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 GSand 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.

HHV‐6 A‐ or B‐specific P41 antigens do not reveal virus variant‐specific IgG or IgM responses in human serum

“…The recombinant protein p41-GST-G was readily detected by immunoblotting using either of the mAb to p41/38 and the mAb to p41, while the recombinant protein p41-GST-Z was detected only with the mAb to p41 [Xu et al, 2001]. The reactivity with monoclonal antibodies was con®rmed in this study using the recombinant p41 proteins as antigens in ELISA.…”

“…The protocols for construction of pGEX-4T-3/U27 plasmids from the HHV-6A strain GS and the HHV-6B strain Z29, expression of the recombinant p41 proteins as glutathione-S-transferase (p41-GST) fusion proteins in Escherichia coli (E. coli), puri®cation of the proteins using glutathione-sepharose 4B, and the reactivities of the recombinant p41 proteins (p41-GST-G and p41-GST-Z) with monoclonal antibodies were previously described [Xu et al, 2001]. p41-GST-G was recognised both by the mAb to p41 [Balachandran et al, 1989] and the mAb to p41/38 [Iyengar et al, 1991].…”

“…p41-GST-Z was recognised by the mAb to p41 but not by the mAb to p41/38 in immunoblotting, while the GST control antigen did not react with any of the two mAbs. The antigenic epitope recognised by the mAb to p41/38 was found within the amino acid sequence of the p41 protein from HHV-6A but not from HHV-6B [Xu et al, 2001]. The recombinant p41 antigens and the GST control antigen were stored at À208C.…”

“…Serum IgM or IgG reactivity to a p41 antigen has been claimed to be a sign of active HHV-6 replication [Iyengar et al, 1991;Patnaik et al, 1995;Ablashi et al, 2000]. However, the protein p38 also recognised by the mAb to p41/38 is likely to be of cellular origin, since the sequence of a peptide fragment of p38 [Iyengar et al, 1994] has a strong similarity to human beta-actin (protein AAH02409 in GenBank) and lacks homology to the open reading frames in HHV-6 [Xu et al, 2001]. An alternative p41 antigen is needed to verify the reported ®nding.…”

“…Distinctive antigenic epitopes have been found in p41 proteins from HHV-6A and HHV-6B [Xu et al, 2001].…”