Internal cleavage of the human cytomegalovirus UL37 immediate-early glycoprotein and divergent trafficking of its proteolytic fragments

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, George Washington University, School of Medicine and Health Sciences, 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 UL37 gene encodes at least three isoforms, which share N-terminal UL37 exon 1 (UL37x1) sequences. UL37 proteins traffic dually into the endoplasmic reticulum (ER) and to mitochondria. Trafficking of the UL37 glycoprotein (gpUL37) in relation to its post-translational processing was investigated. gpUL37 is internally cleaved in the ER and its products traffic differentially. Its C-terminal fragment (UL37COOH) is ER-localized and N-glycosylated. Unlike conventional ER signal sequences, its N-terminal () fragment is stable and traffics to mitochondria. Inhibition of N-glycosylation did not block pUL37 cleavage and dramatically decreased the levels of but not of UL37COOH. pUL37M, which differs from gpUL37 by the lack of residues 178–262 and hence the UL37x3 consensus signal peptidase cleavage site, traffics into the ER and mitochondria, but is neither cleaved nor N-glycosylated. This finding of a relationship between ER processing and mitochondrial importation of UL37 proteins is unique for herpesvirus proteins.


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The UL37 exon 1 (UL37x1) open reading frame (ORF) is essential for human cytomegalovirus (HCMV) growth in humans (Hayajneh et al., 2001a) and in vitro (Dunn et al., 2003; Yu et al., 2003). While non-essential for HCMV growth in cultured human fibroblasts (HFFs) (Borst et al., 1999), the UL37 exon 3 (UL37x3) C-terminal transmembrane (TM) domain and cytosolic tail diverge minimally in primary strains and predictably play important roles in HCMV growth in vivo (Hayajneh et al., 2001b). Analogously, the mouse UL37x3 homologue, M37, is not required for mouse cytomegalovirus growth in culture but clearly plays a role in its pathogenesis in vivo (Lee et al., 2000). Since virus growth relies upon the proper localization and function of its key proteins, a thorough understanding of the processing and trafficking of UL37 proteins is invaluable to the HCMV field.

The UL37x1 (pUL37x1), UL37 glycosylated (gpUL37) and UL37 medium (pUL37M) proteins are products of alternatively processed RNAs (Fig. 1a) (Adair et al., 2003; Al-Barazi & Colberg-Poley, 1996; Goldmacher et al., 1999; Kouzarides et al., 1988; Mavinakere & Colberg-Poley, 2004; Tenney & Colberg-Poley, 1991). The UL37x1 ORF, common to these UL37 proteins, contains two anti-apoptotic domains (Hayajneh et al., 2001a; McCormick et al., 2003). The first, spanning aa 5–34, includes a prominent hydrophobic leader and downstream basic residues. This bipartite signal targets UL37 proteins into the endoplasmic reticulum (ER) and to mitochondria (Mavinakere & Colberg-Poley, 2004) where they prevent cytochrome c release following apoptotic signals (Goldmacher et al., 1999). The second UL37 anti-apoptotic domain (aa 118–147) is required for the association of pUL37x1 with Bax, resulting in the tight association of Bax with the mitochondrial outer membrane and inhibition of its pro-apoptotic function (Poncet et al., 2004).



