Definition of a Divergent Epitope That Allows Differential Detection of Early Protein p41 from Human Herpesvirus 6 Variants A and B

Abstract

The human herpesvirus 6 (HHV-6) early protein, p41, encoded by the U27 gene has been detected in oligodendrocytes of multiple sclerosis (MS) patients by using a monoclonal antibody (MAb to p41/38). We here report the antigenic epitope of HHV-6 p41 recognized by this MAb. First, we established that the MAb to p41/38 recognizes a nuclear antigen in HHV-6A strain GS-infected cells but not in HHV-6B strain Z29-infected cells. Secondly, we compared the reactivity of the MAb to p41/38 to that of another p41-specific MAb (MAb to p41) on immunoblots with purified p41-glutathione S-transferase fusion protein from strains GS and Z29 and GS- and Z29-infected-cell lysates. The two MAbs were tested in an enzyme-linked immunosorbent assay against a panel of synthetic peptides covering the amino acid substitutions between the GS- and Z29-derived p41 proteins, as determined by DNA sequencing of our cloned isolates of the U27 gene. The MAb to p41/38 reacted specifically with a peptide comprising p41 residues 321 to 340 from strain GS. The critical residue in this peptide was serine 328, as the substitution S328N in the Z29 strain rendered the corresponding peptide nonreactive. The p41 S328 marker was present in three of three HHV-6A strains, while four of four sequenced p41 genes from HHV-6B strains had N328. Our findings are of value for the interpretation of previous findings of p41 expression in brains of MS patients and may allow a more detailed analysis of the role of HHV-6 variants in other disorders.

Human herpesvirus 6 (HHV-6) is a lymphotropic herpesvirus which was first isolated from the peripheral blood lymphocytes of patients with lymphoproliferative disorders and AIDS (29). All HHV-6 isolates can be classified into either of the two major viral variants, designated HHV-6A and HHV-6B, with distinctive genetic, immunologic, and biological traits (2). HHV-6A comprises strains GS (1) and U1102 (12), while HHV-6B strains include HST (34) and Z29 (24). HHV-6B infection is ubiquitous, and primary HHV-6B infection has been etiologically linked to exanthema subitum (34), whereas the clinical consequences of HHV-6A infection, as well as its epidemiology, are less well known. Like human immunodeficiency virus (HIV), HHV-6A and HHV-6B preferentially replicate in CD4⁺ T lymphocytes in vitro, but unlike HIV, CD4 is not a receptor for HHV-6 (25). Instead, the human membrane cofactor protein (CD46) was shown to function as a receptor for HHV-6A and HHV-6B (30). The complete DNA sequences of HHV-6A strain U1102 (14) and HHV-6B strains Z29 (11) and HST (19) have been determined. Although the overall nucleotide sequences of HHV-6A and HHV-6B are 90% identical, clinical data indicate that HHV-6A and HHV-6B may have distinct pathogenic properties, with HHV-6B being associated with the majority of HHV-6 related diseases. HHV-6A and HHV-6B may therefore represent different species within the roseolovirus genus.

The HHV-6 early protein p41 is encoded by the U27 gene, which is conserved in HHV-6A and HHV-6B (9). p41 is a DNA-binding protein and a putative DNA polymerase stimulatory factor (4), similar to the human cytomegalovirus UL44 gene. The protein binds to viral DNA polymerase, thereby greatly increasing the rate of DNA synthesis (23). An additional role for p41 as a transcription factor is suggested by experiments showing that cotransfection of plasmid constructs expressing p41 enhance transcription of chloramphenicol acetyl transferase from reporter plasmids containing the HIV long terminal repeats (35). Two monoclonal antibodies (MAbs), MAb to p41 (7) and MAb to p41/38 (20), have been described to react with both HHV-6A and HHV-6B. Using the MAb to p41/38, expression of p41 was observed in oligodendrocytes of multiple sclerosis (MS) patients but not of controls, suggesting an association of HHV-6 with the etiology or pathogenesis of MS (8). A p41 protein antigen, purified using the MAb to p41/38, has been employed for detection of specific antibodies in studies of MS and other HHV-6-associated syndromes (3, 20, 27, 31). However, there are data indicating that the two proteins, p41 and p38, recognized by the MAb to p41/38 are not both encoded by the viral genome. A synthetic probe for the p38 protein reacts with uninfected cellular DNA, and the sequence of a peptide fragment of p38 (21) shows a strong similarity to human beta-actin (protein AAH02409 in GenBank) but lacks homology to the open reading frames in HHV-6.

