochondrial fractions; rather it was predominantly localized in the cytosolic fraction (Fig. 3A top). The purity of the mitochondrial and ER fractions was verified by the respective presence of GRP75 or DPM1 (Fig. 3A middle, bottom). These findings establish that ER translocation and mitochondrial importation of pUL37x1{Delta}2–23 are blocked. These results further establish the requirement of the UL37x1 hydrophobic leader for pUL37x1 ER translocation and, unconventionally, for mitochondrial importation. Moreover, the inability of the cytosolic mutant to traffic to mitochondria, in spite of its remaining NH2-terminal basic residues, suggests that the pUL37x1 targeting signal is bipartite.



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Fig. 3. (A) A hydrophobic signal sequence deletion mutant, pUL37x1{Delta}2–23-myc, does not traffic into the ER or mitochondria. HeLa cells, lipofected with empty vector (p790, lanes 1–4) or vector expressing pUL37x1{Delta}2–23-myc (p856, lanes 5–8), were fractionated into endoplasmic reticulum (ER), mitochondria (mito) and cytosol (cyto). Fractionated proteins (20 µg) were resolved by SDS-PAGE, blotted onto a PVDF membrane and reacted with Ab against UL37x1-myc (anti-Myc, 1 : 1000) (top), subsequently with anti-GRP75 (1 : 2000, mitochondrial marker) (middle) and then with anti-DPM1 (1 : 500, ER marker) (bottom). Lanes: 1 and 5, banded mitochondria (mito); 2 and 6, ER; 3 and 7, total; 4 and 8, cytosol (cyto). The arrowheads indicate the migration of the pUL37x1 mutant protein. (B) pUL37x1{Delta}23–34-myc, lacking NH2-terminal basic residues but retaining the hydrophobic leader, is translocated into the ER but imported very weakly into mitochondria. HeLa cells, lipofected with empty vector (p790, lanes 1–4) or vector expressing pUL37x1{Delta}23–34-myc (p857, lanes 5–8), were fractionated into ER, mitochondria (mito) and cytosol (cyto). Fractionated proteins were resolved by SDS-PAGE, blotted onto a PVDF membrane and reacted with Ab against UL37x1-myc (anti-Myc, top), subsequently with Ab against a mitochondrial marker (anti-GRP75, middle) and then against an ER marker (anti-DPM1, bottom). Lanes: 1 and 5, banded mitochondria (mito); 2 and 6, ER; 3 and 7, cytosol (cyto); 4 and 8, total. (C) pUL37x1 is ER-translocated and mitochondrially imported in transfected HeLa cells. HeLa cells, lipofected with a vector (p790, lanes 1–4) or vector expressing wild-type pUL37x1 (p327, lanes 5–8, Colberg-Poley et al., 1992), were fractionated into ER, mitochondria (mito) and cytosol (cyto). Fractionated proteins were resolved by SDS-PAGE, blotted and sequentially reacted with Ab1064 (top), anti-GRP75 (middle) and anti-DPM1 (bottom). Lanes: 1 and 5, banded mitochondria (mito); 2 and 6, ER; 3 and 7, total; 4 and 8, cytosol (cyto).

 
To determine whether the UL37x1 NH2-terminal basic residues (23R, 27RKR) are required for targeting pUL37x1 to mitochondria, we examined the localization of pUL37x1{Delta}23–34 in transfected cells (Fig. 3B top). pUL37x1{Delta}23–34, which lacks these basic residues, was translocated into the ER but only weakly imported into mitochondria. The markers indicated the proper fractionation of mitochondria and ER (middle, bottom). This result indicates that the UL37x1 NH2-terminal basic residues, in combination with the upstream hydrophobic signal, play a role in targeting pUL37x1 to mitochondria.

As a control for these studies, the localization of wild-type pUL37x1 in transfected cells was examined by Western blot analysis (Fig. 3C top). Similar to the findings in HCMV-infected cells (Fig. 2) and in transfected cells (Al-Barazi & Colberg-Poley, 1996; Colberg-Poley et al., 2000; Goldmacher et al., 1999; Hayajneh et al., 2001a, b; McCormick et al., 2003), pUL37x1 was detected in mitochondrial and ER fractions in transfected cells. The markers indicated proper fractionation of these subcellular compartments (Fig. 3C middle, bottom).

All known UL37 IE proteins share an NH2-terminal hydrophobic signal and juxtaposed basic residues. In combination, these sequences appear to serve as a bipartite mitochondrial targeting sequence. Bipartite signals have been described for the major mitochondrial translocase protein Tom20, in which its NH2-terminal transmembrane domain is closely followed by multiple positively charged residues (Kanaji et al., 2000). The number of positive charges proximal to the COOH-terminus of the TM domain is important for Tom20 mitochondrial targeting. In contrast to Tom20, pUL37x1 is targeted to the ER and mitochondria by its hydrophobic signal. Similar to Tom20, however, the UL37x1 ORF has basic residues just downstream of its hydrophobic leader and these are invariant in primary HCMV strains (Hayajneh et al., 2001a). These basic residues (23R, 27RKR) downstream of the hydrophobic leader are also conserved in the chimpanzee CMV UL37x1 ORF (Davison et al., 2003).

Some mitochondrial proteins are present in both the ER and the outer mitochondrial membrane. For example, cytochrome b5 has ER and outer mitochondrial membrane isoforms (Kuroda et al., 1998). The anti-apoptotic protein bcl-2 is located in the ER, nuclear envelope and mitochondrial outer membranes (Krajewski et al., 1993). pUL37x1 is analogously localized in the secretory and mitochondrial compartments in HCMV-infected cells. The mechanism of this dual trafficking of pUL37x1 is not currently known. Nonetheless, selective trafficking of cytochrome P4502B1 into ER or mitochondria can result from differential phosphorylation of its chimeric NH2-terminal mitochondrial and ER targeting signal (Anandatheerthavarada et al., 1999).

The targeting of pUL37x1 to the ER and mitochondria suggests that targeting of pUL37x1 to these locations is critical for its function(s) during HCMV infection. pUL37x1 has known anti-apoptotic activity in mitochondria (Goldmacher et al., 1999). We hypothesize that HCMV pUL37x1 is targeted to the ER, where it regulates cellular gene expression, including that of ER chaperones, which regulate the processing of cargo proteins. Consistent with this possibility is the finding that pUL37x1 transactivates activity of a human heat-shock protein 70 promoter construction (Colberg-Poley et al., 1992). Induction of ER chaperones by pUL37x1 could facilitate the efficient processing of the HCMV glycoproteins required for its growth in the infected cell.


   ACKNOWLEDGEMENTS
 
The authors thank Drs Richard Adair and Nancy DiFronzo for their critical comments on the manuscript. The authors are grateful to Dr Judy Tevethia for providing the pCMV-T1-708 expression vector. This work was supported by Children's Research Institute Discovery Funds to A. C. -P. and a grant from the Board of Lady Visitors to M. S. M.


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Received 21 August 2003; accepted 7 October 2003.