The Human Herpesvirus 6 G Protein-Coupled Receptor
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病菌学杂志 2005年第18期
Departments of Microbiology and Immunology
Pharmacology and Physiology
Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
ABSTRACT
Human herpesvirus 6 (HHV-6) is a ubiquitous T-lymphotropic betaherpesvirus that encodes two G protein-coupled receptor homologs, U12 and U51. HHV-6A U51 has been reported to bind to CC chemokines including RANTES, but the biological function of U51 remains uncertain. In this report, we stably expressed short interfering RNAs (siRNAs) specific for U51 in human T cells and then infected these cells with HHV-6. Viral DNA replication was reduced 50-fold by the U51 siRNA, and virally induced cytopathic effects were also inhibited. In contrast, viral replication and syncytium formation were unaltered in cells that expressed a scrambled derivative of the siRNA or an irrelevant siRNA and were restored to normal when a human codon-optimized derivative of U51 was introduced into cells containing the U51 siRNA. To examine the mechanism whereby U51 might contribute to viral replication, we explored the signaling characteristics of U51. None of the chemokines and opioids tested was able to induce G protein coupling by U51, and no evidence for opioid ligand binding by U51 was obtained. The effect of U51 on cell-cell fusion was also evaluated; these studies showed that U51 enhanced cell fusion mediated by the G protein of vesicular stomatitis virus. However, a U51-specific antiserum had no virus-neutralizing activity, suggesting that U51 may not be involved in the initial interaction between the virus particle and host cell. Overall, these studies suggest that HHV-6 U51 is a positive regulator of virus replication in vitro, perhaps because it may promote membrane fusion and facilitates cell-cell spread of this highly cell-associated virus.
INTRODUCTION
Human herpesvirus 6 (HHV-6) was first isolated in 1986 from patients with lymphoproliferative disorders (43) and later was identified as the causative agent of roseola infantum (56) and of acute febrile illness (41, 58) in young children. Following primary infection, the virus is able to establish a highly successful state of coexistence with the host, resulting in persistent infection with occasional but generally nonsymptomatic reactivation (13, 24). However, the virus can cause rare, serious complications in immunocompromised hosts or in the context of stem cell transplantation, including encephalitis, hepatitis, and bone marrow suppression (14, 54, 57). There are two variants of this virus, 6A and 6B, which have characteristic differences in their cell tropism and biological properties (1, 4, 16, 44) as well as approximately 10% overall sequence divergence at the genomic level (18, 23, 25).
The U51 gene is one of the two 7-transmembrane (7-tm) receptors carried by HHV-6 (23). It has been shown to be most closely related to the UL78 gene family from human cytomegalovirus (CMV), and gene knockout experiments using the rat CMV have revealed that this gene (R78) is necessary for efficient virus replication in vivo, suggesting that R78 (and perhaps U51 as well) may play a role in virus replication and virulence (6). Direct analyses of U51 itself have revealed that HHV-6 U51 can bind certain CC chemokines such as RANTES with nanomolar affinity (33), but no signaling activities have as yet been associated with this interaction.
To date, U51 has been studied largely in isolation using plasmid expression vectors. As a consequence, its functional significance within the context of the intact virus remains uncertain. To address this question, we decided to employ RNA interference (RNAi) technology (45) to selectively knock down U51 expression in HHV-6-susceptible T cells prior to exposing the cells to infectious HHV-6. As a positive control, we also designed and expressed a short interfering RNA (siRNA) specific for the HHV-6 glycoprotein B (gB), since this protein's gene is known to be essential for the replication and attachment of other human herpesviruses (47). Several negative controls were also included in these experiments, such as scrambled versions of our U51-specific siRNAs, as well as an irrelevant siRNA. In addition, "add-back" experiments were also performed, using siRNA-containing cells that coexpressed a degradation-resistant derivative of the U51 gene (i.e., a human codon-optimized version of U51, lacking homology to the sequences contained within the siRNA). Using these complementary approaches, we examined the role of U51 in HHV-6 replication and cytopathic effect in vitro. The results revealed that U51 makes an important contribution to viral DNA replication and syncytium formation. Finally, studies were performed to examine the mechanism of action of U51. These experiments showed that U51 can enhance the intrinsic cell fusion activity of the vesicular stomatitis virus G (VSV-G) protein, suggesting the possibility that U51's positive effect on HHV-6 replication may occur as a consequence of U51's ability to enhance the cell-cell spread of this highly cell-associated human herpesvirus.
MATERIALS AND METHODS
Vector construction. The U51 wild-type genes (U51nco) were amplified by standard PCR methods. HHV-6A U51 was cloned from strain U1102. A simian virus 5 (SV5) epitope tag was introduced at the N terminus of U51, and KpnI-EcoRV restriction sites were added to facilitate cloning into the expression vector pcDNA3 (Invitrogen). The primer sets used for adding the SV5 tag was 5'-GAGGTACCGCCACCATGGAGGGCAAGCCCATCCCCAACCCCCTGCTGGGCCTGGACAGCACCGGAG-3' and 5'-GGGCCTGGACAGCACCGGAGGCGGCAGCAAAGAAACGAAGTCTTTGGCT-3'.
The human codon-optimized (CO) U51 genes were assembled from synthetic oligonucleotides and cloned into pPCRScript (Geneart, Regensburg, Germany), as previously described (9). Note that the amino acid sequences encoded by these CO genes are identical to their wild-type counterparts (9). HHV-6A U51co was then restricted with HindIII and ApaI and cloned into pLEGFP-N1 retroviral vector (Clontech).
A truncated version of HHV-6A gB without the putative N-terminal signal peptide and C-terminal transmembrane region (nucleotide positions 23 to 652) was amplified from the corresponding HHV-6A strain U1102 cosmid DNA clone (36) and then inserted at the SmaI-PstI sites of pDisplay plasmid vector (Invitrogen), which contains a signal peptide and a hemagglutinin (HA) epitope tag at the N terminus and a platelet-derived growth factor receptor transmembrane domain at the C terminus. The following primer sets were used for amplification: 5'-TACCCGGGAGATCTCCGGATCATTATATCAGAGCGGGCTA-3' and 5'-CGCTGCAGAGAATTAATCCCATTAACATACGAAGGTG-3'.
To construct the 19- to 21-nucleotide hairpin siRNA cassettes, two cDNA oligonucleotides were chemically synthesized, annealed, and inserted between the SalI (XhoI) and XbaI sites immediately downstream of the U6 promoter in pSuppressorRetro vector (Imgenex): 5'-TCGA-19nt-AACG-19nt-TTTTT-3' and 5'-CTAGAAAAA-19nt-CGTT-19nt-3'. The target sequences for each of the genes were as follows: si6U51-130, 5'-GTCGGTCGAGAATACGCTGTG-3', corresponding to nucleotide positions 130 to 148 within the U51 open reading frame (ORF); si6U51-136, 5'-GAATACGCTGTGTTTACAT-3', corresponding to nucleotide positions 136 to 154; si6U51-646, 5'-ATAGCGCATCTGCCGAAAG-3', corresponding to nucleotide positions 646 to 664; si6U51-812, 5'-GTATCTGGCTGGTCAATTT-3', corresponding to nucleotide positions 812 to 830; si6U51-812Scramble, 5'-ACGCGTATTGTCTATTTGG-3', corresponding to a randomly arranged (scrambled) version of the sequences corresponding to nucleotide positions 812 to 830; and si6gB-A861, 5'-ATCGGTGTGTATGCTAAAG-3', and si6gB-B1517, 5'-GTGAAACGATGTGTTATAA-3', corresponding to nucleotide positions 861 to 879 and 1517 to 1535 within the gB ORF, respectively. A similar vector containing an irrelevant sequence that does not show significant homology to any human gene sequence was provided by a company (Imgenex) and used as a negative control (siNeg.Ctrl.; 5'-tcgaTCAGTCACGTTAATGGTCGTTttcaagagaAACGACCATTAACGTGACTGAttttt-3' and 5'-ctagaaaaaTCAGTCACGTTAATGGTCGTTtctcttgaaAACGACCATTAACGTGACTGA-3'; nucleotides in uppercase letters represent stem structure of siRNA).
The knockdown efficiency of each siRNA construct was tested by cotransfecting the corresponding DNA plasmid into human embryonic kidney 293 (HEK293) cells together with a U51- or gB-expressing plasmid (as appropriate). Forty-eight hours after transfection, protein expression levels were assessed by Western blotting.
Antibodies and Western blotting. Mouse monoclonal antibodies to the SV5 (paramyxovirus SV5, simian virus 5) or HA (hemagglutinin) epitopes and ?-tubulin were purchased from Serotec (MCA1360P) and Santa Cruz (sc-7392 and sc-9104), respectively.
For Western blotting, HEK293 cells were lysed with radioimmunoprecipitation assay buffer (Upstate) and then mixed with loading buffer containing 200 mM 2-mercaptoethanol without heating. Protein concentration was measured by a Bradford assay. Equal amounts of protein (25 μg) were loaded per lane and separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis prior to transfer to nitrocellulose. After incubation with appropriate primary antibodies (above) and washing, anti-rabbit or anti-mouse immunoglobulin G conjugated with horseradish peroxidase (Amersham Biosciences) was then added. The blot was developed with enhanced chemiluminescence reagent (Amersham Biosciences) and quantitated by National Institutes of Health Image software.
Retrovirus generation. HEK-293T cells were cotransfected with 5 μg of retrovirus vector plasmid (containing the siRNA of interest in pSuppressor or HHV-6A U51-CO in pLEGFP-N1) plus 5 μg p10A1 or pVSV-G, respectively, in a 100-mm culture dish by using the lipofectamine transfection method. The culture medium was replaced 16 h later, and the viruses were collected from the culture supernatants 48 h posttransfection. For U51 add-back experiment, the retroviruses expressing HHV-6A U51-CO were concentrated by centrifugation of the virus supernatant at 50,000 x g for 90 min at 4°C, and the pellet was then resuspended in 1% of the original volume in TNE (50 mM Tris-HCl [pH 7.8], 130 mM NaCl, 1 mM EDTA) buffer. Titers for the U51-CO expression constructs were about 107 CFU/ml.