Antigenic differences between p41 isolates from different HHV-6 variants have not been reported, and HHV-6 variant-specific serological techniques are not available at present. In this study, we have identified the epitope in p41 which is responsible for the exclusive reactivity of the MAb to p41/38 with HHV-6A. This finding may be of importance to assess the role of HHV-6A and -B in virus-associated diseases.

MATERIALS AND METHODS

Viruses and DNA.

HHV-6 strain GS (HHV-6A GS) (29) was kindly provided by R. Gallo, and HHV-6 strain Z29 (HHV-6B Z29) was from the American Type Culture Collection (VR-1467). The two strains of HHV-6 were propagated in suspension cultures of the human T-cell lines HSB-2 (kindly provided by R. Gallo) and Molt-3 (kindly provided by D. V. Ablashi), respectively. Infected cells and culture supernatants were harvested when 80% of the cells displayed a cytopathic effect. Cell pellets and supernatants were frozen at −70°C. Stock DNAs were prepared from the HHV-6-infected cells and from virus, pelleted from supernatant, using standard techniques (6).

MAbs.

The MAb to p41 (9A5D12, supernatant of cell culture) was kindly provided by Bala Chandran (Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center). The MAb to p41 reacts with both HHV-6A and HHV-6B early protein p41 (7). The MAb to p41/38 (Advanced Biotechnologies Inc., Columbia, Md.) was previously described to react with both HHV-6A and HHV-6B early protein p41 (20).

Patients and CSF samples.

Cerebrospinal fluid (CSF) samples were obtained from three patients with acute HHV-6 infection. Patient 1 (male, 3 years of age) had meningitis. Patient 2 (male, 63 years of age) was a stem cell transplant recipient who presented with a B-cell lymphoma after the transplantation. Patient 3 (male, 5 months of age) had fever and deafness. CSF 1 from patient 1 was positive for HHV-6A, and CSF 2 and 3 from the other two patients were positive for HHV-6B when tested by an HHV-6-subtyping PCR (33) using modified primers. The outer primers were 5′-CAA(G/A)CCCTAACTGTGTA(T/G)GT-3′ and 5′-T(C/T)TGCAATGTAATCA(G/A)TTTC-3′; the inner primers were 5′-CTGGGCGGCCCT(A/G)ATAACTT-3′ and 5′-ATCGCTTTCACTCTCATAAG-3′. The CSF samples were stored at −20°C and were sequenced for the HHV-6 U27 DNA fragment.

Amplification of HHV-6 U27 gene DNA by PCR (PCR 1).

Synthetic primers derived from the U27 gene of HHV-6 strain U1102 (nucleotide X83413 in GenBank) (14) were obtained from Life Technologies, Paisley, England. The corresponding primer sequence for the HHV-6B strain HST (nucleotide AB021506 in GenBank) (19) was the same. Restriction endonuclease recognition sites for BamHI and XhoI were contained in the primers SU27-1 and AU27-1, respectively (Table 1). The PCR cycle was 94°C for 5 min, four cycles of 94°C for 15 s, 52°C for 30 s, and 72°C for 2 min, followed by 26 cycles of 94°C for 30 s and 72°C for 2 min followed by a 7-min final extension at 72°C. PCR DNA products were digested with the restriction endonucleases BamHI and XhoI in Buffer B (Roche Diagnostic Systems, Mannheim, Germany) at 37°C for 4 h. The digested PCR DNA fragments were purified with QIA Quick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.

TABLE 1.

Primers used in PCR for amplifying HHV-6 U27 genes or fragments

PCR round no.	Primer direction	Name	Sequence (5′ to 3′)a	Nucleotide no. in HHV-6 complete DNA sequence
1	Sense	SU27-1	CGTCCTGGATCCATGTGTTGGTCATTTCATTTG	38978-38958, HHV-6A U1102; 40093-40073, HHV-6B Z29
	Antisense	AU27-1	CGTCCTCTCGAGTCAGACGACGCATCTCTG	37796-37814, HHV-6A U1102; 38918-38936, HHV-6B Z29
2	Sense	SU27-2	CGTCCTAAGCTTCCACCATGTGTTGGTCATTTC ATTG	38978-38958, HHV-6A U1102; 40093-40073, HHV-6B Z29
	Antisense	AU27-2	CGTCCTATCGATTCAGACGACGCATCTCTG	37797-37814, HHV-6A U1102; 38919-38936, HHV-6B Z29
3 (outer primers)	Sense	SU27-3O	CCGCTAAGGACTGTAAATTG	38134-38115, HHV-6A U1102; 39250-39231, HHV-6B Z29
	Antisense	AU27-3OA (HHV-6A)	CTCTTGGGATTGGGATTAG	37816-37834, HHV-6A U1102
		AU27-3OB (HHV-6B)	ATGCATGGAATCGTCGCTC	38964-38982, HHV-6B Z29
3 (inner primers)	Sense	SU27-3I	TAACTTCCAAAAGCACGTTGTTGAC	38110-38086, HHV-6A U1102; 39226-39202, HHV-6B Z29
	Antisense	AU27-3IA (HHV-6A)	GTAGTCAAAATGCATGGAATCGTCA	37839-37863, HHV-6A U1102
		AU27-3IB (HHV-6B)	TCGCTCTCGCTATCTTCTTTCTCA	38977-39000, HHV-6B Z29

