View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Virology 365 (2007) 125–135 www.elsevier.com/locate/yviro

Human herpesvirus-6A and -6B encode viral immunoevasins that downregulate class I MHC molecules ⁎ Nicole L. Glosson, Amy W. Hudson

Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701, Watertown Plank Road, Milwaukee, WI 53226, USA Received 2 January 2007; returned to author for revision 8 February 2007; accepted 21 March 2007 Available online 30 April 2007

Abstract

Like all other members of the herpesvirus family, the closely related human herpesviruses-6 and -7 (HHV-6,7) persist in their host throughout life. In so doing, without exception, every member of the herpesvirus family has evolved mechanisms to avoid detection by the . In particular, human cytomegalovirus (HCMV), mouse cytomegalovirus (MCMV), human herpesvirus-8 (HHV-8), and herpes simplex (HSV) all encode multiple that interfere with proper MHC class I . The mechanisms employed by these to effect removal of MHC class I from the cell surface vary. The U21 open reading frame from HHV-7 diverts class I MHC molecules to an endolysosomal compartment using an as-yet unknown mechanism. The two variants of HHV-6, HHV-6A and -6B, both possess a U21 open reading frame which contain only ∼30% amino acid identity to the U21 sequence from HHV-7. Here we describe the characterization of the U21 products from HHV-6A and HHV-6B. Like HHV-7 U21, both of the HHV-6 U21 molecules bind to and divert class I MHC molecules to an endolysosomal compartment, effectively removing them from the cell surface, and providing a possible means of escape from immune detection. © 2007 Elsevier Inc. All rights reserved.

Keywords: Immune evasion; HHV-7; HHV-6; Human herpesvirus-7; Human herpesvirus-6; Class I MHC; U21

Introduction a wider host tissue range and cell tropism than HHV-7 (De Bolle et al., 2005a,b). Of the two viruses, HHV-6 has been more Human herpesvirus-6 (HHV-6) is a β-herpesvirus most frequently and conclusively identified as the causative agent of closely related to human herpesvirus-7 (HHV-7). HHV-6 and clinically serious disease, while less is known about the clinical HHV-7 possess almost entirely colinear genomes and share manifestations of HHV-7 (Ward, 2005). The two viruses also many biological properties. Both viruses are T-lymphotropic, use different receptors for cell entry: The receptor for HHV-6 is but they can also infect other cell types, and both can cause the complement regulator CD46, while HHV-7 uses the exanthem subitum (roseola), although HHV-6 is the most co-receptor CD4 (Lusso et al., 1989, 1994; Santoro et al., 1999). common cause of this childhood disease (Zerr, 2006). Two distinct variants of HHV-6 exist, HHV-6A and HHV- Approximately 90% of the population is seropositive for both 6B, and although the genomes of the two variants are 90% HHV-6 and HHV-7 by the age of 3. Primary with these identical at the nucleotide level (the range among individual viruses occurs between 12 and 24 months with HHV-6, and is 31–98%), they differ significantly in their structure and somewhat later (∼36 months) with HHV-7 (for a review, see epidemiology (Braun et al., 1997; De Bolle et al., 2005a,b; Dewhurst, 2004; Ward, 2005). Dominguez et al., 1999). The vast majority of primary Despite their similarities, many of the clinical, epidemiolo- in children are attributed to the HHV-6B variant gical, and biological features of these viruses differ. HHV-6 has (Zerr et al., 2005). Approximately 40–50% of hematopoietic stem cell transplant patients and ∼32% of solid organ transplant recipients develop HHV-6 infection several weeks after ⁎ Corresponding author. Fax: +1 414 456 6535. transplantation, and 98% of these cases are due to the 6B E-mail address: [email protected] (A.W. Hudson). variant (Humar et al., 2002; Ljungman et al., 2000; Yoshikawa