Viruses and cells: preparation of HHV-6 virus stocks. The U1102 strain of HHV-6A was used throughout this study. JJhan cells infected with HHV-6A were cocultivated with uninfected cells at a ratio of 1:13 for 7 days. Virus stocks were prepared by centrifugation of the culture fluids at 2,000 x g for 10 min, and the supernatant was stored at –80°C. The 50% tissue culture infectious dose (TCID50) was calculated using the Spearman-Karber formula. SupT1 cells were maintained in RPMI 1640 containing 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator.
Virus infection: retrovirus transduction and generation of a stable siRNA-expressing cell line. SupT1 cells were transduced with siRNA-expressing retrovirus supernatant at a 1 to 2 dilution in the presence of 6 μg/ml polybrene (Sigma). Supernatant was removed after 24 h and replaced with fresh growth medium. Forty-eight hours after transduction, cells were passaged and selected for stable transformants in medium containing geneticin (1,000 μg/ml). Three weeks after selection, cell colonies that were resistant were transferred to 96-well plates and expanded. Cells (5 x 105) were mixed with 200 μl virus preparation at a multiplicity of infection (MOI) of 0.1 TCID50/cell, and virus was then centrifugally adsorbed onto the cells to enhance the efficiency of infection (2,000 x g, 30 min). The infected cells were then washed once and suspended in 10 ml RPMI 1640 medium containing 10% fetal calf serum.
RNA extraction and real-time PCR. Total RNA was prepared from SupT1 cells that had been infected with HHV-6 by using High Pure RNA Isolation kits (Roche). Primer extension reactions were performed with SuperScript II First-strand cDNA Synthesis kits (Invitrogen) using oligo(dT) primer, in accordance with the manufacturer's instructions. mRNA expression levels of each gene were quantitated by TaqMan real-time reverse transcription-PCR (RT-PCR) using U51-specific primers and probe and normalized with GAPDH mRNA. The U51-specific primer set was 5'-CCAAGGCTCTGGCAAAGGT-3' (sense) and 5'-TCAGCATCTGAAGAGCTTGCA-3' (antisense). The TaqMan probe used was 5'-TTTCCCGATAGTTTGGATCATA-3'. GAPDH primers and probes (assay-on-demand reagent) were obtained from a commercial supplier (ABI).
Real-time quantitative DNA-PCR. The viral DNA load in HHV-6A U1102-infected cells was quantitated by TaqMan real-time PCR. The HHV-6A U38 polymerase gene was chosen as a target gene for this purpose, and primer sets used for amplification of U38 were 5'-TGCTTCTGTAACGTGTCTTGGAA-3' (sense) and 5'-TCGGACTGCATCTTGGAATTAA-3' (antisense). The TaqMan probe used was 5'-ATGCTTTGTTCCACGGTGGAT-3'. A standard curve for U38 DNA quantitation was generated by using serially 10-fold-diluted plasmid DNA containing the relevant gene sequence. Culture supernatants of virally infected cells were treated with Proteinase K, and DNA was extracted using Wizard DNA extraction kits (Promega). This was used as the template in our experimental assays and was analyzed with a Bio-Rad iCycler. Amplification of standard and sample DNAs was conducted in the same 96-well reaction plate (Bio-Rad) under the following conditions: 2 min at 50°C and 10 min at 95°C, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. The detection limit is about 10 copies/reaction. All standards and samples were assayed in triplicate.
Neutralization assay. The U51-specific antiserum we used was a polyclonal rabbit antiserum directed against HHV-6B U51 (raised against a purified synthetic peptide spanning the third predicted extracellular loop of HHV-6B U51 [CHLPKAALSEIESDK]; there is only a single amino acid difference between HHV-6A and -6B within this region, which is denoted by the underlined residue; note that this same peptide was previously used by Menotti and colleagues to generate a U51-specific antiserum in rabbits [31]). The 15mer peptide was synthesized by SigmaGenosys and injected into rabbits for antibody production. After affinity purification using a peptide-conjugated column, the purified antibody was able to detect both HHV-6A and HHV-6B U51 effectively (down to a dilution of 1:1,000) in an indirect immunofluorescent assay on virus-infected cell cultures (unpublished data). Purified U51 antiserum was incubated with 200 μl of HHV-6A U1102 virus supernatant in a total volume of 500 μl at 37°C for 1 h. After that, infection was performed as described above. Note that the antiserum was not heat inactivated and thus would have been expected to be capable of mediating complement-directed lysis of virus particles in the event that complement-fixing antibodies were bound to cell-free virions.
Opioid receptor binding assay. To determine if HHV-6B U51 bound opioids, CHO-CAR cells were infected with a recombinant adenovirus that expressed the human codon-optimized HHV-6B U51 open reading frame (HHV6BCOwt) using methods previously described (59). Membranes from these cells were then prepared and incubated with opioids that were selective for the μ ([3H]DAMGO, 5 nM), ([3H]naltrindole, 1 nM; [3H]DPDPE, 10 nM), and ([3H]U69,593, 5 nM; [3H]bremazocine) receptors. Also, the nonselective antagonist [3H]diprenorphine was tested to determine if HHV-6B U51 would bind this nonselective high-affinity opioid. Nonspecific binding was measured by the inclusion of either 10 μM naloxone or 10 μM of the unlabeled compound. After a 60-min incubation, binding was terminated by filtering the samples through Schleicher & Schuell no. 32 glass fiber filters (Keene, NH) using a Brandel 48-well cell harvester. Filters were soaked for at least 60 min in 0.25% polyethylenimine for [3H]naltrindole and [3H]U69,593 binding experiments. After filtration, filters were washed three times with 3 ml of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 ml of ecoscint A scintillation fluid.
Establishment of a Tet-inducible cell line expressing U51. The T-Rex expression system (Invitrogen) was used to create a HEK293 cell line that inducibly expressed U51 upon addition of tetracycline (Tet). To do this, the native (non-codon-optimized) HHV-6B U51 open reading frame bearing an N-terminal SV5 tag (described in reference 9) was excised from a parental pcDNA3 vector with KpnI and EcoRV and inserted into pcDNA4/TO. pcDNA4/TO-U51 was then cotransfected with the pcDNA6/TR regulatory vector in a 1:6 ratio into HEK293 cells. After 48 h, cells were selected with 2 μg/ml blasticidin and 60 μg/ml zeocin. Selection of subclones for use in future experiments was based upon the induction profile of U51 expression following treatment of cells with tetracycline, as assessed by Western blot and flow cytometric analyses (representative results for one highly inducible subline are shown in Fig. 6). Cells treated for 24 to 48 h with Tet showed optimal U51 expression.
[35S]GTPS binding assay to measure coupling to G proteins. Three different sets of HEK293 cell membranes were used in experiments, including those from native cells and cells stably transfected with a Tet-inducible mammalian expression plasmid (Invitrogen) encoding an SV5 epitope-tagged derivative of the HHV-6B U51 protein (note that this construct was based on the native, non-codon-optimized viral sequence encoding U51). The latter cells were examined both in their native, uninduced state (in which U51 was expressed at a low level) and following induction (1 μg/ml tetracycline for 24 h), which resulted in a roughly 50- to 100-fold up-regulation of U51 expression at both the RNA and protein levels (as measured by quantitative RT-PCR analysis as well as immunoblot analysis and flow cytometry; see Fig. 6).
Cells were scraped from tissue culture plates and then centrifuged at 1,000 x g for 10 min at 4°C. The cells were resuspended in phosphate-buffered saline, pH 7.4, containing 0.04% EDTA. After centrifugation at 1,000 x g for 10 min at 4°C, the cell pellet was resuspended in membrane buffer, which consisted of 50 mM Tris-HCl, 3 mM MgCl2, and 1 mM EGTA, pH 7.4. The membranes were vortexed, followed by centrifugation at 40,000 x g for 30 min at 4°C. The membrane pellet was resuspended in membrane buffer, and the centrifugation step was repeated. The membranes were then resuspended in assay buffer, which consisted of 50 mM Tris-HCl, 3 mM MgCl2, 100 mM NaCl, and 0.2 mM EGTA, pH 7.4. The protein concentration was determined by the Bradford assay (10) using bovine serum albumin as the standard. The membranes were frozen at –80°C until use. HEK293 cell membranes as described above (15 μg of protein per tube) were incubated with 11 different ligands (ICI, 1 μM; RANTES 100 ng/ml; MCP-3, 1 ng/ml; lymphotactin, 100 ng/ml; interleukin-8, 100 ng/ml; the μ-opioid morphine, 1 μM; the -selective peptide DPDPE, 1 μM; the -selective antagonist naltrindole, 1 μM; and the μ-selective peptide DAMGO, 1 μM) in assay buffer for 60 min at 30°C in a final volume of 0.5 ml. The reaction mixture contained 3 μM GDP and 80 pmol of [35S]GTPS. Basal activity was determined in the presence of 3 μM GDP and in the absence of an agonist, and nonspecific binding was determined in the presence of 10 μM unlabeled GTPS. The membranes were then filtered onto glass fiber filters by vacuum filtration, followed by three washes with 3 ml of ice-cold 50 mM Tris-HCl, pH 7.5. Samples were counted in 2 ml of ecoscint A scintillation fluid. Data represent the percent of agonist stimulation [35S]GTPS binding of the basal activity, defined as (specific binding/basal binding) x 100. All experiments were repeated at least three times and were performed in triplicate.
Cell fusion assay. A cell fusion assay was devised, which relies upon the expression of a transcriptional activator protein (HIV-1 Tat) in one population of cells and the presence of a transcriptional reporter for Tat in a second population of cells (a plasmid containing the luciferase reporter gene, placed under the transcriptional control of the HIV-1 long terminal repeat [LTR], was used for this purpose). When the two populations of cells fuse, Tat will activate the HIV-1 LTR, resulting in high levels of luciferase production.