^aThe underlined parts of the primers are identical to the complete DNA sequences (GenBank) of HHV-6 strains U1102 (nucleotide X83413) and/or Z29 (nucleotide AF157706). 

Construction of pGEX-4T-3/U27 plasmids for expression in Escherichia coli.

The glutathione S-transferase (GST) fusion vector pGEX-4T-3 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used to construct the recombinant plasmid (pGEX-4T-3/U27) and express recombinant p41 in E. coli. The plasmid DNA was digested with the restriction endonucleases BamHI and XhoI at 37°C for 4 h and treated by freezing on dry ice and thawing at 65°C for three cycles, followed by incubating on ice for 30 min. An equal volume of 13% polyethylene glycol 8000–0.8 M NaCl was added, and the DNA was pelleted by centrifugation and dissolved in TE (10 mM Tris-HCl, 1 mM EDTA [pH 7.4]).

The digested U27 PCR DNA fragments from HHV-6 GS or HHV-6 Z29 were inserted into the digested pGEX-4T-3 vector and transformed into E. coli XL-1 Blue cells (Stratagene, La Jolla, Calif.). Positive colonies were selected for U27 inserts by PCR and further screened by restriction enzyme cleavage. Recombinant pGEX-4T-3/U27 plasmids were prepared with a QIAGEN Plasmid Mega kit.

GST fusion protein expression.

E. coli BL-21 cells (E. coli Genetic Stock Center, New Haven, Conn.) were transformed with the recombinant pGEX-4T-3/U27 plasmids from the HHV-6 GS or HHV-6 Z29 strains, and positive colonies were isolated as described above. For GST fusion protein expression, an overnight stationary-phase culture of BL-21 cells harboring the recombinant pGEX-4T-3/U27 was diluted 100-fold in Luria-Bertani medium containing glucose and ampicillin (100 μg/ml) and propagated at 25°C for 2 h. After induction of GST fusion protein expression by adding isopropyl-β-d-thiogalactopyranoside to a final concentration of 0.1 mM, the incubation was continued at 25°C overnight. The bacterial cells were harvested by centrifugation at 2,430 × g for 10 min at 4°C, and the pellets were frozen at −70°C overnight. The cells from 1 liter of culture were thawed, resuspended in 25 ml of lysis buffer (50 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA) supplemented with protease inhibitors (Complete; Roche Diagnostic Systems). The suspension was sonicated in 10-s pulses with intermittent cooling. The sonicated cell lysate was centrifuged at 43,152 × g at 4°C for 20 min, and GST fusion protein was purified from supernatants and pellets, respectively, using glutathione-Sepharose 4B (Amersham Pharmacia Biotech AB) as described previously (18). For GST-p41 fusion protein expression in E. coli in this study, bacterial propagation at 25°C induced the fusion protein, which was expressed mainly in a soluble form and only partly in inclusion bodies. In contrast, incubation at 37°C resulted in p41 fusion protein expression in inclusion bodies.

Immunoblotting.

HHV-6 GS-infected HSB-2 cells, HHV-6 Z29-infected Molt-3 cells, GST-p41 fusion proteins, HSB-2 cell and Molt-3 cell controls, and a GST protein control were lysed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and the samples were processed for immunoblotting as described previously (17). Kaleidoscope prestained standards (Bio-Rad, Hercules, Calif.) were included to determine relative molecular masses. Each sample was loaded on duplicate gels. After SDS-PAGE, the separated proteins were transferred electrophoretically to nitrocellulose membranes. One sheet was reacted with the MAb to p41 and another sheet was reacted with the MAb to p41/38 overnight at 4°C. The two sheets were reacted with goat anti-mouse immunoglobulin G (IgG) (heavy plus light chains) horseradish peroxidase conjugate (Bio-Rad) for 30 min at room temperature (RT). The sheets were reacted with luminol reagents (ECL; Amersham Life Science, Little Chalfont, Buckinghamshire, United Kingdom), and bound secondary antibody was detected by exposing it to X-ray film (Hyperfilm; Amersham Life Science). For detection of GST fusion proteins, a goat anti-GST antibody (Amersham Pharmacia Biotech Inc., Piscataway, N.J.) was also used.