0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2007.03.048 126 N.L. Glosson, A.W. Hudson / Virology 365 (2007) 125–135 et al., 1992, 2000). Moreover, it is now becoming clear that Results HHV-6B is also responsible for severe central nervous system (CNS) disease (HHV-6-encephalitis) in transplant recipients Amino acid sequence comparison of HHV-6A, -6B, and -7 U21 (for a detailed review, see Zerr, 2006). The clinical manifesta- tions of HHV-6A remain ill-defined; although it is clearly both The sequence alignments of HHV-7, -6A, and -6B U21 neuro- and T-lymphotropic, it has not yet been etiologically molecules are presented in Figs. 1a and b. The nucleotide linked to any disease. HHV-6A contains 9 fewer open reading sequences of HHV-6A and -6B U21 molecules are 90% frames (ORFs) than HHV-6B, and 9 more ORFs in HHV-6A do identical to each other, but HHV-6B U21 possesses an not possess counterparts in HHV-6B (Dominguez et al., 1999; additional 67-amino acid extension at its N-terminus (Fig. 1a). Gompels et al., 1995). We used the SignalP 3.0 server to predict the signal peptide Like all other herpesviruses, HHV-6 and -7 remain latent or cleavage sites for the three U21s (Bendtsen et al., 2004). There establish persistent infections. To do so, they must avoid is a clear consensus for signal peptide cleavage after glycine at detection and elimination by the immune system. Viral immune position 14 in HHV-7 U21 that is conserved in all three U21 evasion strategies include restriction of viral gene expression, sequences. In the HHV-6B N-terminal extension, there is also a infection at immunoprivileged sites, obstruction of antiviral weak consensus cleavage site between residues 33 and 34. cytokine function, and interference with antigen presentation However, the deglycosylated products of both HHV-6A (for a review, see Tortorella et al., 2000). Notably, all of the and -6B U21 migrate at the same position in SDS–PAGE gels, herpesviruses thus far examined employ the latter strategy of suggesting that the weak consensus site is not used, and that all interfering with viral antigen presentation to cytotoxic T three U21s are cleaved at the same position (residue 14) in the lymphocytes (CTLs). conserved consensus sequence (Fig. 6, lanes 6 and 8). To present peptide antigens to CTLs, major histocompat- Of the four N-linked glycans utilized in HHV-7 U21, two are ibility complex (MHC) class I heavy chains must form a conserved in HHV-6B U21, at positions 142 and 164, while complex with the light chain, β2-microglobulin (β2-m), and HHV-6A U21 conserves only one glycosylation site, at position peptide to acquire a stable conformation. MHC class I- 164. HHV-6B U21 appears to utilize one of these glycosylation associated peptides are generated by the proteasome in the sites only partially, as it migrates as a doublet in SDS–PAGE and are transported into the gels, and treatment with endoglycosidase F results in a (ER) by the TAP (transporter associated with antigen proces- migratory shift commensurate with the trimming of both N- sing) transporter. Once in the ER, the peptides are loaded onto linked glycans (Fig. 6, lanes 5 and 6). MHC class I molecules. Stable class I/β2-m/peptide complexes The HHV-6 U21 molecules share only 30% identity with then traverse the Golgi en route to the cell surface, where they HHV-7 U21. The cytoplasmic tails of HHV-6 and -7 U21 are can interact with CD8+ CTLs. almost entirely dissimilar (Fig. 1b), but all three cytoplasmic Herpesvirus gene products influence MHC class I antigen tails contain a conserved di-leucine or di-leucine-like (Ile-Ile) presentation in numerous and diverse ways. Some herpesviral sequence at position 412. We have previously demonstrated that proteins interfere with proteolysis of antigens or peptide this Ile-Ile sequence may serve as an internalization signal for transport into the ER (Ahn et al., 1997; Hill et al., 1995; HHV-7 U21 (Hudson et al., 2003). Tomazin et al., 1996; York et al., 1994). Others retain or destroy class I molecules (Ahn et al., 1996; Jones et al., 1996; Wiertz et HHV-6A U21 expression results in redistribution of class I al., 1996; Ziegler et al., 1997), enhance the internalization of MHC molecules to class I molecules, or divert class I molecules to lysosomes for degradation (Coscoy and Ganem, 2000; Hudson et al., 2001; In U373 astrocytoma cells stably transduced to express Ishido et al., 2000; Reusch et al., 1999; Stevenson et al., 2000). HHV-7 U21, class I MHC molecules are located in a punctate Judging from the number and molecular diversity of these perinuclear location, consistent with the endolysosomal com- strategies, the removal of MHC class I-peptide complexes from partment, and class I MHC labeling of the plasma membrane is the cell surface must be evolutionarily advantageous to these greatly diminished (compare Fig. 2a with Fig. 2b; Hudson et al., viruses as a means of escaping immune detection. 2001). To determine whether HHV-6A U21 molecules could HHV-7 encodes at least one such viral immunoevasin, U21. also divert class I MHC molecules to a lysosomal compartment, HHV-7 U21 binds to class I MHC molecules in the ER and we performed immunofluorescence microscopy to examine the diverts them to a lysosomal compartment, where they are intracellular localization of class I molecules in HHV-6 U21- degraded, removing them from the cell surface (Hudson et al., expressing cells. Expression of HHV-6A U21 in U373 cells 2001). HHV-6A, but not HHV-6B, has also been shown to results in the dramatic redistribution of class I molecules to a remove class I MHC molecules from the cell surface of HHV-6- punctate perinuclear compartment (Fig. 2c). infected cells (Hirata et al., 2001). The mechanism for this latter downregulation, however, was not examined in detail (Hirata et HHV-6A U21 associates with properly folded class I MHC al., 2001). Here we demonstrate that the U21 open reading molecules frames encoded by HHV-6A and HHV-6B, which share only ∼30% identity with HHV-7 U21, function similarly to divert To determine whether HHV-6 U21 associates with class I class I molecules to the lysosomal compartment. molecules, we performed co-immunoprecipitation experiments, ..Gosn ..Hdo iooy35(07 125 (2007) 365 Virology / Hudson A.W. Glosson, N.L. – 135

Fig. 1. Sequence alignment of HHV-6A, -6B, and -7 U21. (a) Sequence comparison of HHV-6A and -6B U21. HHV-6A and -6B U21 are 90% identical at the amino acid level. HHV-6B U21 codes for an additional 67 N- terminal amino acids. (b) Sequence comparison of HHV-6A and -7 U21. HHV-6A and -7 U21 share 30% amino acid identity. Identical amino acids are shaded yellow, and conserved residues are shaded green. The red boxes denote N-glycosylation consensus sites, the purple box indicates the predicted transmembrane domains, and black hash marks denote the predicted signal sequence cleavage sites. 127 128 N.L. Glosson, A.W. Hudson / Virology 365 (2007) 125–135

identification of these polypeptides is in progress. In com- parison, the 55-kDa HHV-7 U21 migrates more slowly, reflecting the presence of its 4 N-linked glycans (lane 1; Hudson et al., 2001).

The luminal domain of HHV-6A and -6B U21 binds to and diverts class l MHC molecules to lysosomes

Because the interaction between U21 and class I molecules is long-lasting, we originally hypothesized that HHV-7 U21 might escort class I molecules to the lysosomal compartment, perhaps by virtue of having a cytoplasmic sorting signal responsible for signaling diversion of the U21-class I MHC protein complex (Hudson et al., 2001). Surprisingly, however, deletion of the transmembrane region and cytoplasmic tail of HHV-7 U21 did not affect its ability to redistribute class I molecules to lysosomes; thus, the lysosomal targeting information must be contained within the lumenal domain of HHV-7 U21 (Hudson et al., 2003). To examine whether the cytoplasmic tails are also dispensable in HHV-6A and -6B U21, we replaced the cytoplasmic tails of HHV-6A and -6B U21 molecules with a C-terminal tandem tag consisting of a streptavidin binding protein (SBP) domain and an HA epitope tag, separated by a tobacco etch virus (TEV) site-specific protease cleavage site (U21SBPHA)(Fig. 4c) (Call et al., 2002). We then examined class l MHC localization in stable cell lines expressing HHV-6A and -6B U21SBPHA. As shown in Fig. 4, both HHV-6A and -6B U21SBPHA diverted class I molecules when their cytoplasmic tails were replaced by the tag, demonstrating that, as for HHV-7 U21, the lumenal domains of HHV-6A and -6B U21 are

Fig. 2. HHV-6 U21 diverts class I MHC molecules to a perinuclear location. Control U373 cells (a), non-tagged HHV-7 U21-expressing U373 cells (b), or non-tagged HHV-6A U21-expressing U373 cells (c) were labeled with W6/32, an antibody directed against properly folded class I MHC molecules, and an Alexa-488-conjugated secondary Ab.