The fusion assay was performed by transfecting equal numbers of subconfluent HEK293 cells with either a HIV-1 Tat expressing plasmid (pcTat) (50) or an HIV-1 LTR:luciferase plasmid (17). All of the cells were also transfected with plasmid expression vectors encoding potential fusion-inducing proteins of interest. For our purposes, these were the VSV-G protein (pVSV-G; 0.3 μg; Clontech) and various 7-transmembrane proteins (human cytomegalovirus [HCMV] US28, the rat kappa opioid receptor, or HHV-6 U51). Four hours after transfection, the two populations of cells (Tat+ and LTR+) were treated with 0.25% trypsin-EDTA and mixed at a 1:1 ratio prior to reseeding in 12-well plates. Forty-four hours thereafter, luciferase assays were performed using commercially available reagents (Promega). Luciferase activity was quantitated with a Packard LumiCount microplate luminometer within the linear range of the detector. Results are presented as relative light units.
RESULTS
siRNA-expressing vectors suppressed U51 protein levels in a transient transfection system. Research on HHV-6 and -7 is presently constrained by the lack of a tractable system for the generation of genetically defined viral mutants. This is partly because there are no full-length molecularly cloned genomes for either virus and also because the viruses replicate most efficiently in suspension cell lines that are difficult to transfect with exogenous DNA; even in these cell lines, cell-free virus titers remain low relative to other human herpesviruses. For these reasons, we elected to use RNA interference technology as a means to examine the functional importance of the U51 open reading frame (ORF) in the in vitro replication of HHV-6. To do this, we used Dharmacon software to identify potential siRNAs that might target the HHV-6 U51 genes. Out of 24 potential target sites that were identified, 4 siRNAs were found that recognized target sequences which were fully conserved between the two viral variants, HHV-6A and HHV-6B. The selected siRNA targeting sequences were then subjected to a BLAST search against the entire nonredundant nucleotide sequence database in order to ensure that only the intended viral gene would be recognized. These siRNAs were then cloned into a linearized pSuppressorRetro (pSR) vector downstream of the U6 promoter. To screen the functional activity of these siRNA constructs, we cotransfected HEK293 cells with a plasmid expression vector encoding an SV5 epitope-tagged derivative of HHV-6A U51 plus the various siRNA-carrying pSR vectors (as well as constructs carrying an irrelevant control siRNA). The U51 protein expression level was then examined by Western blot analysis using a monoclonal antibody directed against the SV5 epitope tag. As shown in Fig. 1A, the expression of U51 protein (around 30 kDa) was markedly down-regulated by both si6U51-812 and si6U51-130 (over 80%) but not by the irrelevant siRNA (siNeg. Ctrl.) or the empty vector alone. These results demonstrate that siRNAs can specifically and efficiently inhibit U51 protein expression in mammalian cells in a transient transfection system.
Since the viral envelope glycoprotein B (gB) is known to be essential for replication of herpes simplex virus type 1, CMV, and other herpesviruses, siRNAs directed against HHV-6 gB were designed for use as a positive control in experiments aimed at testing the effect of U51-specific siRNAs on viral replication. The gB-specific siRNAs were tested using a similar approach to that described for the U51 siRNAs. As shown in Fig. 1B, transient expression of HHV-6 gB was efficiently blocked (over 90%) by both of the gB siRNAs that were tested. We therefore selected the HHV-6A gB-specific siRNA (si6gB-A861) for use in our subsequent experiments.
Cell lines stably expressing siRNA-U51 suppressed U51 expression upon virus infection. In order to examine the role of U51 in HHV-6 replication, we set out to derive stable cell lines that expressed one of our U51 siRNAs (si6U51-812 and si6U51-130). For these experiments, we elected to use a cell line that would be highly susceptible to HHV-6A infection; we therefore chose to work with SupT1 cells. These lymphoid suspension cells are difficult to transfect by standard means (electroporation or lipid-mediated DNA transfer), and we therefore created retroviral vectors that expressed a short hairpin RNA which would be expected to direct the generation of U51-specific short interfering RNA. SupT1 cells were then transduced with recombinant retrovirus particles and subjected to G418-mediated selection, and single colonies were picked and expanded. To confirm the specific gene silencing effect of siRNA-U51 in SupT1 cells, we then infected the cells with HHV-6A, and U51 mRNA levels were quantified 24 h postinfection (Fig. 2). After normalization of U51 expression data (using GAPDH mRNA levels as an internal control), we determined that U51 mRNA was decreased by over 90% in cells stably expressing si6U51-812 or si6U51-130 relative to unmodified SupT1 cells or SupT1-siNeg.Ctrl. cells that were infected with HHV-6A. Moreover, the growth properties of the clonal, siU51-expressing SupT1 sublines were found to be indistinguishable from parental SupT1 cells (data not shown).
U51-specific siRNA inhibited HHV-6A replication and virally induced syncytium formation. To test whether expression of a U51-specific siRNA would have any effect on virus replication in vitro, a panel of siRNA-expressing SupT1 sublines was infected with HHV-6A strain U1102 at an MOI of 0.1 TCID50/cell. These experiments were performed using several independent clonal SupT1 cell lines, each of which stably expressed a U51-specific siRNA (si6U51-812 or si6U51-130), as well as cells stably expressing si6gB and cells that expressed an irrelevant control siRNA (siNeg. Ctrl.). Six days later, when these cultures were examined under the light microscope, a significant reduction was detected in virally induced cytopathic effects (syncytium formation) in those cultures which expressed either the U51-specific siRNA or the gB-specific siRNA; no change in virally induced syncytium formation was detected in cells that expressed the irrelevant control siRNA (Fig. 3A to D).
Virus replication in these cultures was also examined by performing a quantitative real-time DNA PCR assay using TaqMan primers and probes specific for the U38 gene (this corresponds to the viral DNA polymerase). As shown in Fig. 3E, virus replication was significantly reduced in the cells that stably expressed either the U51 or the gB-specific siRNA but not in cells that expressed the irrelevant siRNA (siNeg. Ctrl.). Analysis of intracellular viral DNA load was also performed, with very similar results (data not shown).
To confirm our result, we made a scrambled derivative of the effective siRNA (si6U51-812) and tested its effect on virus replication. Viral replication and syncytium formation were unaltered in cells that expressed this scrambled siRNA (data not shown), confirming that the result we saw is a sequence-specific effect due to the expressed siRNA.
Expression of a codon-optimized form of U51 can restore virus replication in SupT1 cells that express a U51-specific siRNA. To determine whether the inhibitory effect of the U51 siRNA on virus replication was indeed due to a specific effect on U51 gene expression, we performed an "add-back" experiment. For this purpose, we took advantage of an available, human codon-optimized (CO) version of the U51 ORF. This synthetic ORF encodes the authentic U51 protein but does so using altered codons relative to the wild-type U51 gene (9). As a result, the expression of the codon-optimized U51 ORF should be resistant to inhibition by our U51 siRNA. We verified this by performing transient transfection experiments analogous to those shown in Fig. 1A; these studies revealed that the expression of the CO-U51 gene was indeed unaffected by the si6U51-812 siRNA (data not shown).
A recombinant retrovirus expressing the U51-CO gene was then constructed and used to transduce SupT1 cells that expressed the si6U51-812 siRNA, at an MOI of 10. This construct has previously been shown to result in high levels of U51 expression, both intracellularly and on the surface of all cell types that we have analyzed (9).
Twenty-four hours after retroviral transduction, the cells were then infected with HHV-6A U1102 at an MOI of 0.1 TCID50/cell. Virally induced cytopathic effects, virus load, and cell growth properties were then measured 6 days later. The results, which are presented in Fig. 4, show that coexpression of the codon-optimized U51 ORF restored virally induced cytopathic effects and viral replication in the SupT1(si6U51-812) cell line.
Virus infectivity was not affected by a U51-specific antibody. Previous studies have shown that 7-transmembrane receptors encoded by the human and mouse cytomegaloviruses (UL33, M28) may be incorporated into enveloped virus particles (30, 37). This suggested to us the possibility that HHV-6 U51 might play a role in virion attachment or entry to target cells. We therefore designed an experiment to test this hypothesis.
Briefly, we mixed HHV-6A virions with an affinity-purified polyclonal antiserum directed against U51 and then tested whether this had any neutralizing effect on virus infectivity. As controls, we used an irrelevant antiserum (directed against a nonconserved peptide from HHV-7 U51) as well as a human antiserum known to contain high levels of virus-neutralizing antibodies. After incubation with these various antisera for 1 h at 37°C, the HHV-6A inoculum was then added to SupT1 cells, and viral load in culture supernatants was then measured 5 days thereafter by quantitative DNA PCR analysis (Fig. 5). As expected, viral replication was essentially abolished in the cultures that received virions premixed with the positive control human serum. In contrast, there was no significant difference in the level of viral replication in cultures that received untreated virus inocula, inocula preincubated with the HHV-6 U51-specific antiserum, or inocula that were treated with the irrelevant antiserum. It is important to note that the U51-specific antiserum was not heat inactivated and thus would have been expected to be capable of mediating complement-directed lysis of virus particles had it bound to cell-free virions. Thus, these data suggest that U51 is most likely not involved in the initial interaction between HHV-6 virions and their target cells. However, this does not rule out the possibility that U51 may be involved either in modulating host cell signaling, so as to favor more efficient virus replication, or in the cell-cell spread of virus, perhaps by promoting fusion of virus-infected cells with virus-negative targets, as has been previously suggested for HCMV US28 (39).
U51-mediated cell signaling. In order to examine whether U51 might contribute to cell signaling events, we performed a series of experiments to examine both ligand binding and G protein coupling. For this set of experiments, we paid particular attention to the possibility that U51 might interact with opioid ligands in light of the previously noted similarity between U51 and human opioid receptors (23). For our initial ligand binding experiments, we transfected cells with recombinant adenovirus vectors that encoded a human codon-optimized form of U51, because it has been shown previously in our lab that codon optimization will enhance U51 expression 10- to 100-fold in mammalian cells (9). As noted previously, use of the codon-optimized constructs permits cell surface expression of U51, even in cell lines that are not of T-cell lineage (9); this contrasts with results reported by Menotti and colleagues, using a non-codon-optimized expression system that probably resulted in lower total levels of protein expression (31).
