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Figure 1 Gene organization of HHV-6 and HHV-7. Gene blocks I—VII containing genes conserved in all herpesviruses are marked in relative positions in HHV-6 and HHV-7. The ORFs encompassed in each block are as follows: I, U27-U37; II, U38-U41; III, U42-U45; IV, U48-U53; V, U56-U58; VI, U60-U77; VII, U81-U82. HHV-6 ori-lyt is indicated by an arrowhead. Representative genes encoded in the conserved and unique regions are listed. Abbreviations are: PPF, DNA polymerase processivity factor; RR, ribonucleotide reductase; mCP, minor capsid protein; Teg, large tegument protein; Pol, DNA polymerase; gB, glycoprotein B; MDBP, major single-stranded DNA-binding protein; Pr, primase; dUT, dUTPase; gH, glycoprotein H; pts, protease/assembly protein; MCP, major capsid protein; PT, phosphotransferase; gM, glycoprotein M; OBP, origin-binding protein; H-P, helicase-primase associated factor; Hel, helicase; UDG, uracil-DNA glycosylase; gL, glycoprotein L; Rep, AAV-2 rep gene homologue.

tion of a rep-deficient AAV-2 genome. There is no rep homologue in HHV-7, suggesting the acquisition of the gene by HHV-6 after the evolutionary divergence between HHV-6 and HHV-7.

Proteins of HHV-6 and HHV-7

In HHV-6, potentially protein-coding open reading frames (ORFs) are numbered by the order occurring in the U and DR regions of the genome from left to right. Since almost all ORFs of HHV-7 encode homo-logues of HHV-6 and are present in the corresponding location in the genome, common numbers have been adopted for naming the homologous HHV-7 ORFs. For example, an ORF encoding the DNA polymerase of HHV-6 is the 38th, numbering from the left of the U, and therefore designated ORF U38, which is also used for indicating that of the DNA polymerase of HHV-7.

Proteins involved in DNA replication

The elements essential and sufficient for lytic HSV-1 DNA replication are an origin for lytic phase replication (ori-lyt) and seven viral gene products: the major single-stranded DNA-binding protein (MDBP), the DNA polymerase, a DNA polymerase processivity factor and an origin binding protein (OBP), as well as a helicase-primase complex composed of three proteins (helicase, primase, helicase-primase associated factor). HHV-6 and HHV-7 encode homologues to all of the seven gene products. In HHV-6 and HHV-7, these genes correspond to ORFs U41, U38, U27, U73,

U77, U43 and U74, respectively. An interesting feature is that HHV-6 and HHV-7 encode a homologue of herpes simplex virus type 1 (HSV-1) OBP, which has been found only in alphaherpesviruses. HHV-6 has an origin for lytic-phase DNA replication that, as in HSV-1, varicella-zoster virus (VSV) and HCMV, is located upstream of the gene encoding the MDBP homologue (U41). The minimal active region of HHV-6B ori-Lyt is about 400 bp. The HHV-6B ori-lyt includes two binding sites, OBP-1 and OBP-2, for the HHV-6B OBP. The OBP sites are arranged with dyad symmetry, flank an AT-rich spacer, and are within the minimum essential origin region. This region is adjacent to a larger AT-rich imperfect direct repeat of about 195 bp that has been proposed to act as a DNA-unwinding element. Both OBP sites are required for DNA replication. Thus, the HHV-6 ori-lyt has features of both beta- and alphaherpesvirus origins, in having a larger set of repeat motifs (beta, 'complex' ori) and sequences which bind the OBP (alpha ori).

HHV-6 and HHV-7 homologues of herpesvirus gene products that are involved indirectly in DNA replication, by providing nucleotide substrates and DNA repair functions, include ribonucleotide reductase (U28), dUTPase (U45), phosphotransferase (U69), alkaline exonuclease (U70) and uracil-DNA glycosylase (U81). Homologues of the thymidine kinase genes found in the alpha- and gammaherpes-viruses are absent in HHV-6 and HHV-7, as well as HCMV. The phosphotransferase encoded by UL97 in HCMV is capable of phosphorylating nucleosides, an activity that accounts for the efficacy of ganciclovir in suppressing HCMV infections. The in vitro susceptibility of HHV-6 to ganciclovir may also be attributed to the function of the phosphotransferase (U69). The dUTPase and uracil-DNA glycosylase homologues of HHV-6 and HHV-7 presumably specify enzymatic activities involved in excision of uridine residues from DNA, by analogy to bacterial and eukaryotic counterparts, although there is notable sequence divergence between the HHV-6 or HHV-7, cellular and other herpesvirus dUTPase homologues.