recovering class l MHC molecules from [35S]-methionine- labeled HHV-6A U21-expressing U373 cells (Fig. 3). U21 molecules were recovered with class I MHC molecules both in HHV-7 U21 cells and HHV-6A U21 cells (Fig. 3, lanes 1 and 3). The singly glycosylated HHV-6A U21 migrates at 46 kDa, slightly slower than the class I MHC heavy chain molecules (lane 3, marked 6A U21). Since an antibody directed against HHV-6A U21 is not available, immunoblotting to confirm that the slower migrating polypeptide in lane 3 is U21 is not possible. However, further conformation that this ∼46-kDa polypeptide corresponds to HHV-6 U21 was obtained by examining the change in immunolocalization of class I MHC molecules in these cells (Fig. 2c). Moreover, in addition to HHV-6 U21, we also recover three polypeptides of high Fig. 3. HHV-6A U21 associates with properly folded class l MHC molecules. molecular weight (300–600 kDa) in the α-class l MHC U373 control cells, HHV-7 U21-expressing cells, and HHV-6A U21-expressing cells were labeled for 40 min with [35S]-methionine and class I molecules were immunoprecipitation from HHV-6 U21-expressing cells (lane recovered with W6/32. HHV-6A U21, at 47 kDa, is noted, as is the 55-kDa 3, asterisks). These are not evident in immunoprecipitates from HHV-7 U21. The asterisks denote unknown high molecular weight polypeptides control U373 cells nor from HHV-7 U21 cells, and the (∼300–600 kDa) also recovered with W6/32. N.L. Glosson, A.W. Hudson / Virology 365 (2007) 125–135 129

Fig. 4. The lumenal domain of HHV-6A and -6B U21 diverts class l MHC molecules to the lysosomal compartment. U373 cells, HHV-7, -6A, and -6B U21SBPHA- expressing U373 cells were labeled with W6/32 and the lysosomal marker protein mAb, α-lamp2. The antibodies used in each panel are indicated. Arrows indicate specific points of colocalization between W6/32 (class I MHC) and lamp2 labeling. (c) Schematic representation of the HHV-6A U21SBPHA construct expressed in panels g–i. (f, i, l) Control U373 cells (green trace), HHV-7 U21SBPHA-expressing cells (orange), HHV-6A U21SBPHA-expressing cells (blue), or HHV-6B U21SBPHA- expressing cells (purple) were incubated with the anti-PE HLA-A,B,C antibody directed against class l MHC molecules and analyzed by flow cytometry. Control cells incubated without anti-PE HLA-A,B,C antibody are indicated with the black trace.

responsible for the diversion of class l MHC molecules (Fig. 4, plasma membrane localization and intense punctate labeling of panels g and j). a compartment that co-localizes with lamp2 (Fig. 4, panels g, h, To determine whether HHV-6A and -6B U21SBPHA-expres- j, and k). Thus, like HHV-7 U21, the lumenal domain of HHV- sing cells could divert class I MHC molecules to the lysosomal 6A and -6B U21 is responsible for rerouting class I MHC compartment, we performed double-label immunofluorescence molecules to a lysosomal compartment, and replacement of the microscopy using an antibody recognizing properly folded class cytoplasmic tail with this tandem epitope tag has no obvious I molecules and an antibody directed against lamp2, a marker effect upon its association with class I MHC molecules, or its for the lysosomal compartment. As shown in Fig. 4, HHV-7 ability to divert class I molecules to the lysosomal compartment. U21SBPHA-expressing cells show a redistribution of class I Therefore, in subsequent experiments, cells expressing the MHC molecules to a punctate perinuclear location that co- SBPHA-tagged versions of each U21 molecule are employed localizes with lamp2 (Fig. 4, panels d and e). Likewise, HHV- when assessing U21's ability to divert class I molecules to the 6A and -6B U21SBPHA-expressing cells also show a decrease in lysosomal compartment. In the absence of an α-HHV-6 U21 130 N.L. Glosson, A.W. Hudson / Virology 365 (2007) 125–135

Fig. 5. HHV-6A and -6B U21 are stabilized by lysosomal protease inhibitors. U373, HHV-7, -6A, and -6B U21SBPHA-expressing cells were pulse-labeled for 1 h and chased for 0 or 6 h in the presence of leupeptin and Concanamycin A. Class I molecules were recovered with W6/32 and HA-tagged U21 molecules were recovered from HHV-7, -6A, and -6B U21SBPHA-expressing cells with the anti-HA antibody 12CA5. Triangles denote polypeptides of high molecular weight that are recovered in HHV-7 and -6B U21SBPHA-expressing cells. PhosphorImager quantitation of the fold difference in the intensities of U21 compared to the class I heavy chain, as indicated by the downward arrow, are noted below the heavy chain in lanes 3–10. The magnitude of difference between the intensities of the class I heavy chain to U21 are denoted below the heavy chains in lanes 15–22, as indicated by the upward arrow, i.e. the class I heavy chain band in lane 8 is 3-fold more intense than the U21 it co-precipitates. antibody, this HA tag allowed us to monitor the progress of and 7; Fig. 7, lanes 3 and 4), suggesting that expression levels U21. do not account for the difference in cell surface class I MHC expression seen between HHV-6A vs. HHV-6B U21SBPHA- HHV-6A and -6B U21 expression results in reduced class l expressing cells. MHC on the plasma membrane

In principle, a successful viral immunoevasin should reduce the number of class I MHC molecules available at the cell surface for recognition by cytotoxic T cells. To examine the presence of class I MHC molecules on the plasma membrane of HHV-6B- and -6B U21SBPHA-expressing cells, we performed flow cytometry experiments using an antibody directed against the extracytoplasmic domain of HLA-A, -B, and -C. Cell surface expression of class I MHC molecules is greatly reduced in HHV-7 U21SBPHA-expressing cells, compared with that of control U373 cells (Fig. 4f). Cell surface expression of class I MHC molecules is also reduced in HHV-6A and -6B U21SBPHA-expressing cells, but to a somewhat lesser extent than in HHV-7 U21SBPHA-expressing cells (compare Figs. 4i and l with Fig. 4f). This variation in cell surface class I expression is in part attributable to differences in U21 expression levels, since subcloning of puromycin-resistant cells resulted in the generation of some clonal cell lines in which the entire population of cells exhibited a more homogeneous reduction in surface expression of class I (data Fig. 6. HHV-6B is partially glycosylated. U373, HHV-7, -6A, and -6B not shown). However, the HHV-6B U21SBPHA cells shown in 35 U21SBPHA-expressing cells were labeled for 40 min with [ S]-methionine and panels j and l are a clonal cell line expressing HHV-6B U21SBPHA class I molecules were recovered with W6/32. Immunoprecipitates were treated at levels indistinguishable from that of the HHV-6A U21SBPHA- with PNGase:F (+ lanes). Class I heavy chains (HC) and HHV-6 U21 molecules expressing cells (see, e.g., Fig. 5, lanes 7 and 11; Fig. 6, lanes 5 +/− N-linked glycans are noted. N.L. Glosson, A.W. Hudson / Virology 365 (2007) 125–135 131