Briefly, our ligand binding studies revealed that membranes from cells which expressed the HHV-6B U51 protein did not specifically bind the μ-selective opioid, [3H]DAMGO, the -selective opioid, [3H]naltrindole or [3H]DPDPE, or the agonist, [3H]U69,593 or [3H]bremazocine. Also, HHV-6B U51 did not specifically bind the nonselective opioid receptor antagonist [3H]diprenorphine (data not shown).
The [35S]GTPS assay was then used to determine if opioids or a selected subset of chemokines could stimulate [35S]GTPS binding mediated by HHV-6B U51. Three different sets of HEK293 cell membranes were used in experiments, including those from wild-type 293 cells and cells stably transfected with a Tet-inducible expression plasmid carrying HHV-6B U51 (membranes were prepared from these cells either in the absence of U51 induction or following addition of 1 μg/ml tetracycline for 24 h, which resulted in a 50- to 100-fold induction of U51 expression at both the RNA and protein levels [Fig. 6]). Membranes from these different sets of HEK293 cells were tested with chemokines and opioids to determine if any chemokines or opioids stimulated the coupling of the U51 protein to G proteins. Table 1 shows that none of the chemokines or opioids tested had a significant effect on [35S]GTPS binding. Overall, we were unable to find any evidence for opioid ligand binding or opioid-induced G protein coupling by HHV-6B U51, and we therefore turned our attention to the possibility that U51 might influence cell membrane fusion events, as has been described previously for HCMV US28 (39).
Coexpression of U51 and vesicular stomatitis virus (VSV) G glycoprotein enhanced cell fusion. Membrane fusion events are important for viral entry into host cells and also for cell-to-cell spread of virus. To examine whether U51 facilitates virus replication and spread by contributing to membrane fusion, we used a luciferase-based gene reporter assay to quantitate cell fusion events. This assay relies on the presence of the HIV-1 transactivating protein (Tat) in one cell and a Tat-inducible reporter gene cassette (firefly luciferase linked to the HIV-1 LTR) in the other cell. Upon fusion of the target and effector cells, Tat will activate luciferase transcription, and luciferase expression can then be detected and quantitated by a luminometer. Because the contents of the effector and target cells must mix in order for the HIV Tat to transcribe the luciferase gene, the level of luciferase activity represents the extent of fusion between the effector and target cells.
Equal numbers of HEK293 cells were transiently transfected with a vector expressing either HIV Tat or luciferase under the transcriptional control of the HIV LTR. All cells also received a plasmid clone encoding pVSV-G, in the presence or absence of expression vectors that carry HHV-6 U51, the rat kappa opioid receptor (as a negative control), or HCMV US28 (as a positive control) (39). Four hours after transfection, the two cell populations were trypsinized and mixed together at a 1:1 ratio. Forty-four hours thereafter, the cell fusion activity was quantitatively determined by measuring luciferase gene expression in the lysates of the cocultured cells (Fig. 7). As expected, cells coexpressing US28 and VSV-G exhibited an increased level of fusion activity (3-fold) compared to cells transfected with VSV-G alone. Cells coexpressing VSV-G plus HHV-6A U51 also showed enhanced high fusion activity (2-fold) compared to cells transfected with VSV-G alone, while the kappa opioid receptor expression plasmid had no effect on cell fusion.
DISCUSSION
Herpesvirus genomes contain homologs of many important cellular genes. Most notable among these are the numerous 7-transmembrane (7-tm), G protein-coupled receptor (GPCR) homologs that are present within human herpesviruses (42, 46). In most cases, the biological function(s) of these proteins remains poorly understood, and this is especially true of the recently discovered human betaherpesviruses, human herpesviruses (HHV) 6 and 7. These viruses encode two GPCRs, U12 and U51, about which very little is known other than the fact that both receptors have the capacity to bind certain ?-chemokines (notably, RANTES) (26, 33, 35).
In order to understand better the role that U51 plays in the life cycle of HHV-6, we decided to use RNA interference (RNAi) technology to selectively inhibit U51 gene expression in virally infected cells. Other researchers have successfully applied this technology to the study of key genes in other herpesviruses (7, 27, 55), indicating that RNAi represents a powerful tool with which to study protein function in the context of virus replication. In order to examine the function of U51 in virus replication, we used a retroviral RNAi system, since this allowed for the efficient and stable suppression of U51 expression in virus-infected cell lines. Silencing of U51 expression was found to reduce viral RNA replication by about 50-fold, and virally induced cytopathic effects were also blocked. Most importantly, virus replication was restored to normal when a human codon-optimized derivative of U51 was introduced into cells containing the U51 siRNA, indicating that the RNAi-mediated effect was specific to U51.
Previous studies on herpesvirus 7-transmembrane (7-tm) receptors have generally concluded that these genes are dispensable for in vitro replication of virus; examples include HCMV US28 (52), HCMV UL33 (30), mouse CMV (MCMV) M33 (15), rat CMV (RCMV) R33 (6), and HCMV UL78 (32). However, deletion of the MCMV M78 gene has been shown to reduce virus replication in cultured fibroblasts (37), and deletion of the RCMV R78 gene also results in attenuation of virus production in cell culture systems (5, 28). Furthermore, deletion studies have revealed that all of the herpesvirus 7-tm proteins exert profound effects on virus replication and pathogenesis in vivo (6, 15) and/or on virally induced effects on host cells (48, 49). Thus, strong precedents exist for the functional importance of HHV-6 U51 in virus replication.
A number of other herpesvirus 7-tm proteins exhibit either constitutive or ligand-mediated signaling characteristics (3, 8, 11, 12, 21, 22, 53). This prompted us to wonder whether U51 might exhibit cell signaling properties that could perhaps influence the efficiency of viral replication. Since U51 has previously been noted to share significant sequence similarity with opioid receptors (23), we elected to focus particular attention on the possibility that U51 might interact with opioid ligands. The panel of opioid ligands that was evaluated for U51 binding included molecules with both broad opioid receptor binding characteristics and compounds with well-defined selectivity for each of the three human opioid receptor subtypes (, , μ). The opioid ligands induced efficient G protein coupling in membrane preparations from CHO cell sublines that overexpressed the relevant cognate receptors (Table 1), but none of the opioid ligands tested was found to bind to U51 (data not shown) or to induce G protein coupling by U51 (Table 1). Similarly, none of the chemokines tested was able to induce G protein coupling by U51, including RANTES. These findings are consistent with previously reported studies, since RANTES has been shown to bind to HHV-6A U51 but not to transduce any intracellular signaling events following such binding (33). Collectively, therefore, these findings suggest that despite the homology between U51 and opioid receptors (23), HHV-6B U51 does not appear to interact functionally with opioid ligands.
While we were unable to obtain any evidence for a functional interaction between HHV-6B U51 and opioid ligands, we did obtain strong evidence to suggest that U51 has the ability to modulate membrane fusion events triggered by other viral proteins. Our finding that the addition of a U51-specific antiserum to virus particles failed to neutralize virus infection suggests U51 may not play a role in the initial interaction between the virus particle and the host cell. This could be because U51 is not present in the virus particle, or it may reflect other possible explanations; further experiments will be needed to resolve this question. Nonetheless, the ability of U51 to enhance viral protein (VSV-G)-mediated cell-cell fusion suggests an alternative mechanism whereby U51 might facilitate the spread and replication of HHV-6. This finding is consistent with previous studies on HCMV US28, in which Pleskoff and coworkers reported that US28 enhanced cell-cell fusion mediated by different viral proteins (including the G protein of vesicular stomatitis virus, VSV-G) (39). These workers concluded that US28 might play a role in the fusion of the HCMV envelope with target cells (39). This view finds precedent in other studies, which show that US28 and cellular 7-tm receptors such as CCR-5 and CXCR-4 can promote fusion events between the HIV-1 envelope and its target cells (2, 19, 20, 40). Nonetheless, the precise mechanism by which HCMV US28 or HHV-6A U51 may enhance cell-cell fusion is uncertain. For example, while we know that U51 is expressed on the cell surface of transfected 293 cells (9), we cannot be certain whether intracellular U51 may also contribute to the fusogenic effects associated with coexpression of U51. We can, however, exclude an effect on the steady-state expression of VSV-G, since flow cytometric analysis revealed no difference in the levels of VSV-G expression on the cell surface, regardless of the coexpression of U51 (data not shown).
Our current working hypotheses are that (i) U51 and/or US28 may interact with cell membrane-tethered chemokine ligands such as fractalkine (29) and (ii) these viral proteins may form a complex with RANTES and cellular glycoaminoglycans, thereby altering viral infectivity and/or promoting cell-cell fusion (51). Since the ligand repertoire and signaling properties of HHV-6A U51 remain largely unknown, it is also possible that U51 may interact with other chemokines or nonchemokine ligands and/or that it may have the capacity to induce signaling events in either a ligand-specific or a constitutive fashion (akin to HCMV US28 and other herpesvirus 7-tm proteins) (3, 8, 11, 12, 22, 53).
Overall, the studies reported here establish that U51 plays an important role in the replication and spread of HHV-6A. Our work also suggests a possible mechanism for this effect, which may reflect the ability of U51 to enhance cell-cell fusion and thus spread of this highly cell-associated human herpesvirus. Further studies will be required to further examine this hypothesis and to unravel the full ligand binding and signaling properties of HHV-6A U51 as well as to determine whether HHV-6B U51 also has similar activity (34).
ACKNOWLEDGMENTS
We thank Frank Neipel for providing HHV-6A cosmid DNAs, Bryan Cullen for providing pcTat, and Caroline Hall, Mary Caserta, and Jennifer Carnahan for providing valuable reagents and advice. We thank Uta Hoepken for the generous gift of US28.
This work was supported by NIH grants RO1 DE14194 (to S.D.), T32 DE007165 (to B.B.-T.), R21 DA14950 and K05 DA000360 (to J.M.B.), and T32 DA07232 (to S.S.).