Glycoproteins

There are several glycoprotein genes conserved in all sequenced herpesviruses. These genes are glycoprotein B (gB), glycoprotein H (gH), glycoprotein M (gM) and glycoprotein L (gL), and also are conserved in HHV-6 and HHV-7. These genes correspond to U39, U48, U72 and U82, respectively. gB and gH are structurally highly conserved between herpesviruses, are membrane-bound glycoproteins, and appear to play roles in virus cell fusion and cellular spread of virus infection. gL forms a physical association with gH precursor protein and may be required for gH transport and/or processing. The gM is an integral membrane protein containing multiple hydrophobic, putative membrane-spanning domains and fairly well conserved between herpesviruses. It has been shown to be a component of the virus particle in HSV-1.

In HHV-6, a highly spliced gene at the right end of the U component of the genome has recently been characterized and shown to encode a glycoprotein species, gp82/105. In strain GS, this gene contains 13 exons. The role of gp82/105 is not known but is likely to be important in the biology of HHV-6, as neutralizing epitopes are present. Three HHV-7 ORFs (U98, U99 and U100) show homology to regions of the gp82/105 amino acid sequence.

Glycoprotein U18 appears to be betaherpesvirus-specific. In HCMV this gene is spliced and encodes an immediate early glycoprotein.

HHV-6 U20 exhibits significant sequence similarity to the immunoglobulin E (IgE) C chain and therefore is a member of the Ig superfamily. The U85 genes of HHV-6 and HHV-7 encode homologues of OX-2 membrane antigen, which is also a member of the Ig family. Members of the Ig family have roles in cell-to-cell contact via protein-protein interactions and it is postulated that expression of these glycoproteins will function in infected cell adhesion to uninfected lymphocytes or monocytes.

Glycoproteins U21, U23, U24 appear to be unique to HHV-6 and HHV-7. There are glycoproteins en coded in a similar location in HCMV, but they share no detectable similarity.

Capsid, tegument and virus assembly proteins

Several of the ORFs identified in HHV-6 and HHV-7 are homologous to herpesvirus genes encoding characterized and candidate structural proteins. These include homologues of the major capsid protein (MCP; U57), minor capsid protein (mCP; U29), large tegument protein (U31) and virion proteins specified by U33, U34, U36, U50, U56 and U76. Betaherpes-virus-specific and conserved structural proteins are homologues of HCMV UL32 (antigenic phosphopro-tein, ppl50) and UL82/83 (tegument transactivator, pp65/72K), which correspond to Ull and U54, respectively. The HHV-6 Ull gene product is called pplOO and has been characterized as a major antigenic phosphoprotein and a component of the virion. The homologues of herpesvirus gene products involved in DNA packaging and capsid assembly have also been identified in HHV-6 and HHV-7. These correspond to U29 (mCP), U30, U53 and U60/U66. These genes appear to be conserved (at least positionally) in all of the sequenced herpesviruses. The U60/U66 ORFs correspond to the two exons of the late spliced gene characterized initially in HSV-1, which is likely to play a role in DNA packaging and capsid assembly. U53 sequences code for the protease/assembly protein (assemblin) and the scaffolding protein. These proteins are derived through proteolytic cleavage (assemblin) and internal initiation (scaffolding protein). The sequences encoding the scaffolding protein are referred to as U53a and correspond to HSV-1 UL26.5. From studies on HSV-1 UL26/26.5 viral mutants, it appears that proteolytic activity is essential for DNA packaging and virus assembly.

Chemokine receptors

HHV-6 and HHV-7 contains two genes (U12 and U51) that encode putative homologues of cellular G-protein-coupled receptors (GCR). GCR homologues have also been identified in HCMV, as well as in the gammaherpesviruses herpesvirus saimiri (HVS) and Kaposi's sarcoma-associated herpesvirus (KHSV or HHV-8). The U12 gene of HHV-6 is expressed late in infection from a spliced mRNA. U12 functionally encodes a calcium-mobilizing receptor for /?-chemo-kines, such as regulated upon activation, normal T expressed and secreted (RANTES), macrophage inflammatory proteins la and If} (MlP-la and MIP-1/5) and monocyte chemoattractant protein 1, but not for the a-chemokine interleukin 8, suggesting that the chemokine selectivity of the U12 product is distinct from that of the known mammalian chemokine receptors. These findings suggest that the product of U12 may play an important role in the pathogenesis of HHV-6 through transmembrane signaling by binding with /J-chemokines.

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