in the cell lysates demonstrates that HHV-7 U21 is expressed at roughly 4-fold higher levels than HHV-6 U21 molecules (Fig. 7, lane 2). Alternatively, this observation could also be explained by differences in the rates of degradation, synthesis, and/or trafficking of class I MHC or U21 molecules. However, even when we inhibit lysosomal degradation with lysosomal protease inhibitors, the ratio of [35S]-labeled class I MHC molecules recovered with HHV-6 U21 appears unchanged (Fig. 5, compare lanes 10 ((U21:class I=∼11:1) with lane 6 (U21: class I=∼2:1)), suggesting that a difference in stability does not explain the observation that fewer labeled class I MHC molecules are recovered with HHV-6 U21. The remaining possibility is consistent with the idea that HHV-6 U21 molecules may associate less efficiently with class I MHC molecules than does HHV-7 U21. The reciprocal immunoprecipitations yield similar informa- tion: When class I MHC molecules were recovered from the Fig. 7. Immunoblot analysis of U21 and class I MHC heavy chain expression. same HHV-7 U21SBPHA and HHV-6B U21SBPHA lysates, the U373, HHV-7, -6A, and -6B U21 -expressing cell lysates were loaded on SBPHA ratio of class I heavy chains to the coprecipitating U21SBPHA is SDS–polyacrylamide gels, transferred to nitrocellulose, and immunoblotted α α more equivalent for HHV-7 U21 than for HHV-6B U21 with an -HA mAb or the -class I MHC heavy chain mAb HC10. The asterisk ∼ denotes an SDS-resistant dimer of HHV-7 U21 , the circle denotes an α- (compare lanes 15, 17, or 18 (class I:U21 = 1:1) to lanes 19, SBPHA ∼ HA-reactive possible degradation product of HHV-7 U21SBPHA. HHV-7, -6A, 21, or 22 (class I:U21= 3:1)). and -6B U21 as well as class I MHC heavy chains are indicated. This set of reciprocal immunoprecipitations, using W6/32 to recover class I and the U21SBPHA molecules that associate with HHV-6A and -6B U21 are stabilized by lysosomal protease it, also suggests that U21 likely remains in complex with the inhibitors class I molecules that are diverted to the lysosomal compart- ment: When U21 molecules are recovered, virtually all of the If U21 acts as a chaperone to escort class l MHC molecules U21 and the class I molecules associated with it were degraded to a lysosomal compartment for degradation, then inhibition of after 6 h of chase (Fig. 5, lanes 4, 8, and 12). However, in lysosomal proteases should result in the stabilization of both immunoprecipitations with W6/32, all of the U21 associating U21 and class I MHC molecules. To examine whether HHV- with class I molecules is degraded, yet a large proportion of 6A and -6B U21 expression resulted in the degradation of class I MHC still remains, suggesting that the class I MHC class l MHC molecules, we performed pulse-chase experi- molecules not bound by U21 are not redirected to the lysosomal ments in the presence of leupeptin, a cysteine protease compartment (lanes 16 and 20). inhibitor, and Concanamycin A, an inhibitor of the vacuolar Although all samples were normalized for TCA-precipitable proton pump. Class I MHC molecules are stable for >6 h in counts before immunoprecipitating, in this experiment we note control U373 cells (Fig. 5, lanes 1 and 2). In cells expressing that for some reason the amount of radioactivity in the HHV-7 U21SBPHA from either HHV-6A, -6B, or -7, however, neither U21 IPs in the absence of lysosomal protease inhibitors (lanes class I MHC nor U21 molecules are recoverable after 6 h of 15 and 16) appears uniformly reduced compared to other chase (Fig. 5, lanes 4, 8, and 12). In the presence of lysosomal samples. The problem does not affect interpretation of the protease inhibitors, both U21 and class I MHC molecules are experiment. stabilized, indicating that in cells expressing U21, both U21 Of note, we also recover an ∼140-kDa coprecipitating and class I MHC molecules are degraded by lysosomal polypeptide with HHV-6B U21SBPHA that is stabilized in the proteases (Fig. 5, lanes 6, 10, and 14). Of note, the tandem presence of lysosomal protease inhibitors (Fig. 5, lanes 9–12, SBPHA tag is approximately 40 amino acids longer than the gray arrowhead). This polypeptide is not recovered with HHV-7 original cytoplasmic tail, resulting in slower migration in U21SBPHA, nor is it apparent in HHV-6A U21SBPHA immune SDS–PAGE than we observed for non-tagged HHV-6 U21 complexes. Similarly, from HHV-7 U21SBPHA-expressing cells, (Fig. 3). we recover a polypeptide of ∼210 kDa, which is stabilized by We recover fewer labeled class I MHC molecules in α-HA lysosomal protease inhibitors (black arrowhead). Both of these immunoprecipitations from HHV-6 U21SBPHA cells than from higher molecular weight polypeptides are also recovered upon HHV-7 U21SBPHA-expressing cells (Fig. 5, compare the ratio of immunoprecipitation of the complex with the W6/32 anti-class I co-precipitating class I molecules to the immunoprecipitated MHC antibody (Fig. 6, lanes 3 and 5), and both are endo- HA-tagged U21 in lanes 7 and 11 (U21:class I=∼12:1) to that glycosidase sensitive (Fig. 6, lanes 4 and 6). In fact, upon in lane 3 (U21:class I=∼2:1)). There are several possible PNGase:F digestion, the HHV-7 U21-associated 210-kDa band explanations for this observation: One possibility is that HHV-6 and the HHV-6B U21-associated 140-kDa band are reduced to U21 may be expressed at higher levels than HHV-7 U21. This is polypeptides that migrate similarly, both at ∼130 kDa in SDS– not the case; immunoblot analysis of total HA immunoreactivity PAGE gels (Fig. 6, lanes 4 and 6, open square). Based on their 132 N.L. Glosson, A.W. Hudson / Virology 365 (2007) 125–135 similar apparent molecular weight and sensitivity to endogly- The cytoplasmic tails of the HHV-6 U21 molecules possess cosidase F, we surmised these slower-migrating polypeptides almost no homology to the cytoplasmic tail of HHV-7 U21, with to be SDS-resistant multimers of the U21 molecules. An α-HA the exception of a conserved di-leucine sequence (Fig. 1b). For immunoblot also confirmed