These authors contributed equally to this work.
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Pharmacology and Physiology
Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
ABSTRACT
Human herpesvirus 6 (HHV-6) is a ubiquitous T-lymphotropic betaherpesvirus that encodes two G protein-coupled receptor homologs, U12 and U51. HHV-6A U51 has been reported to bind to CC chemokines including RANTES, but the biological function of U51 remains uncertain. In this report, we stably expressed short interfering RNAs (siRNAs) specific for U51 in human T cells and then infected these cells with HHV-6. Viral DNA replication was reduced 50-fold by the U51 siRNA, and virally induced cytopathic effects were also inhibited. In contrast, viral replication and syncytium formation were unaltered in cells that expressed a scrambled derivative of the siRNA or an irrelevant siRNA and were restored to normal when a human codon-optimized derivative of U51 was introduced into cells containing the U51 siRNA. To examine the mechanism whereby U51 might contribute to viral replication, we explored the signaling characteristics of U51. None of the chemokines and opioids tested was able to induce G protein coupling by U51, and no evidence for opioid ligand binding by U51 was obtained. The effect of U51 on cell-cell fusion was also evaluated; these studies showed that U51 enhanced cell fusion mediated by the G protein of vesicular stomatitis virus. However, a U51-specific antiserum had no virus-neutralizing activity, suggesting that U51 may not be involved in the initial interaction between the virus particle and host cell. Overall, these studies suggest that HHV-6 U51 is a positive regulator of virus replication in vitro, perhaps because it may promote membrane fusion and facilitates cell-cell spread of this highly cell-associated virus.
INTRODUCTION
Human herpesvirus 6 (HHV-6) was first isolated in 1986 from patients with lymphoproliferative disorders (43) and later was identified as the causative agent of roseola infantum (56) and of acute febrile illness (41, 58) in young children. Following primary infection, the virus is able to establish a highly successful state of coexistence with the host, resulting in persistent infection with occasional but generally nonsymptomatic reactivation (13, 24). However, the virus can cause rare, serious complications in immunocompromised hosts or in the context of stem cell transplantation, including encephalitis, hepatitis, and bone marrow suppression (14, 54, 57). There are two variants of this virus, 6A and 6B, which have characteristic differences in their cell tropism and biological properties (1, 4, 16, 44) as well as approximately 10% overall sequence divergence at the genomic level (18, 23, 25).
The U51 gene is one of the two 7-transmembrane (7-tm) receptors carried by HHV-6 (23). It has been shown to be most closely related to the UL78 gene family from human cytomegalovirus (CMV), and gene knockout experiments using the rat CMV have revealed that this gene (R78) is necessary for efficient virus replication in vivo, suggesting that R78 (and perhaps U51 as well) may play a role in virus replication and virulence (6). Direct analyses of U51 itself have revealed that HHV-6 U51 can bind certain CC chemokines such as RANTES with nanomolar affinity (33), but no signaling activities have as yet been associated with this interaction.
To date, U51 has been studied largely in isolation using plasmid expression vectors. As a consequence, its functional significance within the context of the intact virus remains uncertain. To address this question, we decided to employ RNA interference (RNAi) technology (45) to selectively knock down U51 expression in HHV-6-susceptible T cells prior to exposing the cells to infectious HHV-6. As a positive control, we also designed and expressed a short interfering RNA (siRNA) specific for the HHV-6 glycoprotein B (gB), since this protein's gene is known to be essential for the replication and attachment of other human herpesviruses (47). Several negative controls were also included in these experiments, such as scrambled versions of our U51-specific siRNAs, as well as an irrelevant siRNA. In addition, "add-back" experiments were also performed, using siRNA-containing cells that coexpressed a degradation-resistant derivative of the U51 gene (i.e., a human codon-optimized version of U51, lacking homology to the sequences contained within the siRNA). Using these complementary approaches, we examined the role of U51 in HHV-6 replication and cytopathic effect in vitro. The results revealed that U51 makes an important contribution to viral DNA replication and syncytium formation. Finally, studies were performed to examine the mechanism of action of U51. These experiments showed that U51 can enhance the intrinsic cell fusion activity of the vesicular stomatitis virus G (VSV-G) protein, suggesting the possibility that U51's positive effect on HHV-6 replication may occur as a consequence of U51's ability to enhance the cell-cell spread of this highly cell-associated human herpesvirus.
MATERIALS AND METHODS
Vector construction. The U51 wild-type genes (U51nco) were amplified by standard PCR methods. HHV-6A U51 was cloned from strain U1102. A simian virus 5 (SV5) epitope tag was introduced at the N terminus of U51, and KpnI-EcoRV restriction sites were added to facilitate cloning into the expression vector pcDNA3 (Invitrogen). The primer sets used for adding the SV5 tag was 5'-GAGGTACCGCCACCATGGAGGGCAAGCCCATCCCCAACCCCCTGCTGGGCCTGGACAGCACCGGAG-3' and 5'-GGGCCTGGACAGCACCGGAGGCGGCAGCAAAGAAACGAAGTCTTTGGCT-3'.
The human codon-optimized (CO) U51 genes were assembled from synthetic oligonucleotides and cloned into pPCRScript (Geneart, Regensburg, Germany), as previously described (9). Note that the amino acid sequences encoded by these CO genes are identical to their wild-type counterparts (9). HHV-6A U51co was then restricted with HindIII and ApaI and cloned into pLEGFP-N1 retroviral vector (Clontech).
A truncated version of HHV-6A gB without the putative N-terminal signal peptide and C-terminal transmembrane region (nucleotide positions 23 to 652) was amplified from the corresponding HHV-6A strain U1102 cosmid DNA clone (36) and then inserted at the SmaI-PstI sites of pDisplay plasmid vector (Invitrogen), which contains a signal peptide and a hemagglutinin (HA) epitope tag at the N terminus and a platelet-derived growth factor receptor transmembrane domain at the C terminus. The following primer sets were used for amplification: 5'-TACCCGGGAGATCTCCGGATCATTATATCAGAGCGGGCTA-3' and 5'-CGCTGCAGAGAATTAATCCCATTAACATACGAAGGTG-3'.
To construct the 19- to 21-nucleotide hairpin siRNA cassettes, two cDNA oligonucleotides were chemically synthesized, annealed, and inserted between the SalI (XhoI) and XbaI sites immediately downstream of the U6 promoter in pSuppressorRetro vector (Imgenex): 5'-TCGA-19nt-AACG-19nt-TTTTT-3' and 5'-CTAGAAAAA-19nt-CGTT-19nt-3'. The target sequences for each of the genes were as follows: si6U51-130, 5'-GTCGGTCGAGAATACGCTGTG-3', corresponding to nucleotide positions 130 to 148 within the U51 open reading frame (ORF); si6U51-136, 5'-GAATACGCTGTGTTTACAT-3', corresponding to nucleotide positions 136 to 154; si6U51-646, 5'-ATAGCGCATCTGCCGAAAG-3', corresponding to nucleotide positions 646 to 664; si6U51-812, 5'-GTATCTGGCTGGTCAATTT-3', corresponding to nucleotide positions 812 to 830; si6U51-812Scramble, 5'-ACGCGTATTGTCTATTTGG-3', corresponding to a randomly arranged (scrambled) version of the sequences corresponding to nucleotide positions 812 to 830; and si6gB-A861, 5'-ATCGGTGTGTATGCTAAAG-3', and si6gB-B1517, 5'-GTGAAACGATGTGTTATAA-3', corresponding to nucleotide positions 861 to 879 and 1517 to 1535 within the gB ORF, respectively. A similar vector containing an irrelevant sequence that does not show significant homology to any human gene sequence was provided by a company (Imgenex) and used as a negative control (siNeg.Ctrl.; 5'-tcgaTCAGTCACGTTAATGGTCGTTttcaagagaAACGACCATTAACGTGACTGAttttt-3' and 5'-ctagaaaaaTCAGTCACGTTAATGGTCGTTtctcttgaaAACGACCATTAACGTGACTGA-3'; nucleotides in uppercase letters represent stem structure of siRNA).
The knockdown efficiency of each siRNA construct was tested by cotransfecting the corresponding DNA plasmid into human embryonic kidney 293 (HEK293) cells together with a U51- or gB-expressing plasmid (as appropriate). Forty-eight hours after transfection, protein expression levels were assessed by Western blotting.
Antibodies and Western blotting. Mouse monoclonal antibodies to the SV5 (paramyxovirus SV5, simian virus 5) or HA (hemagglutinin) epitopes and ?-tubulin were purchased from Serotec (MCA1360P) and Santa Cruz (sc-7392 and sc-9104), respectively.
For Western blotting, HEK293 cells were lysed with radioimmunoprecipitation assay buffer (Upstate) and then mixed with loading buffer containing 200 mM 2-mercaptoethanol without heating. Protein concentration was measured by a Bradford assay. Equal amounts of protein (25 μg) were loaded per lane and separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis prior to transfer to nitrocellulose. After incubation with appropriate primary antibodies (above) and washing, anti-rabbit or anti-mouse immunoglobulin G conjugated with horseradish peroxidase (Amersham Biosciences) was then added. The blot was developed with enhanced chemiluminescence reagent (Amersham Biosciences) and quantitated by National Institutes of Health Image software.
Retrovirus generation. HEK-293T cells were cotransfected with 5 μg of retrovirus vector plasmid (containing the siRNA of interest in pSuppressor or HHV-6A U51-CO in pLEGFP-N1) plus 5 μg p10A1 or pVSV-G, respectively, in a 100-mm culture dish by using the lipofectamine transfection method. The culture medium was replaced 16 h later, and the viruses were collected from the culture supernatants 48 h posttransfection. For U51 add-back experiment, the retroviruses expressing HHV-6A U51-CO were concentrated by centrifugation of the virus supernatant at 50,000 x g for 90 min at 4°C, and the pellet was then resuspended in 1% of the original volume in TNE (50 mM Tris-HCl [pH 7.8], 130 mM NaCl, 1 mM EDTA) buffer. Titers for the U51-CO expression constructs were about 107 CFU/ml.