Construction of pLNCX/U27 plasmids for expression in human cells.

HHV-6 U27 DNA (from strain GS or strain Z29) was amplified by PCR (PCR 2) using primers SU27-2 and AU27-2 (Table 1). Primer SU27-2 contains the recognition site of restriction endonuclease HindIII followed by a canonical ribosomal binding motif, CCACC, and primer AU27-2 contains the recognition site for restriction endonuclease ClaI. PCR cycling conditions and DNA fragment preparation were as described above. The U27 PCR DNA fragment was cloned in the pGEM-T vector (Promega, Madison, Wis.), and positive clones were identified as described above. Recombinant pGEM-T/U27 plasmids (from the GS or Z29 strains) were digested with HindIII and ClaI, separated by electrophoresis on 1% agarose (NuSieve GTG agarose; BioWhittaker, Rockland, Maine) containing 10 μg of crystal violet (Sigma, St. Louis, Mo.) per ml, and isolated for cloning as described previously (K. N. Rand, Technical Tips Online, T40022, 1996 [http://tto.biomednet.com]). The U27 DNA fragment was inserted into the pLNCX vector (26) digested with HindIII and ClaI. The resulting recombinant pLNCX/U27 plasmid and pLNCX plasmid control were prepared and purified with the EndoFree Plasmid Maxi kit (Qiagen) according to the manufacturer's instructions.

Expression of recombinant p41 in human cells.

Human embryonic kidney (HEK-293) cells (15) were transfected with the recombinant pLNCX/U27 DNA (from the HHV-6 GS or Z29 strains) and pLNCX plasmid control by calcium-phosphate coprecipitation (10) using piperazine-N,N′-bis(2-ethanesulfonic acid) in place of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer. The cells were incubated at 37°C with 5% CO₂, and medium was changed 12 h after the transfection. The cells were fixed for immunofluorescence with cold methanol-acetone (20:80) at 48 h after the transfection.

Detection of p41 expression by immunofluorescence assay (IFA) using MAbs.

Fixed HHV-6 GS-infected HSB-2 cells, HHV-6 Z29-infected Molt-3 cells, recombinant pLNCX/U27 plasmid (HHV-6 GS or Z29)-transfected HEK-293 cells, HSB-2 and Molt-3 cell controls, and control pLNCX-transfected HEK-293 cells on glass slides were reacted with the MAb to p41 or the MAb to p41/38 at 37°C for 30 min in a moist chamber. The bound MAb was then detected with goat anti-mouse IgG-fluorescein isothiocyanate conjugate at 37°C for 30 min using Evans Blue as a counterstain. Fluorescence was observed using a Nikon fluorescence microscope.

U27 DNA sequencing.

Sequencing reactions were performed with the ABI PRISM BigDye Terminator kit and synthetic oligonucleotide primers, using a GeneAmp PCR System 2400 (Perkin-Elmer, Norwalk, Conn.). Reaction products were precipitated with ethanol-potassium acetate, dissolved in 15 μl of template suppression reagent, and run on an ABI 310 capillary sequencer (Perkin-Elmer, Foster City, Calif.). The U27 DNA sequences of HHV-6 GS and HHV-6 Z29 were translated into amino acid sequences and aligned.

Synthesis of peptides.

To determine the epitope identified by the MAb to p41/38, based on the alignment of the amino acid sequences of HHV-6 p41 proteins determined in this study, six pairs of peptides were synthesized that covered the regions where the amino acid sequence differed between the p41 proteins of HHV-6A GS and HHV-6B Z29. Two additional peptides were synthesized to determine the critical residue in the antigenic peptide recognized by the MAb to p41/38. The amino acid sequences of the 14 synthetic peptides are given in Table 2. The peptides were synthesized by a solid-phase method using 9-fluorenylmethoxy carbonyl reagents. Peptide stock solutions (1 mg/ml) were prepared in phosphate-buffered saline (PBS; pH 7.4) and stored at −20°C.

TABLE 2.

