Sequence requirements for interaction of human herpesvirus 7 origin binding protein with the origin of lytic replication.
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).
Krug
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Microbiology and Molecular Genetics Program, Emory University, Atlanta, Georgia, USA.
Inoue
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Pellett
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D016384:Consensus Sequence / Q000235:genetics; D004261:DNA Replication; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016199:Herpesvirus 7, Human / Q000235:genetics / Q000378:metabolism; D020180:Heteroduplex Analysis; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D018741:Replication Origin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013816:Thermodynamics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012367:RNA, Viral; D014764:Viral Proteins; C055789:origin-binding proteins, viral