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Fig. 1. (a) UL37 isoforms. The known UL37 isoforms share N-terminal hydrophobic signal (cylinder), basic (+) and acidic (–, rectangle) residues as well as two anti-apoptotic domains (aa 5–34 and aa 118–147; Hayajneh et al., 2001a; McCormick et al., 2003). In addition, gpUL37 and pUL37M contain multiple N-glycosylation sites (spanning aa 206–391), downstream basic residues (+), a TM domain (pyramid) and a compact cytosolic tail. The previously unrecognized hydrophobic domain (oval, aa 178–196) and the UL37x3 consensus signal peptidase cleavage site (aa 193/194) contained exclusively within gpUL37 were detected using HMMTOP 1.1 (Tusnády & Simon, 1998) and SIGNAL IP 1.1 (Nielsen et al., 1997), respectively. The UL37x3 hydrophobic residues (aa 178–196) from HCMV (Chee et al., 1990; Kouzarides et al., 1988) and CCMV (aa 166–184) (Davison et al., 2003) are shown. Identical residues (lines), conservative substitutions (double dots) and non-conservative, hydrophobic, neutral substitutions (single dots) between the HCMV and CCMV sequences are indicated. Also shown is the predicted consensus cleavage site (arrowhead) within both UL37x3 sequences. The epitopes reactive with polyvalent rabbit anti-UL37 antisera (Ab1064, aa 27–40) or anti-Flag (C-terminal Flag tag) Ab are indicated. (b–e) Differential trafficking of the gpUL37 cleavage products, and UL37COOH. HeLa cells were transfected as described previously (Mavinakere & Colberg-Poley, 2004) with plasmid DNA (p816, 20 µg) expressing gpUL37–Flag and an SV40 T antigen expression vector (pCMV-T1-708, 5 µg) using Lipofectamine 2000 (Invitrogen) (lanes 1–6) or were untransfected (Un; lanes 7–9). Cells were harvested 24 h after transfection. After pelleting of the crude mitochondria at 15 000 g, the loosely associated bound membranes on top of the compact mitochondrial pellet were collected as mitochondrial wash (Mito wash; lane 3). The mitochondrial pellet was further fractionated on discontinuous sucrose gradients (Colberg-Poley et al., 2000; Mavinakere & Colberg-Poley, 2004) and the banded mitochondria isolated (Mito; lanes 1 and 7). The broken membranes at the top of the gradient were collected as the mitochondrial/lysosomal fraction (Mito lyso; lane 2). The supernatant from the first pelleting at 15 000 g was further fractionated to isolate ER on a discontinuous sucrose gradient at 100 000 g (lanes 4 and 8) (Mavinakere & Colberg-Poley, 2004). The supernatant from above was collected as the cytosolic fraction (lane 6). Fractionated proteins (20 µg) and total proteins (lanes 5 and 9) were separated by 10 % SDS-PAGE and transferred onto PVDF membranes (Bio-Rad) as described previously (Al-Barazi & Colberg-Poley, 1996; Mavinakere & Colberg-Poley, 2004). Blotted proteins were reacted with rabbit anti- aa 27–40 (Ab1064, 1 : 1000) (b). The blots were stripped and reprobed with Ab against the UL37COOH (mouse anti-Flag, M2, 1 : 2000; Sigma) (c), ER (goat anti-DPM1, I-20, 1 : 500; Santa Cruz Biotechnology) (d) and mitochondrial (mouse anti-GRP75, SPA-825, 1 : 1000; StressGen Biotechnologies) (e) markers and with the corresponding horseradish peroxidase-conjugated secondary Ab (1 : 2500). Reactivity was detected using the chemiluminescent method (Amersham Pharmacia Biotech). Images were digitized using SCANWIZARD PRO version 1.21 and imported into Adobe PHOTOSHOP 5.0 LE and Microsoft POWERPOINT 2000.

 
In addition to the UL37x1 and exon 2 ORFs, gpUL37 and pUL37M contain 17 and 11 consensus N-glycosylation signals, respectively, while pUL37M lacks aa 178–262 (Chee et al., 1990; Goldmacher et al., 1999; Kouzarides et al., 1988). Finally, gpUL37 and pUL37M share the TM domain and cytosolic tail.

ER targeting signals of secretory proteins are defined by a hydrophobic segment of the nascent polypeptide, which is often removed by cleavage following ER translocation (Johnson & van Waes, 1999). According to the (–3, –1) rule, the UL37x1 hydrophobic leader is predicted to be non-cleavable because of the presence of two large residues (Phe and Tyr) in the critical –3 and –1 positions (Kouzarides et al., 1988; Nielsen et al., 1997). pUL37x1, the predominant UL37 protein during permissive HCMV infection, is ER-translocated and mitochondrially imported (Goldmacher et al., 1999; Mavinakere & Colberg-Poley, 2004). gpUL37 is produced at exceedingly low amounts in HCMV-infected HFFs and traffics through the ER, where it is N-glycosylated, and then to the Golgi apparatus (Al-Barazi & Colberg-Poley, 1996). We have previously detected gpUL37 similarly trafficking into the secretory apparatus and into mitochondria in transiently transfected HFFs and HeLa cells (Colberg-Poley et al., 2000).