Amino acid sequences of synthetic peptides derived from U27 genes of HHV-6A GS and HHV-6B Z29

Name of peptide	Sequence of amino acid residues (NH2 terminus to COOH terminus)	HHV-6 strain	Location in p41 amino acid sequencea
G-p41-1	HLFFKAHKARVGAR	GS	6–19
Z-p41-1	HLFFKAQKARVGAR	Z29	6–19
G-p41-2	TINKSLKAFAKLLKENATVT	GS	61–80
Z-p41-2	TINKPLKAFTKLLKENTTVT	Z29	61–80
G-p41-3	LFLSDTDRFLTKTI	GS	107–120
Z-p41-3	LFLSDTEHFLTKTI	Z29	107–120
G-p41-4	KVIFYDVKNMRFNL	GS	244–257
Z-p41-4	KVIFYDVNNMRFNL	Z29	244–257
G-p41-5	DDGKGDRSHKNEDESALASK	GS	321–340
Z-p41-5	DDGKGDRNHKNEDGSALASK	Z29	321–340
G-p41-6	DDSMHFDYSSNPNPKRQ	GS	373–389
Z-p41-6	DDSMHFEYSSNPKRQ	Z29	373–387
G-p41-7b	DDGKGDRSHKNEDGSALASK
G-p41-8b	DDGKGDRNHKNEDESALASK

^aThe U27 genes of HHV-6 strain GS and strain Z29 were sequenced and translated into amino acid sequences in this study. 

^bSynthesized to further determine which amino acid residue (S328 or E334) was critical for the antigenic site in peptide G-p41-5. 

ELISA using synthetic peptide antigens.

Enzyme-linked immunosorbent assay (ELISA) plates (Nunc-Immuno plate; NUNC A/S, Roskilde, Denmark) were coated with synthetic peptides in PBS (pH 7.4) and incubated at RT overnight. The plates were blocked with 1% bovine serum albumin in PBS at RT for 1.5 h. The MAb to p41/38 and the MAb to p41 diluted in ELISA dilution buffer (PBS containing 10% fetal bovine serum and 0.05% Tween 20) were added to different wells and incubated at 37°C for 1.5 h. Goat anti-mouse IgG-horseradish peroxidase conjugate was used at a dilution of 1:1,000 with 0.1% (wt/wt) o-phenylenediamine hydrochloride as substrate. The reaction was stopped by adding 2.5 M H₂SO₄, and the absorbance was measured at 490 nm using an automated ELISA reader (E Max; Molecular Devices, Sunnyvale, Calif.).

DNA sequencing of amplified HHV-6 U27 fragments by PCR (PCR 3) from CSF.

The amplified HHV-6 U27 DNA fragments obtained from a nested PCR from CSF samples were sequenced and translated into amino acid sequences to see whether the antigenic peptide recognized by the MAb to p41/38 is conserved in HHV-6A isolates but not conserved in HHV-6B isolates. CSF samples were treated by heating at 95°C for 15 min before 5 μl of CSF was used as the template in the first-round PCR. The outer and inner primers used in the nested PCR are given in Table 1. Thirty cycles were performed in both rounds. Two microliters of PCR product from the first round was used as the template in the second round. The second-round PCR product was ligated into pGEM-T vector, and the recombinant pGEM-T/U27 DNA was used to transfect XL-1 Blue cells. White colonies were tested for inserts by PCR using the inner primers of the nested PCR (PCR 3; Table 1). Purified pGEM-T/U27 plasmid DNA was used as the template for sequencing of the HHV-6 U27 DNA fragments. The primers SU27-31 and AU27-31A were used for HHV-6A and the primers SU27-3I and AU27-3IB were used for HHV-6B.

RESULTS

Selective reactivity of the MAb to p41/38 with U27 gene product from HHV-6A but not from that of HHV-6B.

Our primary IFA experiment indicated that the MAb to p41/38 recognized a nuclear antigen in the HHV-6A GS-infected HSB-2 cells but not in the HHV-6B Z29-infected Molt-3 cells, while the MAb to p41 recognized a nuclear antigen from both strains. The recombinant pLNCX/U27 (from HHV-6A GS or HHV-6B Z29)-transfected HEK-293 cells were tested by IFA using the same MAbs. A nuclear fluorescence was observed using the MAb to p41/38 in the recombinant pLNCX/U27 of HHV-6A GS-transfected HEK-293 cells but not in the recombinant pLNCX/U27 of HHV-6B Z29-transfected HEK-293 cells. Using the MAb to p41, specific nuclear fluorescence was observed in transfected HEK-293 cells expressing pLNCX/U27 from the HHV-6A GS strain as well as from the the HHV-6B Z29 strain, confirming our observation in the infected cells. No fluorescence was observed with either the MAb to p41 or the MAb to p41/38 in HEK-293 cell, HSB-2 cell, or Molt-3 cell controls.