the presence of high molecular HHV-7 U21, we originally hypothesized that its cytoplasmic tail weight α-HA-reactive polypeptides (Fig. 7, lane 2). Treatment might contain lysosomal targeting information to divert the of the cells with the alkylating agent N-ethyl maleimide U21-class I MHC complex to the lysosomal compartment. To (NEM) before lysis altogether abolished the appearance of test this hypothesis, we replaced the cytoplasmic tail of the these higher molecular weight polypeptides, suggesting that plasma membrane-localized CD4 molecule with that of HHV-7 they are indeed dimers of U21 that form post-lysis (data not U21. We demonstrated that the cytoplasmic tail of U21 was shown). sufficient to confer intracellular localization upon this chimera, and that this conserved di-leucine-like sequence was respon- Steady-state levels of class I MHC heavy chain molecules are sible for intracellular sequestration. Interestingly, the intracel- reduced in U21-expressing cells lular localization of this CD4-U21 chimera was not lysosomal; rather, it appeared to be localized to the Golgi (Hudson et al., To examine the steady-state expression levels of the 2003). And, as we have discussed, the cytoplasmic tail of U21 is U21SBPHA and class I MHC molecules, we performed not necessary for its ability to redirect class I MHC molecules to immunoblot analysis on NP-40 lysates of U21SBPHA-transduced the lysosomal compartment. However, the conservation of this U373 cells. To ensure that all immunoreactive bands would di-leucine motif in the cytoplasmic tails of HHV-6A, -6B, and appear within linear range of detection, we loaded half as much -7 U21 molecules, together with the clear demonstration that the HHV-7 U21SBPHA cell lysate as U373, and HHV-6A and -6B di-leucine-like sequence in HHV-7 U21 acts as a potent U21SBPHA lysates. HHV-7 U21SBPHA, with its four N-linked intracellular sequestration motif, suggests that the cytoplasmic glycans and complex-type oligosaccharide modifications, tails of these molecules may not be entirely without function. appears at steady state to be present at 4- to 8-fold higher We have not yet pursued this possibility further with HHV-6A levels than either HHV-6A or -6B U21 (Fig. 7, lane 2). As and -6B U21, since it is clear that the cytoplasmic tail of U21 is discussed earlier, an α-HA-reactive polypeptide of approxi- dispensable for U21-mediated diversion of class I MHC mately twice the size of HHV-7 U21SBPHA is present in these molecules to lysosomes. However, it is worth noting that the lysates (Fig. 7, lane 2, asterisk), and we believe this to be an tailless and hybrid HHV-7 U21SBPHA molecules may be slightly SDS-resistant dimer of U21 that forms post-lysis. It is usually less efficient at preventing class I molecules from arriving at the visible in HHV-6B U21 immunoprecipitations as well, but it is cell surface, and we hypothesize that this internalization signal not apparent in this immunoblot at this exposure (see Figs. 5 and may act redundantly to remove any U21-class I complexes that 6). Additionally, we observe an α-HA-reactive degradation do arrive at the cell surface in error (data not shown; Hudson et product of HHV-7 U21SBPHA in these lysates (Fig. 7, lane 2). al., 2003). This degradation product must necessarily contain the C- In examining the abilities of the HHV-6A, -6B, and -7 U21 terminal SBPHA tag and transmembrane domain, and interest- molecules to divert class I MHC molecules to lysosomes, we ingly, since we do not recover this product in class I MHC show representative immunofluorescence of single cells (Fig. 4, immunoprecipitation experiments, this fragment must exclude a panels d, g, j, arrowheads), as well as flow cytometric analysis portion of the lumenal domain necessary for binding to class I of the population cells engineered to express the various U21 MHC molecules (Fig. 6, lane 3). As discussed previously, HHV- molecules (panels f, i, l). We consistently observe less class I 6B U21 migrates as a doublet due to its partial use of a second MHC immunoreactivity at the cell surface in HHV-7 glycosylation site (Fig. 7, lane 3). Steady-state expression levels U21SBPHA-expressing cells than in either HHV-6A or -6B of HHV-6A and HHV-6B U21 appear similar (Fig. 7, lanes 3 U21SBPHA cells, suggesting that HHV-7 U21 may be a more and 4). effective immunoevasin. It remains possible, however, that U21 from HHV-7 appears more efficient because its expression level Discussion of HHV-7 U21SBPHA in the cells is ∼4- to 8-fold higher (Figs. 5–7). We have not obtained HHV-6A or -6B U21 cell lines that Here we describe two new viral immunoevasins from HHV-6 express levels of U21 equal to that expressed in our HHV-7 that share homology with and function similarly to the U21 U21SBPHA cells. It is also possible that replacement of the immunoevasin from HHV-7. Although HHV-6A and -6B U21 cytoplasmic tails of the U21 molecules with the SBPHA tag proteins share only 30% amino acid identity with HHV-7 U21, might differentially affect their function. However, without both HHV-6A and HHV-6B encode U21 proteins that, like U21 specific antisera directed against HHV-6A and -6B U21, we from HHV-7, can divert class I MHC molecules to a lysosomal must utilize tagged molecules so that we may confirm the compartment. From the information we have gathered, we may expression of these molecules in cells. now employ sequence alignment to infer possible regions of the We also demonstrate that HHV-6A U21 appears to be more U21 molecule that might be important either in its association effective than HHV-6B U21 at removing class I molecules from with class I MHC molecules, or in its association with the the cell surface (Fig. 4, panels g–l). In this instance, the cellular mechanism for lysosomal sorting that is hijacked by expression levels of HHV-6A and -6B U21SBPHA in the cells are these viral proteins. similar and cannot account for the observed variation in class I N.L. Glosson, A.W. Hudson / Virology 365 (2007) 125–135 133