In this communication, we show a unique pattern of differential trafficking coupled with post-translational processing of HCMV gpUL37. We observed a previously unreported internal consensus signal peptidase cleavage site in UL37x3 (aa 193/194) using SIGNAL IP 1.1 (Nielsen et al., 1997) within a compact hydrophobic domain identified using HMMTOP 1.1 (Tusnády & Simon, 1998) (Fig. 1a). Therefore, we anticipated that gpUL37 might be internally cleaved. This consensus cleavage site is well conserved in the chimpanzee cytomegalovirus (CCMV) UL37 ORF (Davison et al., 2003) and is reminiscent of the internal cleavage site of influenza CM2 glycoprotein (Perkosz & Lamb, 1998).

To gain further insight into its processing and trafficking, HeLa cells expressing gpUL37–Flag were fractionated as described previously (Mavinakere & Colberg-Poley, 2004). To determine whether gpUL37 is internally cleaved, we used antibodies (Abs) against the N (Ab1064) (Fig. 1b) and C (anti-Flag) termini (Fig. 1c) of gpUL37–Flag. Ab1064, which recognizes UL37x1 aa 27–40, reacted with (~27 kDa) in banded mitochondria (Fig. 1b, lane 1) and intermediate as well as ER fractions (Fig. 1b, lanes 2–4). The molecular mass of corresponded well with cleavage at UL37x3 aa 193/194. A minor, non-specific band (~28 kDa) was weakly detected by Ab1064 in ER and total protein (Fig. 1b, lanes 8 and 9) but not in purified mitochondria (Fig. 1b, lane 7) of untransfected cells. Anti-Flag Ab detected cleaved gpUL37COOH–Flag (~70 kDa), which was larger than predicted, in purified ER (Fig. 1c, lane 4) and in intermediate fractions (Fig. 1c, lanes 2 and 3). Surprisingly, neither Ab1064 nor anti-Flag Ab readily detected the uncleaved UL37 precursor, with predicted masses of ~60 kDa (unglycosylated) or ~98 kDa (N-glycosylated) in either ER or intermediate fractions. This latter finding suggests that proteolytic cleavage of the UL37 precursor is rapid and efficient and that the Western blot analyses were not sufficiently sensitive to detect the short-lived, intermediate species of gpUL37 protein processing, as it was weakly observed in radiolabelled immunoprecipitates from tunicamycin-treated, HCMV-infected HFFs (Al-Barazi & Colberg-Poley, 1996). Reactivity of purified and intermediate fractions with Abs against ER (anti-dolichyl phosphate mannose synthase 1, anti-DPM1) and mitochondrial (anti-glucose regulated protein 75, anti-GRP75) markers indicated the enrichment of the corresponding markers (Fig. 1d and e, lanes 1–9). In addition to the DPM1 subunit (~30 kDa), a large band (>~100 kDa) from the ER of gpUL37-expressing cells reacted with anti-DPM1 Ab (Fig. 1d, lane 4). Because of its size, this species likely contains non-denatured complexes containing DPM1. Taken together, these results suggested that gpUL37 is proteolytically cleaved in the ER and that its products are stable in transfected cells. Moreover, and UL37COOH dissociate and traffic to distinct subcellular compartments.

As UL37COOH was larger than predicted following proteolytic cleavage and contains multiple N-glycosylation sites, we examined whether N-glycosylation accounted for its increased molecular mass. Purified mitochondrial and ER fractions from the transfected HeLa cells expressing gpUL37–Flag and treated with tunicamycin, an inhibitor of N-glycosylation, or untreated were examined by reactivity with Ab1064, anti-Flag, anti-DPM1 and anti-GRP75 Abs (Fig. 2). Ab1064 detected in the mitochondria of untreated transfected cells but in trace amounts in tunicamycin-treated cells (Fig. 2, top panel, lanes 3 and 4), suggesting that from unglycosylated UL37 protein is unstable or that its epitope reactive with Ab1064 is unavailable. migrated more slowly than pUL37x1 purified from the ER of HCMV-infected HFFs (Fig. 2, top panel, lanes 3 and 9), consistent with pUL37 cleavage at the consensus peptidase site within UL37x3, approximately 30 residues downstream of the termination site of the UL37x1 ORF. UL37COOH from the ER of tunicamycin-treated cells detected by anti-Flag Ab was reduced in molecular mass (~32–33 kDa) compared with that from untreated cells (~70 kDa; Fig. 2, second panel, lanes 7 and 8). The molecular mass of the unglycosylated UL37COOH corresponded well with cleavage at the predicted site. Purified mitochondria (Fig. 2, third panel, lanes 1–4) reacted well with anti-GRP75 but not detectably with DPM1 Ab (Fig. 2, bottom panel, lanes 1–4). Conversely, purified ER fractions (Fig. 2, bottom panel, lanes 5–9) reacted primarily with anti-DPM1 Ab and weakly with anti-GRP75 Ab (Fig. 2, third panel, lanes 5–9). These results showed that UL37COOH is N-glycosylated in the ER and that pUL37 proteolytic cleavage does not require prior N-glycosylation. The abundance of UL37COOH carrying the N-glycosylation domain was minimally affected by tunicamycin treatment. In contrast, the abundance of was dramatically reduced in tunicamycin-treated cells. Probable explanations for this unexpected finding include the possibilities that, in absence of N-glycosylation of the full-length UL37 precursor, is misfolded and targeted for degradation or that its epitope for Ab1064 is masked by misfolding or protein–protein interactions and hence unavailable.