Recombinant GST-p41 fusion proteins (of HHV-6A GS and HHV-6B Z29) purified from E. coli were tested with immunoblotting by using the MAb to p41/38 (Fig. 1A) and the MAb to p41 (Fig. 1B). Two polypeptides of about 67 and 61 kDa were observed by using the MAb to p41 from the GST-p41 fusion protein of HHV-6A GS or HHV-6B Z29. The two polypeptides were observed only with the GST-p41 fusion protein of HHV-6A strain GS and not with that of the HHV-6B strain Z29 when using the MAb to p41/38. This suggests that the epitope recognized by the MAb to p41 was present in the GST-p41 fusion proteins derived from both the HHV-6A GS and the HHV-6B Z29 strains, while the epitope recognized by the MAb to p41/38 was present in the GST-p41 fusion protein of HHV-6A GS but absent in that of HHV-6B Z29. Using a goat anti-GST antibody, the same two polypeptides were detected in SDS-PAGE and immunoblotting experiments, indicating that both fusion proteins were present in roughly the same amounts on the filters.

FIG. 1.

SDS-PAGE and immunoblotting analysis of GST-p41 fusion proteins and HHV-6-infected cell lysates. Molecular sizes are marked on the left. Each sample was loaded on duplicate gels, separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose sheets. Sheets were reacted with the MAb to p41/38 (A) or the MAb to p41 (B). MAb bound to separated polypeptides was detected with goat anti-mouse IgG-horseradish peroxidase conjugate and with a luminol detection system.

HHV-6A GS- and HHV-6B Z29-infected cells were tested by using SDS-PAGE and immunoblotting with the MAb to p41/38 (Fig. 1A) and the MAb to p41 (Fig. 1B). A polypeptide of about 41 kDa was detected using either of the two MAbs in HSB-2 cells infected with HHV-6A GS, and a less intense band of 41-kDa polypeptide was detected in Molt-3 cells infected with HHV-6B Z29 at day 2 postinfection (p.i.) by using the MAb to p41 but not by using the MAb to p41/38. An intense band of 41-kDa polypeptide was detected using the MAb to p41 from the HHV-6B Z29-infected Molt-3 cells at day 7 p.i., but the 41-kDa polypeptide was still not detectable at this time using the MAb to p41/38. Either of the two MAbs detected an intense band at 41-kDa and additional smaller polypeptides, possibly representing p41 degradation products in the HHV-6A GS-infected HSB-2 cells at day 7 p.i. No bands were detected using either of the two MAbs from the GST protein, HSB-2 cell, or Molt-3 cell controls. Thus, the reactivity of the two MAbs with the HHV-6-infected-cell lysates and the recombinant pLNCX/U27-transfected cells in the immunoblotting experiments was consistent with the observations in IFA.

In view of the fact that the MAb to p41/38 can coimmunoprecipitate a p38 antigen (21) that is likely to be beta-actin, we tested this interaction with an ELISA using a highly purified complex of beta-actin and tropomyosin (generously donated by U. Lindberg). Although binding of the MAb to the antigen could be demonstrated at high concentrations of antibody, we concluded that the interaction was weak in comparison to the reactivity with HHV-6 p41 and did not significantly influence our assays on whole-cell lysates from infected cells.

Genetic divergence of HHV-6 U27 gene between HHV-6 strains A and B.

The U27 genes of HHV-6A strain GS and HHV-6B strain Z29 were 95% (1,124 of 1,182) and 94% (373 of 393) identical on the DNA and amino acid levels, respectively. The DNA sequences of the GS and Z29 U27 genes determined in this study were almost identical to the reported sequences for the HHV-6A strain U1102 (1,175 of 1,182 nucleotides identical) and the HHV-6B strain HST (1,172 of 1,176 nucleotides identical). There was a deletion of six base pairs (two amino acid residues) in the 3′ end of the U27 gene of HHV-6B strain Z29 compared to that of the HHV-6A strain GS.

Identification of the epitope recognized by MAb to p41/38.