MHC molecules at the cell surface (Figs. 5–7). These data similarly to HHV-7 U21, the U21 from HHV-6B, in particular, suggest that, although HHV-6B U21 shares 89% amino acid does not appear to remove class I MHC molecules from the cell identity to HHV-6A U21, it may be a less-effective immunoe- surface as efficiently as its counterpart from HHV-7, calling in vasin than the U21 from HHV-6A. Interestingly, Hirata et al. to question whether, like HCMV and MCMV, HHV-6 and -7 (2001) examined the cell-surface expression of class I MHC may encode more than one mechanism devoted to removing molecules in the context of HHV-6A and -6B virus infection class I MHC molecules from the cell surface to escape detection and found that class I MHC molecules were downregulated by cytotoxic T lymphocytes. from HHV-6A but not HHV-6B-infected cells. Taken together, these results suggest that U21 provides a mechanism to remove Materials and methods class I MHC molecules from the cell surface in HHV-6A- infected cells. Although HHV-6B U21 can clearly bind to and Cell lines divert class I MHC molecules (Figs. 4–6), on the whole, its role in this process in the cell and during virus infection appears U373 astrocytoma cells were cultured in Dulbecco's reduced, in comparison to HHV-6A. modified Eagle medium (DMEM)–5% fetal bovine serum– Our working model for U21-mediated diversion of class I 5% newborn calf serum in the presence or absence of MHC molecules incorporates the following observations: U21 puromycin [375 ng/ml final] (Sigma-Aldrich, St. Louis, MO, binds to class I molecules in the ER, soon after both molecules USA) or geneticin (G418) [400 ng/ml final] (Gibco, Grand are synthesized. It accompanies class I at least as far as the trans- Island, NY, USA). Expression of viral proteins in cells was Golgi (Hudson et al., 2001), thus we hypothesize that U21 acts carried out via retrovirus-mediated gene transfer, using the as an escort to divert class I molecules to the lysosomal vector pLNCX, which carries the selectable marker for compartment. Since the interaction between U21 and class I neomycin resistance. U373 astrocytoma cells were infected molecules is lasting (Hudson et al., 2001), and both U21 and with retroviruses encoding HHV-6A U21, or HHV-6A or -6B class I molecules are stabilized in the presence of lysosomal U21 with a streptavidin binding protein (SBP) domain and an protease inhibitors (Fig. 5), we favor the idea that the two HA epitope tag separated by a tobacco etch virus (TEV) molecules travel together to the lysosomal compartment by an protease cleavage site (SBPHA), as described by Hudson et al. as-yet unknown mechanism. (2001, 2003). Stably expressing cell clones were isolated after Is U21 the only mechanism employed by HHV-6 to remove selection in G418. class I molecules from the cell surface, or, like the other β- herpesviruses, are there also multiple mechanisms encoded by Antibodies HHV-6 to impair CTL recognition of virus-infected cells? If not, how does HHV-6B, which appears to have a less effective U21, W6/32 is a monoclonal antibody (mAb) that recognizes survive in the face of a fully functioning immune system? assembled, β2m-associated HLA-A, -B, or -C molecules which Interestingly, the most closely related viruses to HHV-6 and was generously provided by Dr. Hidde Ploegh (Whitehead HHV-7, the β-herpesviruses HCMV and MCMV, each encode Institute, Cambridge, MA) (Barnstable et al., 1978). α-Giantin at least four different immunoevasins that affect the cell surface was purchased from Covance (San Diego, CA). Immunofluor- localization of class I MHC molecules (Tortorella et al., 2000). escence studies were performed either with a polyclonal In HCMV, these gene products may have arisen by gene antibody directed against the HA epitope tag (Affinity duplication of a common ancestor; US2, 3, 6, and 11, are all BioReagents, Golden, CO) or the α-HA mAb 12CA5. α-HA encoded within the US region of the genome, and all of them immunoprecipitation experiments were performed with the share sequence and/or structural homology with one another 12CA5 mAb. Alexa-Fluor 488 and 594-conjugated secondary (Gewurz et al., 2001; Jones and Muzithras, 1992; Jones et al., antibodies were purchased from Molecular Probes, Eugene, 1991). Of these gene products, US2 and US11 function OR, USA. In some instances, Alexa 594-conjugated FAb similarly to cause the retrotranslocation of class I MHC fragments (Zenon, Molecular Probes) were used to label lamp2 molecules. In addition to US2, 3, 6, and 11, the US region of mAbs. α-lamp2 MAb was the generous gift of Dr. T. August the genome encodes several other gene products, US7, 8, 9, and (Johns Hopkins Medical School, Baltimore, MD). A phycoer- 10, which are all predicted to contain structurally similar Ig ythrin (PE)-conjugated anti-HLA-A,B,C mAb (BD Pharmin- folds (Gewurz et al., 2001), and some (US8, 9, and 11) share as gen, San Jose, CA) was used in flow cytometry experiments. much as 30% sequence identity. However, no function, other than the ability to bind to class I, has been attributed to these Pulse-chase experiments viral glycoproteins (Furman et al., 2002; Huber et al., 2002; Jones and Muzithras, 1992; Jones et al., 1991; Tirabassi and Cells were detached with trypsin and incubated with Ploegh, 2002). Can a virus truly afford to faithfully encode a methionine- and cysteine-free DMEM for 30 min at 37 °C. protein that does not abet its infectivity or growth, or are these The cells were labeled with 500 μCi/ml of [35S]-Express label herpesvirus proteins serving other, as-yet unidentified func- (1175 Ci/mmol; PerkinElmer, Boston, MA) at 37 °C for the tions? The data we report here describes the function of two new indicated times and chased with complete DMEM supplemen- viral immunoevasins, the U21 molecules from HHV-6A and ted with non-radiolabeled methionine and cysteine to a final HHV-6B. Although the HHV-6 U21s appear to function concentration of 1 mM at 37 °C. Cells were lysed in Nonidet P- 134 N.L. Glosson, A.W. Hudson / Virology 365 (2007) 125–135