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Fig. 2. Internal cleavage of the UL37 precursor does not require prior N-glycosylation. HeLa cells were transfected with gpUL37–Flag vector (p816, lanes 3, 4, 7 and 8) or vector lacking insert (p790, lanes 1, 2, 5 and 6). Transfected cells were untreated (C) or treated with tunicamycin (Tn; 2 µg ml–1; Sigma) at the time of transfection to inhibit N-glycosylation. Mitochondrial (Mito) and ER fractions were purified as described in Fig. 1(b–e). The Western blot was reacted and reprobed with Ab against (Ab1064, top panel), UL37COOH (anti-Flag, second panel), mitochondria (anti-GRP75, third panel) and the ER (anti-DPM1, bottom panel) as detailed in Fig. 1(b–e). The arrowheads indicate migration of , glycosylated UL37COOH (gpUL37COOH) and unglycosylated UL37COOH (pUL37COOH). The pUL37x1 doublet in purified ER from HCMV-infected HFFs at 56 h post-infection (lane 9) is indicated by double dots.

 
To determine whether cleavage of UL37 occurs in the UL37x3 consensus signal peptidase site, we examined the processing and localization of pUL37M (Fig. 3). This naturally occurring HCMV UL37 variant lacks aa 178–262 and therefore the UL37x3 hydrophobic domain at aa 178–196, which includes the consensus signal peptidase site at aa 193/194. Transfected HeLa cells expressing pUL37M were fractionated into mitochondrial and ER fractions. Ab1064 detected pUL37M in both mitochondrial and ER fractions (Fig. 3, left panel, lanes 3 and 4). To determine whether pUL37M, which includes 11 N-glycosylation sites, is N-glycosylated, the fractionated proteins were treated with PNGase (Fig. 3, left panel, lanes 5 and 6). The molecular mass (~50 kDa) of pUL37M corresponded well with its predicted primary sequence (Chee et al., 1990; Goldmacher et al., 1999) and was not reduced by PNGase treatment (Fig. 3, left panel, lanes 3–6). A non-specific band of 75 kDa, which reacted with Ab1064, was detected in the ER (Fig. 3, left panel, lanes 2, 4 and 6) but not in mitochondria (Fig. 3, left panel, lanes 1, 3 and 5) of vector-transfected cells or of transfected cells expressing pUL37M. Taken together, these results suggested that pUL37M traffics both into the ER and mitochondria but is neither proteolytically cleaved nor N-glycosylated. These results further suggested that the gpUL37 cleavage site is within UL37x3 aa 178–262, consistent with gpUL37 cleavage at the consensus signal peptidase site in UL37x3.



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Fig. 3. pUL37M is neither proteolytically cleaved nor N-glycosylated. HeLa cells were transfected with empty vector (p790, lanes 1 and 2) or vector expressing pUL37M (lanes 3–10; Goldmacher et al., 1999). Transfected cells were fractionated into mitochondria (Mito, lanes 1, 3, 5, 7 and 9) or ER (lanes 2, 4, 6, 8 and 10) as described in Fig. 1(b–e). Fractionated proteins were untreated (–PNGase, lanes 3 and 4) or PNGase-treated (+PNGase, lanes 5 and 6) to deglycosylate UL37 proteins. PNGase was used as recommended by the manufacturer (New England Biolabs). Briefly, following denaturation, protein (20 µg) was digested with PNGase (500 units) for 30 min at 37 °C. Deglycosylated proteins were precipitated using 80 % cold acetone and resuspended in 1xSDS loading buffer for subsequent gel electrophoresis. Blotted proteins were reacted with Ab1064, anti-DPM1 or anti-GRP75 as described in Fig. 1(b–e). The arrowhead indicates migration of pUL37M and the asterisk indicates a non-specific band present in the ER fractions.