Viruses and cells: preparation of HHV-6 virus stocks. The U1102 strain of HHV-6A was used throughout this study. JJhan cells infected with HHV-6A were cocultivated with uninfected cells at a ratio of 1:13 for 7 days. Virus stocks were prepared by centrifugation of the culture fluids at 2,000 x g for 10 min, and the supernatant was stored at –80°C. The 50% tissue culture infectious dose (TCID50) was calculated using the Spearman-Karber formula. SupT1 cells were maintained in RPMI 1640 containing 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator.
Virus infection: retrovirus transduction and generation of a stable siRNA-expressing cell line. SupT1 cells were transduced with siRNA-expressing retrovirus supernatant at a 1 to 2 dilution in the presence of 6 μg/ml polybrene (Sigma). Supernatant was removed after 24 h and replaced with fresh growth medium. Forty-eight hours after transduction, cells were passaged and selected for stable transformants in medium containing geneticin (1,000 μg/ml). Three weeks after selection, cell colonies that were resistant were transferred to 96-well plates and expanded. Cells (5 x 105) were mixed with 200 μl virus preparation at a multiplicity of infection (MOI) of 0.1 TCID50/cell, and virus was then centrifugally adsorbed onto the cells to enhance the efficiency of infection (2,000 x g, 30 min). The infected cells were then washed once and suspended in 10 ml RPMI 1640 medium containing 10% fetal calf serum.
RNA extraction and real-time PCR. Total RNA was prepared from SupT1 cells that had been infected with HHV-6 by using High Pure RNA Isolation kits (Roche). Primer extension reactions were performed with SuperScript II First-strand cDNA Synthesis kits (Invitrogen) using oligo(dT) primer, in accordance with the manufacturer's instructions. mRNA expression levels of each gene were quantitated by TaqMan real-time reverse transcription-PCR (RT-PCR) using U51-specific primers and probe and normalized with GAPDH mRNA. The U51-specific primer set was 5'-CCAAGGCTCTGGCAAAGGT-3' (sense) and 5'-TCAGCATCTGAAGAGCTTGCA-3' (antisense). The TaqMan probe used was 5'-TTTCCCGATAGTTTGGATCATA-3'. GAPDH primers and probes (assay-on-demand reagent) were obtained from a commercial supplier (ABI).
Real-time quantitative DNA-PCR. The viral DNA load in HHV-6A U1102-infected cells was quantitated by TaqMan real-time PCR. The HHV-6A U38 polymerase gene was chosen as a target gene for this purpose, and primer sets used for amplification of U38 were 5'-TGCTTCTGTAACGTGTCTTGGAA-3' (sense) and 5'-TCGGACTGCATCTTGGAATTAA-3' (antisense). The TaqMan probe used was 5'-ATGCTTTGTTCCACGGTGGAT-3'. A standard curve for U38 DNA quantitation was generated by using serially 10-fold-diluted plasmid DNA containing the relevant gene sequence. Culture supernatants of virally infected cells were treated with Proteinase K, and DNA was extracted using Wizard DNA extraction kits (Promega). This was used as the template in our experimental assays and was analyzed with a Bio-Rad iCycler. Amplification of standard and sample DNAs was conducted in the same 96-well reaction plate (Bio-Rad) under the following conditions: 2 min at 50°C and 10 min at 95°C, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. The detection limit is about 10 copies/reaction. All standards and samples were assayed in triplicate.
Neutralization assay. The U51-specific antiserum we used was a polyclonal rabbit antiserum directed against HHV-6B U51 (raised against a purified synthetic peptide spanning the third predicted extracellular loop of HHV-6B U51 [CHLPKAALSEIESDK]; there is only a single amino acid difference between HHV-6A and -6B within this region, which is denoted by the underlined residue; note that this same peptide was previously used by Menotti and colleagues to generate a U51-specific antiserum in rabbits [31]). The 15mer peptide was synthesized by SigmaGenosys and injected into rabbits for antibody production. After affinity purification using a peptide-conjugated column, the purified antibody was able to detect both HHV-6A and HHV-6B U51 effectively (down to a dilution of 1:1,000) in an indirect immunofluorescent assay on virus-infected cell cultures (unpublished data). Purified U51 antiserum was incubated with 200 μl of HHV-6A U1102 virus supernatant in a total volume of 500 μl at 37°C for 1 h. After that, infection was performed as described above. Note that the antiserum was not heat inactivated and thus would have been expected to be capable of mediating complement-directed lysis of virus particles in the event that complement-fixing antibodies were bound to cell-free virions.
Opioid receptor binding assay. To determine if HHV-6B U51 bound opioids, CHO-CAR cells were infected with a recombinant adenovirus that expressed the human codon-optimized HHV-6B U51 open reading frame (HHV6BCOwt) using methods previously described (59). Membranes from these cells were then prepared and incubated with opioids that were selective for the μ ([3H]DAMGO, 5 nM), ([3H]naltrindole, 1 nM; [3H]DPDPE, 10 nM), and ([3H]U69,593, 5 nM; [3H]bremazocine) receptors. Also, the nonselective antagonist [3H]diprenorphine was tested to determine if HHV-6B U51 would bind this nonselective high-affinity opioid. Nonspecific binding was measured by the inclusion of either 10 μM naloxone or 10 μM of the unlabeled compound. After a 60-min incubation, binding was terminated by filtering the samples through Schleicher & Schuell no. 32 glass fiber filters (Keene, NH) using a Brandel 48-well cell harvester. Filters were soaked for at least 60 min in 0.25% polyethylenimine for [3H]naltrindole and [3H]U69,593 binding experiments. After filtration, filters were washed three times with 3 ml of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 ml of ecoscint A scintillation fluid.
Establishment of a Tet-inducible cell line expressing U51. The T-Rex expression system (Invitrogen) was used to create a HEK293 cell line that inducibly expressed U51 upon addition of tetracycline (Tet). To do this, the native (non-codon-optimized) HHV-6B U51 open reading frame bearing an N-terminal SV5 tag (described in reference 9) was excised from a parental pcDNA3 vector with KpnI and EcoRV and inserted into pcDNA4/TO. pcDNA4/TO-U51 was then cotransfected with the pcDNA6/TR regulatory vector in a 1:6 ratio into HEK293 cells. After 48 h, cells were selected with 2 μg/ml blasticidin and 60 μg/ml zeocin. Selection of subclones for use in future experiments was based upon the induction profile of U51 expression following treatment of cells with tetracycline, as assessed by Western blot and flow cytometric analyses (representative results for one highly inducible subline are shown in Fig. 6). Cells treated for 24 to 48 h with Tet showed optimal U51 expression.
[35S]GTPS binding assay to measure coupling to G proteins. Three different sets of HEK293 cell membranes were used in experiments, including those from native cells and cells stably transfected with a Tet-inducible mammalian expression plasmid (Invitrogen) encoding an SV5 epitope-tagged derivative of the HHV-6B U51 protein (note that this construct was based on the native, non-codon-optimized viral sequence encoding U51). The latter cells were examined both in their native, uninduced state (in which U51 was expressed at a low level) and following induction (1 μg/ml tetracycline for 24 h), which resulted in a roughly 50- to 100-fold up-regulation of U51 expression at both the RNA and protein levels (as measured by quantitative RT-PCR analysis as well as immunoblot analysis and flow cytometry; see Fig. 6).
Cells were scraped from tissue culture plates and then centrifuged at 1,000 x g for 10 min at 4°C. The cells were resuspended in phosphate-buffered saline, pH 7.4, containing 0.04% EDTA. After centrifugation at 1,000 x g for 10 min at 4°C, the cell pellet was resuspended in membrane buffer, which consisted of 50 mM Tris-HCl, 3 mM MgCl2, and 1 mM EGTA, pH 7.4. The membranes were vortexed, followed by centrifugation at 40,000 x g for 30 min at 4°C. The membrane pellet was resuspended in membrane buffer, and the centrifugation step was repeated. The membranes were then resuspended in assay buffer, which consisted of 50 mM Tris-HCl, 3 mM MgCl2, 100 mM NaCl, and 0.2 mM EGTA, pH 7.4. The protein concentration was determined by the Bradford assay (10) using bovine serum albumin as the standard. The membranes were frozen at –80°C until use. HEK293 cell membranes as described above (15 μg of protein per tube) were incubated with 11 different ligands (ICI, 1 μM; RANTES 100 ng/ml; MCP-3, 1 ng/ml; lymphotactin, 100 ng/ml; interleukin-8, 100 ng/ml; the μ-opioid morphine, 1 μM; the -selective peptide DPDPE, 1 μM; the -selective antagonist naltrindole, 1 μM; and the μ-selective peptide DAMGO, 1 μM) in assay buffer for 60 min at 30°C in a final volume of 0.5 ml. The reaction mixture contained 3 μM GDP and 80 pmol of [35S]GTPS. Basal activity was determined in the presence of 3 μM GDP and in the absence of an agonist, and nonspecific binding was determined in the presence of 10 μM unlabeled GTPS. The membranes were then filtered onto glass fiber filters by vacuum filtration, followed by three washes with 3 ml of ice-cold 50 mM Tris-HCl, pH 7.5. Samples were counted in 2 ml of ecoscint A scintillation fluid. Data represent the percent of agonist stimulation [35S]GTPS binding of the basal activity, defined as (specific binding/basal binding) x 100. All experiments were repeated at least three times and were performed in triplicate.
Cell fusion assay. A cell fusion assay was devised, which relies upon the expression of a transcriptional activator protein (HIV-1 Tat) in one population of cells and the presence of a transcriptional reporter for Tat in a second population of cells (a plasmid containing the luciferase reporter gene, placed under the transcriptional control of the HIV-1 long terminal repeat [LTR], was used for this purpose). When the two populations of cells fuse, Tat will activate the HIV-1 LTR, resulting in high levels of luciferase production.