40 lysis buffer (50 mM Tris–HCl [pH 7.4], 0.5% NP-40, 5 mM amplification of both U21 DNAs was performed from the MgCl2, 150 mM NaCl). Lysates were centrifuged for 5–10 min gDNA, which yielded PCR products of the predicted sizes. The at 16,000×g at 4 °C in an Eppendorf centrifuge to pellet nuclei resulting PCR products were cloned into the vector pCR-TOPO and debris and precleared with standardized Pansorbin (Invitrogen, Carlsbad, CA), and the sequences were confirmed. (Staphylococcus aureus) cells (EMD Biosciences, Inc., San PCR was performed from the cDNAs using the following Diego, CA) and 3 μl of normal mouse serum, followed by primers corresponding to the 5′ and 3′ ends of the HHV-6A immunoprecipitation with specific antiserum and protein A and -6B U21 open reading frames: -6A U21 (for C-terminally agarose (RepliGen Corporation, Waltham, MA, USA). Immu- HA-tagged construct), 5′-gccaccATGATGATCTGCCTT-3′ noprecipitates were normalized to equal trichloroacetic acid- and 5′-TCAAATTTTTAATTCACGTCG-3′;-6AU21(for precipitable 35S-labeled protein. The immunoprecipitates were C-terminally SBPHA-tagged construct), 5′-gccaccATGAT- washed five times with NP-40 wash buffer (50 mM Tris–HCl GATCTGCCTT-3′ and 5′-CTCCTTCAGTCCTACAAAACA- [pH 7.4], 0.5% NP-40, 5 mM EDTA, 150 mM NaCl) and GGAAATACC-3′; -6B U21 (for C-terminally SBPHA-tagged subjected to sodium dodecyl sulfate–polyacrylamide gel construct), 5′-gccaccATGAGCGCTTTGTTTATTAC-3′ and 5′- electrophoresis (SDS–PAGE). Leupeptin (EMD Biosciences, CTCCTTCAGTCCTACAAAACAGGAAATACC-3′. The U21 Inc.) was added to the pulse and chase medium at 200 μM final cDNAs were then subcloned into the lentiviral expression concentration and Folimycin (Concanamycin A)(EMD Bios- vector pHAGE, which was the generous gift from Drs. Gustavo ciences, Inc.) was added to the pulse and chase medium at Mostoslavsky and Richard Mulligan (Harvard Medical School, 20 nM final concentration. In some experiments, peptide:N- Boston, MA). C-terminally HA-tagged HHV-6A U21 was Glycosidase F (PNGase F) (New England Biolabs, Beverly, amplified using a primer containing the additional HA MA) was added to the immunoprecipitates according to the sequence: GCCTACCCATACGACGTACCAGACTACGCA. manufacturer's instructions. C-terminally SBPHA-tagged -6A and -6B U21 were amplified from primers containing the additional peptide tag sequence Immunoblots (single letter code): SBP (DEKTTGWRGGHVVEGLAGE- LEQLRARLEHHPQGQREP); TEV peptide (ENLY); and HA Cell lysates (as described above) were assayed for protein peptide (YPYDVPDYA), as described in (Call et al., 2002). concentration using the Pierce BCA protein assay kit (Pierce, Rockford, IL). The specified amounts of protein were subjected Acknowledgments to SDS–PAGE. The gels were transferred to BA-85 nitrocellu- lose membrane (Whatman, Florham Park, NJ), and probed with The authors would like to thank Dr. Paula Traktman for the α-HA mAb HA.11 (Covance), followed by goat α-mouse critical reading of the manuscript and Tina Schneider and Grace HRP-conjugated secondary Ab (Bio-Rad, Hercules, CA). Ouyang for thoughtful discussion and technical assistance. Bands were visualized using the Pierce Supersubstrate Chemi- luminescence reagents (Pierce) and in some cases quantitated References with an Alpha Imager (AlphaInnotech, San Leandro, CA). Ahn, K., Angulo, A., Ghazal, P., Peterson, P.A., Yang, Y., Fruh, K., 1996. Immunofluorescence microscopy Human cytomegalovirus inhibits antigen presentation by a sequential multistep process. Proc. Natl. Acad. Sci. U.S.A. 93 (20), 10990–10995. Cells on coverslips were washed with PBS and fixed with Ahn, K., Gruhler, A., Galocha, B., Jones, T.R., Wiertz, E.J., Ploegh, H.L., 4% paraformaldehyde for 20 min, permeabilized with 0.5% Peterson, P.A., Yang, Y., Fruh, K., 1997. The ER-luminal domain of the HCMV glycoprotein US6 inhibits peptide translocation by TAP. Immunity 6 saponin in PBS and 3% BSA, incubated with primary (5), 613–621. antibodies for 30 min, washed, and followed by Alexa 488- Barnstable, C.J., Bodmer, W.F., Brown, G., Galfre, G., Milstein, C., Williams, or Alexa 594-conjugated secondary antibodies. A.F., Ziegler, A., 1978. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for – Flow cytometry genetic analysis. Cell 14 (1), 9 20. Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340 (4), 783–795. Cells were removed from the plates with trypsin, washed Braun, D.K., Dominguez, G., Pellett, P.E., 1997. Human herpesvirus 6. Clin. with ice-cold PBS, incubated with PE anti-HLA-A,B,C for Microbiol. Rev. 10 (3), 521–567. 30 min on ice, washed thoroughly, and resuspended in ice- Call, M.E., Pyrdol, J., Wiedmann, M., Wucherpfennig, K.W., 2002. The – cold PBS. Cell surface expression of class l MHC molecules organizing principle in the formation of the T cell receptor CD3 complex. Cell 111 (7), 967–979. was then evaluated with a Beckman FACScalibur flow Coscoy, L., Ganem, D., 2000. Kaposi's sarcoma-associated herpesvirus encodes cytometer. two proteins that block cell surface display of MHC class I chains by enhancing their . Proc. Natl. Acad. Sci. U.S.A. 97 (14), Cloning of U21 from HHV-6A and -6B-infected cells 8051–8056. De Bolle, L., Naesens, L., De Clercq, E., 2005a. Update on human herpesvirus 6 biology, clinical features, and therapy. Clin. Microbiol. Rev. 18 (1), The J-JHAN T cell line was infected with strain U1102 of 217–245. HHV-6A, and the Molt-3 T cell line with strain Z29 of HHV-6B, De Bolle, L., Van Loon, J., De Clercq, E., Naesens, L., 2005b. Quantitative and genomic DNA was purified from infected cells. PCR analysis of human herpesvirus 6 cell tropism. J. Med. Virol. 75 (1), 76–85. N.L. Glosson, A.W. Hudson / Virology 365 (2007) 125–135 135

