Sequence Requirements for Interaction of Human Herpesvirus 7 Origin Binding Protein with the Origin of Lytic Replication

Abstract

As do human herpesvirus 6 variants A and B (HHV-6A and -6B), HHV-7 encodes a homolog of the alphaherpesvirus origin binding protein (OBP), which binds at sites in the origin of lytic replication (oriLyt) to initiate DNA replication. In this study, we sought to characterize the interaction of the HHV-7 OBP (OBP H7 ) with its cognate sites in the 600-bp HHV-7 oriLyt. We expressed the carboxyl-terminal domain of OBP H7 and found that amino acids 484 to 787 of OBP H7 were sufficient for DNA binding activity by electrophoretic mobility shift analysis. OBP H7 has one high-affinity binding site (OBP-2) located on one flank of an AT-rich spacer element and a low-affinity site (OBP-1) on the other. This is in contrast to the HHV-6B OBP (OBP H6B ), which binds with similar affinity to its two cognate OBP sites in the HHV-6B oriLyt. The minimal recognition element of the OBP-2 site was mapped to a 14-bp sequence. The OBP H7 consensus recognition sequence of the 9-bp core, BRTYCWCCT (where B is a T, G, or C; R is a G or A; Y is a T or C; and W is a T or A), overlaps with the OBP H6B consensus YGWYCWCCY and establishes YCWCC as the roseolovirus OBP core recognition sequence. Heteroduplex analysis suggests that OBP H7 interacts along one face of the DNA helix, with the major groove, as do OBP H6B and herpes simplex virus type 1 OBP. Together, these results illustrate both conserved and divergent DNA binding properties between OBP H7 and OBP H6B . Human herpesvirus 7 (HHV-7) is a widely prevalent betaherpesvirus with an in vitro tropism for CD4ϩ T lymphocytes (reviewed in reference 5). HHV-7 is usually acquired during early childhood after HHV-6 infection and is likely transmitted via saliva (29). HHV-7 has been associated with febrile illnesses in children, neurological manifestations during primary infection, and clinical complications in organ transplant patients.The betaherpesvirus subfamily consists of the cytomegaloviruses and the roseoloviruses (HHV-7 and HHV-6 variants A and B [HHV-6A and HHV-6B]). Their genomes are genetically colinear, with origins of lytic replication (oriLyts) in analogous positions upstream from U41, the gene encoding the major DNA binding protein (1,7,11,23,27). However, sequence features of their oriLyt regions indicate substantial differences between cytomegaloviruses and roseoloviruses in their mechanisms for initiating viral replication. The minimal oriLyt regions of the cytomegaloviruses are long (Ͼ1.3-kb) complex structures consisting of multiple inverted and direct repeats, in addition to numerous transcription factor recognition sites that are believed to mediate activation of the replication origin (2,20,21,28). Unlike the cytomegaloviruses, oriLyts of HHV-6A and HHV-6B have features in common with the replication origins of alphaherpesviruses; these origins are less complex and are centered around binding sites for a virus-encoded replication initiator protein, the OBP.Roseoloviruses each encode a homolog of the alphaherpesvirus OBP (14,16,23), which has no homolog in the cytomegaloviru...

Characterization of the Lymphotropic Amplicons-6 and Tamplicon-7 Vectors Derived from HHV-6 and HHV-7

“…The DRs are approximately 8 to 10 Kb in size, and contain the pac-1 and pac-2 cleavage-packaging signals, as well as imperfect and perfect reiterations of telomeric-like GGGTTA sequence [Berneman et al, 1992a;Frenkel, 1995;Martin et al, 1991;Nicholas, 1996;Pellett, 2002;Secchiero et al, 1995;Singer and Frenkel, 1997;Thomson, Dewhurst, and Gray, 1994]. The viral genome contains a single DNA replication origin designated oriLyt, found to operate during the lytic viral replication phase [Dewhurst et al, 1993a;Dewhurst, Krenitsky, and Dykes, 1994;Krug, [Levine et al, 1992] PBMC Cuende et al, 1994;Jarrett et al, 1990] macrophages and monocytes [Kondo et al, 1991] CD4+/CD8-and CD3+/CD4+ mature T lymphocytes [Takahashi et al, 1989] kidney tubule endothelial cells [Okuno et al, 1990] salivary glands Fox et al, 1990;Jarrett et al, 1990;Krueger et al, 1990;Ranger-Rogez et al, 1995] CNS, neurons and oligodenrocytes [Challoner et al, 1995b;Luppi et al, 1994;Luppi et al, 1995] HHV-6 lesions of Langerhans cell histiocytosis [Leahy et al, 1993] CD4+ lymphocytes (rescue from latent infection). [Frenkel et al, 1990c] Persistent infection of salivary glands in > 80% of healthy adults.…”

“…In similarity with other herpesviruses a major control of DNA replication involves the binding of the roseolovirus origin binding proteins (OBPs) to the oriLyt, initiating the process of DNA replication [Inoue et al, 1994;Krug, Inoue, and Pellett, 2001]. Significant data are available with regards to this process.…”

“…The genes, which have functions homologous to the 7 HSV-1 essential DNA replication functions, are indicated and placed on the map. Inoue, and Pellett, 2001;van Loon et al, 1997]. Defective genomes produced from engineered amplicons are approximately full size.…”

“…Coordinates are derived from HHV-7 (RK) and HHV-6B (Z29). This Figure is based on [Borenstein et al, 2004;Dewhurst et al, 1993a;Dewhurst, Krenitsky, and Dykes, 1994;Dykes et al, 1997;Inoue et al, 1994;Inoue and Pellett, 1995;Krug, Inoue, and Pellett, 2001;Nicholas, 1996;Romi et al, 1999;Stamey et al, 1995;van Loon et al, 1997].…”

Stepwise Evolution of the Herpes Simplex Virus Origin Binding Protein and Origin of Replication

“…A number of studies have attempted to define amino acids involved in DNA binding (18 -21). In addition, sequence comparisons between alphaherpesviruses and roseoloviruses have helped to identify amino acids in OBP potentially involved in DNA binding as well as corresponding recognition sequences in origins of DNA replication (22)(23)(24)(25). However, a comprehensive and quantitative study of evolutionarily conserved amino acids required for DNA binding is still lacking.…”