School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, UK1
Author for correspondence: Joe Conner.Fax +44 141 331 3208. e-mail J.Conner{at}gcal.ac.uk
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Amongst the alpha- and gammaherpesviruses, the R1 and R2 subunits are highly conserved and key catalytic residues are readily identifiable (Willoughby et al., 1997 ). In protein R1 certain residues are absolutely conserved including five catalytically active cysteines, three that are grouped in the active site and two that are located at the C terminus, and a pair of tyrosines, involved in electron transfer from the radical centre in R2 to the R1 active site cysteines (Ekberg et al., 1996
). DNA sequencing of the human betaherpesviruses, human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7) has identified an ORF with homology to R1 (Chee et al., 1990
; Gompels et al., 1995
; Nicholas, 1996
; Megaw et al., 1998
) but an ORF for R2 is absent, suggesting that these viruses do not express a functional RR enzyme. We report here on the cloning, expression and characterization of the proposed R1 protein (U28) from HHV-7 and our results demonstrate that the polypeptide is not a functional R1 protein.
Submission of the U28 amino acid sequence to database searches identifies the protein as an R1 homologue with the closest matches occurring amongst other herpesvirus R1 subunits. Initially therefore, we aligned the amino acid sequence of U28 with that of the equine herpesvirus-4 (EHV-4) R1, chosen as a representative of herpesvirus R1 proteins (Fig. 1). Although a 22% amino acid identity exists between the two sequences the alignment demonstrates that essential catalytic residues are absent from the HHV-7 polypeptide. Significantly, only one of the five essential cysteines (EHV-4 amino acid 223) is conserved with the remaining four (EHV-4 amino acids 452, 462, 784 and 787) absent from the U28 sequence. The proposed GxGxxG nucleotide-binding site (EHV-4 amino acids 517522) and the C- terminal tyrosine pair (EHV-4 amino acids 764 and 765) are not conserved in U28. All of these residues are conserved absolutely in Class 1 R1 proteins including those from the alpha- and gammaherpesviruses for which sequencing information is available. The catalytic roles of these residues have been defined in the E. coli RR but their precise functions in herpesvirus RR have not been addressed directly. Two HSV-1 R1 amino acids, Pro-1090 and Ala-1091, are involved directly in its interaction with R2 (Bonneau et al ., 1996
), forming part of the subunit interface helix. Although the role of the equivalent residues in EHV-4 R1, Pro-743 and Ala-744 (in bold and underlined in Fig. 1
), has not been defined directly, they are well-conserved in other herpesvirus R1 proteins suggesting a common purpose. Importantly, these amino acids are absent from U28. Thus, amino acid comparisons suggest that U28 is most unlikely to be the functional equivalent of R1 and we confirmed this by its cloning, expression and characterization.
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Our results clearly demonstrate that the U28 protein is not the functional equivalent of the R1 subunit of herpesvirus RR. The cloned and bacterially expressed protein was unable to complex with the R2 subunits from HSV-1 and EHV-4 in both RR activity and subunit interaction assays. The sites of subunit interaction, particularly at the R2 C terminus, are well-conserved amongst alpha- and gammaherpesviruses and in our assay system both HSV-1 and EHV-4 R1 were able to form hetero-subunit complexes with EHV-4 and HSV-1 R2 (see Fig. 3A). Importantly, any interaction of U28 with a R2 subunit, including the remote possibility of interaction with the host cell R2, would not result in a functional RR as most of the key catalytic residues essential for R1 activity are absent from U28. It is highly improbable also that U28 forms a single subunit RR as other single subunit enzymes are restricted to bacteria and archaea and we could detect no RR activity with U28 in our assay system. However, interaction with other mammalian cell proteins (known or unknown) to form an active RR complex cannot be completely ruled out.
We analysed U28 of HHV-7 as a representative of the R1 homologues identified also in HCMV and HHV-6 and we conclude that betaherpesviruses have no requirements for RR activity. The absence of this key nucleotide metabolizing enzyme is most probably a consequence of the biological properties of this group of viruses: in particular, their restricted host ranges and long replication cycles. We propose that during the evolution of the betaherpesvirus subfamily there was minimal requirement for a virus-encoded RR and, consequently, the R2 ORF was lost, allowing the R1 ORF to mutate and develop another, as yet unidentified, function. Interestingly, HSV-1 R1 possess a unique N- terminal extension of 300 amino acids that plays an additional role in virus replication (Conner et al., 1995 ; Smith et al., 1998
) and the function of the U28 protein may be related to this. The availability of the bacterially expressed and purified U28 will allow its further characterization and the development of specific antibody reagents to address more fully the function of this protein during HHV-7 infection.
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References |
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Received 17 May 1999;
accepted 7 July 1999.