The fusion assay was performed by transfecting equal numbers of subconfluent HEK293 cells with either a HIV-1 Tat expressing plasmid (pcTat) (50) or an HIV-1 LTR:luciferase plasmid (17). All of the cells were also transfected with plasmid expression vectors encoding potential fusion-inducing proteins of interest. For our purposes, these were the VSV-G protein (pVSV-G; 0.3 μg; Clontech) and various 7-transmembrane proteins (human cytomegalovirus [HCMV] US28, the rat kappa opioid receptor, or HHV-6 U51). Four hours after transfection, the two populations of cells (Tat+ and LTR+) were treated with 0.25% trypsin-EDTA and mixed at a 1:1 ratio prior to reseeding in 12-well plates. Forty-four hours thereafter, luciferase assays were performed using commercially available reagents (Promega). Luciferase activity was quantitated with a Packard LumiCount microplate luminometer within the linear range of the detector. Results are presented as relative light units.
RESULTS
siRNA-expressing vectors suppressed U51 protein levels in a transient transfection system. Research on HHV-6 and -7 is presently constrained by the lack of a tractable system for the generation of genetically defined viral mutants. This is partly because there are no full-length molecularly cloned genomes for either virus and also because the viruses replicate most efficiently in suspension cell lines that are difficult to transfect with exogenous DNA; even in these cell lines, cell-free virus titers remain low relative to other human herpesviruses. For these reasons, we elected to use RNA interference technology as a means to examine the functional importance of the U51 open reading frame (ORF) in the in vitro replication of HHV-6. To do this, we used Dharmacon software to identify potential siRNAs that might target the HHV-6 U51 genes. Out of 24 potential target sites that were identified, 4 siRNAs were found that recognized target sequences which were fully conserved between the two viral variants, HHV-6A and HHV-6B. The selected siRNA targeting sequences were then subjected to a BLAST search against the entire nonredundant nucleotide sequence database in order to ensure that only the intended viral gene would be recognized. These siRNAs were then cloned into a linearized pSuppressorRetro (pSR) vector downstream of the U6 promoter. To screen the functional activity of these siRNA constructs, we cotransfected HEK293 cells with a plasmid expression vector encoding an SV5 epitope-tagged derivative of HHV-6A U51 plus the various siRNA-carrying pSR vectors (as well as constructs carrying an irrelevant control siRNA). The U51 protein expression level was then examined by Western blot analysis using a monoclonal antibody directed against the SV5 epitope tag. As shown in Fig. 1A, the expression of U51 protein (around 30 kDa) was markedly down-regulated by both si6U51-812 and si6U51-130 (over 80%) but not by the irrelevant siRNA (siNeg. Ctrl.) or the empty vector alone. These results demonstrate that siRNAs can specifically and efficiently inhibit U51 protein expression in mammalian cells in a transient transfection system.
Since the viral envelope glycoprotein B (gB) is known to be essential for replication of herpes simplex virus type 1, CMV, and other herpesviruses, siRNAs directed against HHV-6 gB were designed for use as a positive control in experiments aimed at testing the effect of U51-specific siRNAs on viral replication. The gB-specific siRNAs were tested using a similar approach to that described for the U51 siRNAs. As shown in Fig. 1B, transient expression of HHV-6 gB was efficiently blocked (over 90%) by both of the gB siRNAs that were tested. We therefore selected the HHV-6A gB-specific siRNA (si6gB-A861) for use in our subsequent experiments.
Cell lines stably expressing siRNA-U51 suppressed U51 expression upon virus infection. In order to examine the role of U51 in HHV-6 replication, we set out to derive stable cell lines that expressed one of our U51 siRNAs (si6U51-812 and si6U51-130). For these experiments, we elected to use a cell line that would be highly susceptible to HHV-6A infection; we therefore chose to work with SupT1 cells. These lymphoid suspension cells are difficult to transfect by standard means (electroporation or lipid-mediated DNA transfer), and we therefore created retroviral vectors that expressed a short hairpin RNA which would be expected to direct the generation of U51-specific short interfering RNA. SupT1 cells were then transduced with recombinant retrovirus particles and subjected to G418-mediated selection, and single colonies were picked and expanded. To confirm the specific gene silencing effect of siRNA-U51 in SupT1 cells, we then infected the cells with HHV-6A, and U51 mRNA levels were quantified 24 h postinfection (Fig. 2). After normalization of U51 expression data (using GAPDH mRNA levels as an internal control), we determined that U51 mRNA was decreased by over 90% in cells stably expressing si6U51-812 or si6U51-130 relative to unmodified SupT1 cells or SupT1-siNeg.Ctrl. cells that were infected with HHV-6A. Moreover, the growth properties of the clonal, siU51-expressing SupT1 sublines were found to be indistinguishable from parental SupT1 cells (data not shown).
U51-specific siRNA inhibited HHV-6A replication and virally induced syncytium formation. To test whether expression of a U51-specific siRNA would have any effect on virus replication in vitro, a panel of siRNA-expressing SupT1 sublines was infected with HHV-6A strain U1102 at an MOI of 0.1 TCID50/cell. These experiments were performed using several independent clonal SupT1 cell lines, each of which stably expressed a U51-specific siRNA (si6U51-812 or si6U51-130), as well as cells stably expressing si6gB and cells that expressed an irrelevant control siRNA (siNeg. Ctrl.). Six days later, when these cultures were examined under the light microscope, a significant reduction was detected in virally induced cytopathic effects (syncytium formation) in those cultures which expressed either the U51-specific siRNA or the gB-specific siRNA; no change in virally induced syncytium formation was detected in cells that expressed the irrelevant control siRNA (Fig. 3A to D).
Virus replication in these cultures was also examined by performing a quantitative real-time DNA PCR assay using TaqMan primers and probes specific for the U38 gene (this corresponds to the viral DNA polymerase). As shown in Fig. 3E, virus replication was significantly reduced in the cells that stably expressed either the U51 or the gB-specific siRNA but not in cells that expressed the irrelevant siRNA (siNeg. Ctrl.). Analysis of intracellular viral DNA load was also performed, with very similar results (data not shown).
To confirm our result, we made a scrambled derivative of the effective siRNA (si6U51-812) and tested its effect on virus replication. Viral replication and syncytium formation were unaltered in cells that expressed this scrambled siRNA (data not shown), confirming that the result we saw is a sequence-specific effect due to the expressed siRNA.
Expression of a codon-optimized form of U51 can restore virus replication in SupT1 cells that express a U51-specific siRNA. To determine whether the inhibitory effect of the U51 siRNA on virus replication was indeed due to a specific effect on U51 gene expression, we performed an "add-back" experiment. For this purpose, we took advantage of an available, human codon-optimized (CO) version of the U51 ORF. This synthetic ORF encodes the authentic U51 protein but does so using altered codons relative to the wild-type U51 gene (9). As a result, the expression of the codon-optimized U51 ORF should be resistant to inhibition by our U51 siRNA. We verified this by performing transient transfection experiments analogous to those shown in Fig. 1A; these studies revealed that the expression of the CO-U51 gene was indeed unaffected by the si6U51-812 siRNA (data not shown).
A recombinant retrovirus expressing the U51-CO gene was then constructed and used to transduce SupT1 cells that expressed the si6U51-812 siRNA, at an MOI of 10. This construct has previously been shown to result in high levels of U51 expression, both intracellularly and on the surface of all cell types that we have analyzed (9).
Twenty-four hours after retroviral transduction, the cells were then infected with HHV-6A U1102 at an MOI of 0.1 TCID50/cell. Virally induced cytopathic effects, virus load, and cell growth properties were then measured 6 days later. The results, which are presented in Fig. 4, show that coexpression of the codon-optimized U51 ORF restored virally induced cytopathic effects and viral replication in the SupT1(si6U51-812) cell line.
Virus infectivity was not affected by a U51-specific antibody. Previous studies have shown that 7-transmembrane receptors encoded by the human and mouse cytomegaloviruses (UL33, M28) may be incorporated into enveloped virus particles (30, 37). This suggested to us the possibility that HHV-6 U51 might play a role in virion attachment or entry to target cells. We therefore designed an experiment to test this hypothesis.
Briefly, we mixed HHV-6A virions with an affinity-purified polyclonal antiserum directed against U51 and then tested whether this had any neutralizing effect on virus infectivity. As controls, we used an irrelevant antiserum (directed against a nonconserved peptide from HHV-7 U51) as well as a human antiserum known to contain high levels of virus-neutralizing antibodies. After incubation with these various antisera for 1 h at 37°C, the HHV-6A inoculum was then added to SupT1 cells, and viral load in culture supernatants was then measured 5 days thereafter by quantitative DNA PCR analysis (Fig. 5). As expected, viral replication was essentially abolished in the cultures that received virions premixed with the positive control human serum. In contrast, there was no significant difference in the level of viral replication in cultures that received untreated virus inocula, inocula preincubated with the HHV-6 U51-specific antiserum, or inocula that were treated with the irrelevant antiserum. It is important to note that the U51-specific antiserum was not heat inactivated and thus would have been expected to be capable of mediating complement-directed lysis of virus particles had it bound to cell-free virions. Thus, these data suggest that U51 is most likely not involved in the initial interaction between HHV-6 virions and their target cells. However, this does not rule out the possibility that U51 may be involved either in modulating host cell signaling, so as to favor more efficient virus replication, or in the cell-cell spread of virus, perhaps by promoting fusion of virus-infected cells with virus-negative targets, as has been previously suggested for HCMV US28 (39).
U51-mediated cell signaling. In order to examine whether U51 might contribute to cell signaling events, we performed a series of experiments to examine both ligand binding and G protein coupling. For this set of experiments, we paid particular attention to the possibility that U51 might interact with opioid ligands in light of the previously noted similarity between U51 and human opioid receptors (23). For our initial ligand binding experiments, we transfected cells with recombinant adenovirus vectors that encoded a human codon-optimized form of U51, because it has been shown previously in our lab that codon optimization will enhance U51 expression 10- to 100-fold in mammalian cells (9). As noted previously, use of the codon-optimized constructs permits cell surface expression of U51, even in cell lines that are not of T-cell lineage (9); this contrasts with results reported by Menotti and colleagues, using a non-codon-optimized expression system that probably resulted in lower total levels of protein expression (31).