 
gpUL37 was cleaved in the ER and the molecular masses of its products corresponded closely with those predicted by cleavage at the UL37x3 consensus site, whereas pUL37M, which lacks the site, was not internally cleaved. This UL37x3 internal consensus signal peptidase cleavage site is conserved in primary HCMV strains (Hayajneh et al., 2001b) and in CCMV (Davison et al., 2003) UL37 sequences (Fig. 1a). Although three of its residues (187L->F, 189S ->N and 192H->N or Y) diverge in primary HCMV strains, the hydrophobic domain (aa 176–198) is maintained. The evolutionary conservation of the internal UL37x3 signal peptidase cleavage site suggests that proteolytic cleavage of gpUL37 may be important for its function during HCMV and CCMV growth in their respective hosts. Moreover, the physical proximity between the UL37x3 hydrophobic domain, the cleavage site and the first N-glycosylation site suggests that pUL37 maturation, including its proteolytic cleavage and N-glycosylation in the ER, may in part be dictated by its internal signal sequence as described previously for myocilin (Rutkowski et al., 2003). Our finding that pUL37M lacking the hydrophobic domain (aa 178–196) and the consensus signal peptidase site therein is not N-glycosylated in spite of its 11 N-glycosylation sites is consistent with this latter possibility.

There are few examples of proteolytic cleavage of internal signal sequences except for proteins that span membranes more than once (Perkosz & Lamb, 1998). The capsid precursors of rubella virus (Oker-Blom et al., 1990) and hepatitis C virus (Grakoui et al., 1993) have internal signal peptidase cleavage sites. Cleavage of the influenza C virus gp42 internal signal peptidase site generates p31 and the glycosylated integral membrane protein CM2 (Perkosz & Lamb, 1998). In contrast to our findings, one of the gp42 fragments, the p31 protein, could not be detected in infected cells. Both UL37 cleavage products ( and UL37COOH) were detected in transfected cells indicating that the N-terminal fragment () is not degraded but rather is imported into mitochondria.

Alternative processing of HCMV UL37 transcripts generates at least 11 different unspliced and spliced transcripts (Adair et al., 2003; Goldmacher et al., 1999; Kouzarides et al., 1988; Su et al., 2003). Alternatively spliced UL37 RNAs are predicted to encode at least three additional UL37 isoforms. The UL37di and dii ORFs include the UL37x3 consensus signal peptidase cleavage site whereas UL37s (sdi and sdii) ORF does not. Thus, in addition to diversity resulting from alternative UL37 RNA processing, other UL37 isoforms are generated by pUL37 precursor cleavage in the ER as demonstrated by these studies. Because of their distinct domains, the UL37 isoforms will likely differ in functions as well.

The HCMV glycoprotein gB is cleaved during its trafficking through the cellular secretory apparatus (Britt & Auger, 1986; Singh & Compton, 2000). However, gB is cleaved by furin as it traffics through the Golgi apparatus and forms inter-fragment dimers (Vey et al., 1995). In contrast to gpUL37, the HCMV gB cleavage fragments are covalently linked by disulfide bridges, traffic jointly through the secretory apparatus and are both incorporated into the viral envelope (Britt & Auger, 1986; Lopper & Compton, 2002). gpUL37 fragments dissociate and traffic differentially. UL37COOH is preferentially ER-localized and extensively N-glycosylated, and the stable traffics from the ER to mitochondria. Although they share most of their ORFs (aa 1–162), their different C termini and, specifically, the second hydrophobic domain at aa 178–196 suggest that is structurally different from pUL37x1. These structural differences may have functional significance for HCMV growth in humans considering their preferential trafficking to mitochondria.


   ACKNOWLEDGEMENTS
 
The authors thank Dr Richard Adair for his critical comments on the manuscript. The authors are grateful to Drs Judy Tevethia and Victor Goldmacher for providing the pCMV-T1-708 and pUL37M expression vectors, respectively. This work was supported by Children's Research Institute Discovery Funds to A. M. C.-P. and grants from the Board of Lady Visitors to M. S. M. and to A. M. C.-P.


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Received 5 March 2004; accepted 8 April 2004.