Twelve synthetic peptides (Table 2) were derived from the regions of U27 where amino acid differences were found by us in this study between HHV-6A strain GS and HHV-6B strain Z29. The synthetic peptides were tested using the MAb to p41 and the MAb to p41/38, respectively, in an ELISA. The MAb to p41/38 reacted only with the synthetic peptide G-p41-5 (DDGKGDRSHKNEDESALASK), which was derived from HHV-6A strain GS, but not with the corresponding synthetic peptide Z-p41-5 (DDGKGDRNHKNEDGSALASK), which was derived from HHV-6B strain Z29 (Fig. 2). The MAb to p41 did not react with any of the 12 synthetic peptides selected in this study, indicating that its recognized peptide is located elsewhere in p41. There were two substitutions (S328N and E334G) in the peptide Z-p41-5 compared to the peptide G-p41-5. To further determine which of the two amino acids (S328 or E334) was critical for the antigenic peptide G-p41-5, we synthesized another two peptides, G-p41-7 and G-p41-8 (Table 2). G-p41-7 differs from G-p41-5 by the substitution E334G, and the G-p41-8 differs from the G-p41-5 by the substitution S328N. The MAb to p41/38 reacted with G-p41-7 but not G-p41-8, demonstrating that S328 is critical for antigenic discrimination by the MAb to p41/38. The amino acid residue S328 is conserved in the p41 amino acid sequences of HHV-6A strains GS (protein JQ2007 in GenBank) (4) and U1102 (protein CAA58407 in GenBank). The substitution S328N is conserved in the p41 amino acid sequences of HHV-6B strains Z29 (protein AAD49641 in GenBank) (10) and HST (protein BAA78248 in GenBank) (19).

FIG. 2.

Reactivity of the MAb to p41/38 and the MAb to p41 with synthetic peptides, as detected by ELISA. Wells were coated with synthetic peptides at 10 μg/ml of PBS (pH 7.4) at RT overnight, followed by blocking with 1% bovine serum albumin in PBS. The MAb to p41 was diluted 1:10 and the MAb to p41/38 was diluted 1:3,000 in PBS containing 10% fetal bovine serum and 0.05% Tween 20. Bound MAb was detected by goat anti-mouse IgG-horseradish peroxidase conjugate. Color was developed using o-phenylenediamine solution and was measured at 490 nm.

HHV-6 U27 sequences from patient isolates.

To test whether the antigenic difference at p41 residue 328, described above, could be observed also in direct isolates of HHV-6, we designed a nested PCR for the U27 gene and sequenced the amplified fragments from three CSF samples, which had been previously typed as HHV-6A or HHV-6B by using the modified HHV-6 subtyping PCR (see Materials and Methods). The CSF from patient 1 was positive for HHV-6A and negative for HHV-6B, while the CSF samples from patient 2 and patient 3 were positive for HHV-6B and negative for HHV-6A in the nested PCR for HHV-6 U27 developed in the present study. The sequences of the PCR fragments showed that S328 was present in the HHV-6A isolate (Fig. 3, CSF1), while the two HHV-6B isolates (CSF 2 and 3) had N328.

FIG. 3.

Parts of nucleotide sequences of the amplified U27 DNA fragments from patient CSF samples and the deduced amino acid sequences. The numbers on the left side show the nucleotide position, in reference to the complete DNA sequences (GenBank) of HHV-6A strain U1102 (nucleotide X83413) or HHV-6B strain Z29 (nucleotide AF157706). The parts in the amino acid sequences that correspond to the synthetic peptides G-p41-5 or Z-p41-5 are underlined. Patient 1 was previously diagnosed with acute HHV-6A infection; patients 2 and 3 were diagnosed with acute HHV-6B infection. Serine 328, which was critical for the antigenic reactivity of the peptide to the MAb to p41/38, was present in the p41 sequence isolated from CSF 1, and asparagine 328 was found in the sequences from CSF 2 and 3.

DISCUSSION

Some studies point to an association between MS and the expression of two HHV-6 antigens in oligodendrocytes of afflicted patients. Challoner et al. (8) observed expression of the HHV-6 nuclear protein p41 and the 101-kDa HHV-6B virion protein p101 in the nuclei of oligodendrocytes in 12 of 15 MS patients, but not in 41 non-MS patients or in 4 stillborn fetal brains. These studies employed indirect immunofluorescence with two MAbs, MAb C5, which is directed against the DNA binding protein p41 (MAb to p41/38) (4, 20), and a MAb to the HHV-6B virion protein p101. The former antibody was here shown to selectively react with p41 from HHV-6A, while the latter reportedly reacts with p101 from HHV-6B but not with the HHV-6A homologue (28). In these MS patients, the nuclear fluorescence was observed around plaques more frequently than in uninvolved white matter. Since destruction of oligodendrocytes is a hallmark of MS, these findings suggested an association of HHV-6 with MS. The study by Challoner et al. (8) concluded that HHV-6B is expressed in the oligodendrocytes of MS patients. The expression of HHV-6B virion protein p101 was also observed by Friedman et al. (13) in 47% (7 of 15) of MS patient brains, while in none of 16 controls. In contrast, another study (A. R. M. Coates and J. Bell, Letter, Nat. Med. 4:537–538, 1998) found no evidence for HHV-6 antigen expression in MS plaques from brain biopsies of 23 MS patients using the MAb to the 101-kDa HHV-6B virion protein p101. The selection of both MS and control patient groups may influence the significance of correlations found between HHV-6 and MS, as discussed by Fillet et al. (A. M. Fillet, P. Lozeron, H. Agut, O. Lyon-Caen, and R. Liblau, Letter, Nat. Med. 4:537, 1998). Three groups (8, 13; Coates and Bell, letter) have studied HHV-6 in brain sections using both PCR-based methods and immunocytochemistry. The fact that two studies reported a positive correlation (8, 13) and one study found no correlation (Coates and Bell, letter) may be due to such a bias in the selection of patients or to differences in the protocols for specimen preparation or immunocytochemical staining.