Briefly, our ligand binding studies revealed that membranes from cells which expressed the HHV-6B U51 protein did not specifically bind the μ-selective opioid, [3H]DAMGO, the -selective opioid, [3H]naltrindole or [3H]DPDPE, or the agonist, [3H]U69,593 or [3H]bremazocine. Also, HHV-6B U51 did not specifically bind the nonselective opioid receptor antagonist [3H]diprenorphine (data not shown).
The [35S]GTPS assay was then used to determine if opioids or a selected subset of chemokines could stimulate [35S]GTPS binding mediated by HHV-6B U51. Three different sets of HEK293 cell membranes were used in experiments, including those from wild-type 293 cells and cells stably transfected with a Tet-inducible expression plasmid carrying HHV-6B U51 (membranes were prepared from these cells either in the absence of U51 induction or following addition of 1 μg/ml tetracycline for 24 h, which resulted in a 50- to 100-fold induction of U51 expression at both the RNA and protein levels [Fig. 6]). Membranes from these different sets of HEK293 cells were tested with chemokines and opioids to determine if any chemokines or opioids stimulated the coupling of the U51 protein to G proteins. Table 1 shows that none of the chemokines or opioids tested had a significant effect on [35S]GTPS binding. Overall, we were unable to find any evidence for opioid ligand binding or opioid-induced G protein coupling by HHV-6B U51, and we therefore turned our attention to the possibility that U51 might influence cell membrane fusion events, as has been described previously for HCMV US28 (39).
Coexpression of U51 and vesicular stomatitis virus (VSV) G glycoprotein enhanced cell fusion. Membrane fusion events are important for viral entry into host cells and also for cell-to-cell spread of virus. To examine whether U51 facilitates virus replication and spread by contributing to membrane fusion, we used a luciferase-based gene reporter assay to quantitate cell fusion events. This assay relies on the presence of the HIV-1 transactivating protein (Tat) in one cell and a Tat-inducible reporter gene cassette (firefly luciferase linked to the HIV-1 LTR) in the other cell. Upon fusion of the target and effector cells, Tat will activate luciferase transcription, and luciferase expression can then be detected and quantitated by a luminometer. Because the contents of the effector and target cells must mix in order for the HIV Tat to transcribe the luciferase gene, the level of luciferase activity represents the extent of fusion between the effector and target cells.
Equal numbers of HEK293 cells were transiently transfected with a vector expressing either HIV Tat or luciferase under the transcriptional control of the HIV LTR. All cells also received a plasmid clone encoding pVSV-G, in the presence or absence of expression vectors that carry HHV-6 U51, the rat kappa opioid receptor (as a negative control), or HCMV US28 (as a positive control) (39). Four hours after transfection, the two cell populations were trypsinized and mixed together at a 1:1 ratio. Forty-four hours thereafter, the cell fusion activity was quantitatively determined by measuring luciferase gene expression in the lysates of the cocultured cells (Fig. 7). As expected, cells coexpressing US28 and VSV-G exhibited an increased level of fusion activity (3-fold) compared to cells transfected with VSV-G alone. Cells coexpressing VSV-G plus HHV-6A U51 also showed enhanced high fusion activity (2-fold) compared to cells transfected with VSV-G alone, while the kappa opioid receptor expression plasmid had no effect on cell fusion.
DISCUSSION
Herpesvirus genomes contain homologs of many important cellular genes. Most notable among these are the numerous 7-transmembrane (7-tm), G protein-coupled receptor (GPCR) homologs that are present within human herpesviruses (42, 46). In most cases, the biological function(s) of these proteins remains poorly understood, and this is especially true of the recently discovered human betaherpesviruses, human herpesviruses (HHV) 6 and 7. These viruses encode two GPCRs, U12 and U51, about which very little is known other than the fact that both receptors have the capacity to bind certain ?-chemokines (notably, RANTES) (26, 33, 35).
In order to understand better the role that U51 plays in the life cycle of HHV-6, we decided to use RNA interference (RNAi) technology to selectively inhibit U51 gene expression in virally infected cells. Other researchers have successfully applied this technology to the study of key genes in other herpesviruses (7, 27, 55), indicating that RNAi represents a powerful tool with which to study protein function in the context of virus replication. In order to examine the function of U51 in virus replication, we used a retroviral RNAi system, since this allowed for the efficient and stable suppression of U51 expression in virus-infected cell lines. Silencing of U51 expression was found to reduce viral RNA replication by about 50-fold, and virally induced cytopathic effects were also blocked. Most importantly, virus replication was restored to normal when a human codon-optimized derivative of U51 was introduced into cells containing the U51 siRNA, indicating that the RNAi-mediated effect was specific to U51.
Previous studies on herpesvirus 7-transmembrane (7-tm) receptors have generally concluded that these genes are dispensable for in vitro replication of virus; examples include HCMV US28 (52), HCMV UL33 (30), mouse CMV (MCMV) M33 (15), rat CMV (RCMV) R33 (6), and HCMV UL78 (32). However, deletion of the MCMV M78 gene has been shown to reduce virus replication in cultured fibroblasts (37), and deletion of the RCMV R78 gene also results in attenuation of virus production in cell culture systems (5, 28). Furthermore, deletion studies have revealed that all of the herpesvirus 7-tm proteins exert profound effects on virus replication and pathogenesis in vivo (6, 15) and/or on virally induced effects on host cells (48, 49). Thus, strong precedents exist for the functional importance of HHV-6 U51 in virus replication.
A number of other herpesvirus 7-tm proteins exhibit either constitutive or ligand-mediated signaling characteristics (3, 8, 11, 12, 21, 22, 53). This prompted us to wonder whether U51 might exhibit cell signaling properties that could perhaps influence the efficiency of viral replication. Since U51 has previously been noted to share significant sequence similarity with opioid receptors (23), we elected to focus particular attention on the possibility that U51 might interact with opioid ligands. The panel of opioid ligands that was evaluated for U51 binding included molecules with both broad opioid receptor binding characteristics and compounds with well-defined selectivity for each of the three human opioid receptor subtypes (, , μ). The opioid ligands induced efficient G protein coupling in membrane preparations from CHO cell sublines that overexpressed the relevant cognate receptors (Table 1), but none of the opioid ligands tested was found to bind to U51 (data not shown) or to induce G protein coupling by U51 (Table 1). Similarly, none of the chemokines tested was able to induce G protein coupling by U51, including RANTES. These findings are consistent with previously reported studies, since RANTES has been shown to bind to HHV-6A U51 but not to transduce any intracellular signaling events following such binding (33). Collectively, therefore, these findings suggest that despite the homology between U51 and opioid receptors (23), HHV-6B U51 does not appear to interact functionally with opioid ligands.
While we were unable to obtain any evidence for a functional interaction between HHV-6B U51 and opioid ligands, we did obtain strong evidence to suggest that U51 has the ability to modulate membrane fusion events triggered by other viral proteins. Our finding that the addition of a U51-specific antiserum to virus particles failed to neutralize virus infection suggests U51 may not play a role in the initial interaction between the virus particle and the host cell. This could be because U51 is not present in the virus particle, or it may reflect other possible explanations; further experiments will be needed to resolve this question. Nonetheless, the ability of U51 to enhance viral protein (VSV-G)-mediated cell-cell fusion suggests an alternative mechanism whereby U51 might facilitate the spread and replication of HHV-6. This finding is consistent with previous studies on HCMV US28, in which Pleskoff and coworkers reported that US28 enhanced cell-cell fusion mediated by different viral proteins (including the G protein of vesicular stomatitis virus, VSV-G) (39). These workers concluded that US28 might play a role in the fusion of the HCMV envelope with target cells (39). This view finds precedent in other studies, which show that US28 and cellular 7-tm receptors such as CCR-5 and CXCR-4 can promote fusion events between the HIV-1 envelope and its target cells (2, 19, 20, 40). Nonetheless, the precise mechanism by which HCMV US28 or HHV-6A U51 may enhance cell-cell fusion is uncertain. For example, while we know that U51 is expressed on the cell surface of transfected 293 cells (9), we cannot be certain whether intracellular U51 may also contribute to the fusogenic effects associated with coexpression of U51. We can, however, exclude an effect on the steady-state expression of VSV-G, since flow cytometric analysis revealed no difference in the levels of VSV-G expression on the cell surface, regardless of the coexpression of U51 (data not shown).
Our current working hypotheses are that (i) U51 and/or US28 may interact with cell membrane-tethered chemokine ligands such as fractalkine (29) and (ii) these viral proteins may form a complex with RANTES and cellular glycoaminoglycans, thereby altering viral infectivity and/or promoting cell-cell fusion (51). Since the ligand repertoire and signaling properties of HHV-6A U51 remain largely unknown, it is also possible that U51 may interact with other chemokines or nonchemokine ligands and/or that it may have the capacity to induce signaling events in either a ligand-specific or a constitutive fashion (akin to HCMV US28 and other herpesvirus 7-tm proteins) (3, 8, 11, 12, 22, 53).
Overall, the studies reported here establish that U51 plays an important role in the replication and spread of HHV-6A. Our work also suggests a possible mechanism for this effect, which may reflect the ability of U51 to enhance cell-cell fusion and thus spread of this highly cell-associated human herpesvirus. Further studies will be required to further examine this hypothesis and to unravel the full ligand binding and signaling properties of HHV-6A U51 as well as to determine whether HHV-6B U51 also has similar activity (34).
ACKNOWLEDGMENTS
We thank Frank Neipel for providing HHV-6A cosmid DNAs, Bryan Cullen for providing pcTat, and Caroline Hall, Mary Caserta, and Jennifer Carnahan for providing valuable reagents and advice. We thank Uta Hoepken for the generous gift of US28.
This work was supported by NIH grants RO1 DE14194 (to S.D.), T32 DE007165 (to B.B.-T.), R21 DA14950 and K05 DA000360 (to J.M.B.), and T32 DA07232 (to S.S.).
These authors contributed equally to this work.
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