Dewhurst, S., 2004. Human herpesvirus type 6 and human herpesvirus type 7 blood leucocytes are correlated to platelet engraftment and disease in infections of the central nervous system. Herpes 11 (Suppl. 2), 105A–111A. allogeneic stem cell transplant patients. Br. J. Haematol. 111 (3), 774–781. Dominguez, G., Dambaugh, T.R., Stamey, F.R., Dewhurst, S., Inoue, N., Pellett, Lusso, P., Gallo, R.C., DeRocco, S.E., Markham, P.D., 1989. CD4 is not the P.E., 1999. Human herpesvirus 6B genome sequence: coding content and membrane receptor for HHV-6. Lancet 1 (8640), 730. comparison with human herpesvirus 6A. J. Virol. 73 (10), 8040–8052. Lusso, P., Secchiero, P., Crowley, R.W., Garzino-Demo, A., Berneman, Z.N., Furman, M.H., Dey, N., Tortorella, D., Ploegh, H.L., 2002. The human Gallo, R.C., 1994. CD4 is a critical component of the receptor for human cytomegalovirus US10 gene product delays trafficking of major histocom- herpesvirus 7: interference with human immunodeficiency virus. Proc. Natl. patibility complex class I molecules. J. Virol. 76 (22), 11753–11756. Acad. Sci. U.S.A. 91 (9), 3872–3876. Gewurz, B.E., Gaudet, R., Tortorella, D., Wang, E.W., Ploegh, H.L., Wiley, D. Reusch, U., Muranyi, W., Lucin, P., Burgert, H.G., Hengel, H., Koszinowski, U. C., 2001. Antigen presentation subverted: structure of the human H., 1999. A cytomegalovirus glycoprotein re-routes MHC class I complexes cytomegalovirus protein US2 bound to the class I molecule HLA-A2. to lysosomes for degradation. EMBO J. 18 (4), 1081–1091. Proc. Natl. Acad. Sci. U.S.A. 98 (12), 6794–6799. Santoro, F., Kennedy, P.E., Locatelli, G., Malnati, M.S., Berger, E.A., Lusso, P., Gompels, U.A., Nicholas, J., Lawrence, G., Jones, M., Thomson, B.J., Martin, 1999. CD46 is a cellular receptor for human herpesvirus 6. Cell 99 (7), M.E., Efstathiou, S., Craxton, M., Macaulay, H.A., 1995. The DNA 817–827. sequence of human herpesvirus-6: structure, coding content, and genome Stevenson, P.G., Efstathiou, S., Doherty, P.C., Lehner, P.J., 2000. Inhibition of evolution. Virology 209 (1), 29–51. MHC class I-restricted antigen presentation by gamma 2-herpesviruses. Hill, A., Jugovic, P., York, I., Russ, G., Bennink, J., Yewdell, J., Ploegh, H., Proc. Natl. Acad. Sci. U.S.A. 97 (15), 8455–8460. Johnson, D., 1995. turns off the TAP to evade host Tirabassi, R.S., Ploegh, H.L., 2002. The human cytomegalovirus US8 immunity. Nature 375 (6530), 411–415. glycoprotein binds to major histocompatibility complex class I products. Hirata, Y., Kondo, K., Yamanishi, K., 2001. Human herpesvirus 6 down- J. Virol. 76 (13), 6832–6835. regulates major histocompatibility complex class I in dendritic cells. J. Med. Tomazin, R., Hill, A.B., Jugovic, P., York, I., van Endert, P., Ploegh, H.L., Virol. 65 (3), 576–583. Andrews, D.W., Johnson, D.C., 1996. Stable binding of the herpes simplex Huber, M.T., Tomazin, R., Wisner, T., Boname, J., Johnson, D.C., 2002. Human virus ICP47 protein to the peptide binding site of TAP. EMBO J. 15 (13), cytomegalovirus US7, US8, US9, and US10 are cytoplasmic glycoproteins, 3256–3266. not found at cell surfaces, and US9 does not mediate cell-to-cell spread. J. Tortorella, D., Gewurz, B.E., Furman, M.H., Schust, D.J., Ploegh, H.L., 2000. Virol. 76 (11), 5748–5758. Viral subversion of the immune system. Annu. Rev. Immunol. 18, 861–926. Hudson, A.W., Howley, P.M., Ploegh, H.L., 2001. A human herpesvirus 7 Ward, K.N., 2005. Human herpesviruses-6 and -7 infections. Curr. Opin. Infect. glycoprotein, U21, diverts major histocompatibility complex class I Dis. 18 (3), 247–252. molecules to lysosomes. J. Virol. 75 (24), 12347–12358. Wiertz, E.J., Jones, T.R., Sun, L., Bogyo, M., Geuze, H.J., Ploegh, H.L., 1996. Hudson, A.W., Blom, D., Howley, P.M., Ploegh, H.L., 2003. The ER-lumenal The human cytomegalovirus US11 gene product dislocates MHC class I domain of the HHV-7 immunoevasin U21 directs class I MHC molecules to heavy chains from the endoplasmic reticulum to the . Cell 84 (5), lysosomes. Traffic 4 (12), 824–837. 769–779. Humar, A., Kumar, D., Caliendo, A.M., Moussa, G., Ashi-Sulaiman, A., Levy, York, I.A., Roop, C., Andrews, D.W., Riddell, S.R., Graham, F.L., Johnson, G., Mazzulli, T., 2002. Clinical impact of human herpesvirus 6 infection D.C., 1994. A cytosolic herpes simplex virus protein inhibits antigen after liver transplantation. Transplantation 73 (4), 599–604. presentation to CD8+ T lymphocytes. Cell 77 (4), 525–535. Ishido, S., Wang, C., Lee, B.S., Cohen, G.B., Jung, J.U., 2000. Downregu- Yoshikawa, T., Suga, S., Asano, Y., Nakashima, T., Yazaki, T., Ono, Y., Fujita, lation of major histocompatibility complex class I molecules by Kaposi's T., Tsuzuki, K., Sugiyama, S., Oshima, S., 1992. A prospective study of sarcoma-associated herpesvirus K3 and K5 proteins. J. Virol. 74 (11), human herpesvirus-6 infection in renal transplantation. Transplantation 54 5300–5309. (5), 879–883. Jones, T.R., Muzithras, V.P., 1992. A cluster of dispensable genes within the Yoshikawa, T., Ihira, M., Suzuki, K., Suga, S., Iida, K., Saito, Y., Asonuma, K., human cytomegalovirus genome short component: IRS1, US1 through US5, Tanaka, K., Asano, Y., 2000. Human herpesvirus 6 infection after living and the US6 family. J. Virol. 66 (4), 2541–2546. related liver transplantation. J. Med. Virol. 62 (1), 52–59. Jones, T.R., Muzithras, V.P., Gluzman, Y., 1991. Replacement mutagenesis of Zerr, D.M., 2006. Human herpesvirus 6: a clinical update. Herpes 13 (1), 20–24. the human cytomegalovirus genome: US10 and US11 gene products are Zerr, D.M., Meier, A.S., Selke, S.S., Frenkel, L.M., Huang, M.L., Wald, A., nonessential. J. Virol. 65 (11), 5860–5872. Rhoads, M.P., Nguy, L., Bornemann, R., Morrow, R.A., Corey, L., 2005. A Jones, T.R., Wiertz, E.J., Sun, L., Fish, K.N., Nelson, J.A., Ploegh, H.L., 1996. population-based study of primary human herpesvirus 6 infection. N. Engl. Human cytomegalovirus US3 impairs transport and maturation of major J. Med. 352 (8), 768–776. histocompatibility complex class I heavy chains. Proc. Natl. Acad. Sci. Ziegler, H., Thale, R., Lucin, P., Muranyi, W., Flohr, T., Hengel, H., Farrell, H., U.S.A. 93 (21), 11327–11333. Rawlinson, W., Koszinowski, U.H., 1997. A mouse cytomegalovirus Ljungman, P., Wang, F.Z., Clark, D.A., Emery, V.C., Remberger, M., Ringden, glycoprotein retains MHC class I complexes in the ERGIC/cis-Golgi O., Linde, A., 2000. High levels of human herpesvirus 6 DNA in peripheral compartments. Immunity 6 (1), 57–66.