However, our present finding that the MAb to p41/38 reacts only with p41 of HHV-6A implies that Challoner et al.'s findings support an association between both HHV-6A and HHV-6B and MS. HHV-6B has been claimed to be a commensal virus in MS since it can be detected in the brains of healthy individuals (8). Some recent evidence also indicates a possible association between HHV-6A and MS. Soldan et al. (32) described a significantly elevated lymphoproliferative response to HHV-6A in MS patients compared with that in healthy controls, whereas the lymphoproliferative responses to HHV-6B in MS patients were similar to those in healthy individuals. Kim et al. (22) reported that HHV-6 DNA sequences were found by PCR in peripheral blood mononuclear cells from 7 of 34 MS patients and 2 of 6 patients with idopathic transverse myelitis, but none of 20 healthy controls. The genomic sequences in these positive cases were all from HHV-6A, as determined by PCR product restriction fragment length polymorphism. Akhyani et al. (5) recently reported that HHV-6A was predominantly detected in cell-free compartments (serum and urine) of patients with MS. Detection of cell-free HHV-6 DNA and the presence of an increased cellular immune response to the HHV-6A variant in patients with MS suggests that frequent reactivation occurs and may contribute to disease pathogenesis. Little is known about the epidemiological distribution of HHV-6A at present, and one study indicates that HHV-6A is more neurotropic and that HHV-6A infection remains uncommon into adulthood (16). To determine the potential role of HHV-6A in the pathogenesis of MS, further investigation is clearly needed.

Antigen purified by the MAb to p41/38 (20) has been used for detection of specific IgG and IgM antibodies in other studies (3, 20, 27, 31). The MAb to p41/38 was developed against HHV-6 GS-infected cells. It has been assumed that the MAb to p41/38 detects p41 from HHV-6A as well as from HHV-6B strains. In the present study, the p41 from the HHV-6 strain GS was readily detected by immunofluorescence and immunoblotting using either the MAb to p41/38 or the MAb to p41. However, the p41 from the HHV-6B strain Z29 was detected only with the MAb to p41 (Fig. 1). Bacterially expressed p41-GST fusion proteins and cell lysates of HHV-6-infected cells showed the same pattern of reactivity with the MAbs, pointing to an antigenic difference between the p41 proteins of the two HHV-6 strains. This finding prompted us to determine the specific epitope in p41 that is recognized by the MAb to p41/38. Of the six pairs of synthetic peptides covering the nonconservative amino acid changes between the p41 proteins of the GS and Z29 strains, only one, G-p41-5, was recognized by the MAb to p41/38. A serine-asparagine substitution at p41 amino acid 328 (S328N) was shown to account for the difference in antigenic reactivity of this peptide to the MAb to p41/38. The reactive serine 328 is present in HHV-6A strains GS and U1102, while asparagine 328 occurs in the HHV-6B strains Z29 and HST. In addition, the asparagine 328 marker was found in p41 sequences amplified from the CSF samples of two patients with acute HHV-6 infection, previously typed as HHV-6B, while the amplified p41 sequence from a child with HHV-6A-associated meningitis showed a serine at position 328. These findings indicate that the antigenic epitope surrounding residue 328 may allow discrimination between HHV-6A and HHV-6B.

Our results also underline the need for reviewing some of the data on p41 antigen expression in MS patients and point to the possibility of developing an HHV-6 variant-specific peptide ELISA as a means to investigate the epidemiology and pathology of HHV-6A and HHV-6B infections, especially in the context of an association with MS and other HHV-6-associated disease syndromes. Our studies also underscore the need for a careful definition of the antigenic specificity of MAbs, particularly when they are used for diagnostic purposes. Our findings specifically suggest the need for a similar investigation of other MAbs currently being used for investigating the link between HHV-6 and MS, such as the MAb to the 101-kDa HHV-6B virion protein p101 (13, 28).