Differences in DNA Binding Specificity among Roseolovirus Origin Binding Proteins

Abstract

The Roseolovirus genus of the Betaherpesvirinae consists of the very closely related viruses, human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) plus the somewhat more distantly related human herpesvirus 7 (HHV-7). The roseoloviruses each encode a homolog of the alphaherpesvirus origin binding protein (OBP) which is required for lytic DNA replication. In contrast, members of the other betaherpesvirus genera, the cytomegaloviruses, initiate DNA replication by a different mechanism. To better understand the basis of roseolovirus OBP sequence specificity, we investigated their ability to recognize each other's binding sites. HHV-6A OBP (OBP(H6A)) and HHV-6B OBP (OBP(H6B)) each bind to both of the HHV-7 OBP sites (OBP-1 and OBP-2) with similar strengths, which are also similar to their nearly equivalent interactions with their own sites. In contrast, HHV-7 OBP (OBP(H7)) had a gradient of binding preferences: HHV-7 OBP-2 > HHV-6 OBP-2 > HHV-7 OBP-1 > HHV-6 OBP-1. Thus, the roseolovirus OBPs are not equally reciprocal in their recognition of each other's OBP sites, suggesting that the sequence requirements for the interaction of OBPH7 at the OBP sites in its cognate oriLyt differ from those of OBPH6A and OBPH6B.

Elephant Endotheliotropic Herpesviruses EEHV1A, EEHV1B, and EEHV2 from Cases of Hemorrhagic Disease Are Highly Diverged from Other Mammalian Herpesviruses and May Form a New Subfamily

“…The likely functionality of the U73(OBP) gene within Proboscivirus genomes is strongly supported by the presence of an Ori-Lyt-like dyad symmetry domain in both EEHV1A and EEHV1B within the small noncoding region just upstream from the U41(MDBP) gene and downstream from the U42(MTA) gene as noted originally by Ehlers et al (8) in the EEHV1B(Kiba) genome. This same feature is present in the Kala, Kumari, and Haji data obtained here, as well as in the Kimba, Raman, and Emelia genomes (24,25) and closely resembles the position and organization of the conserved Ori-Lyt domains in the Roseolovirus genus and less so those of typical alphaherpesviruses (33). However, the predicted EEHV1 Ori-Lyt domains at Kimba equivalent map coordinates 67,980 to 68,170 (unlike the HHV6 and HHV7 versions) are all tandemly duplicated with each copy encompassing four potential OBP binding GGTGGAACG motifs (box I/box II) distributed over a 192-bp palindromic struc- ture (not shown).…”

“…Overall, the entire CD-III block extends over 8.3 kb and displays 37% DNA level differences for both Kiba and Haji relative to Kumari, Kala, and Kimba, compared to 44% divergence here for EEHV2 versus EEHV1A. Remarkably, the EEHV1B versions of ORF-M, UDG, gL, ORF-O, ORF-P, and ORF-Q differ by 16,28,31,41,47, and 51% at the DNA level, respectively, and by 14,29,33,34,54, and 70% at the protein level, respectively, from their EEHV1A counterparts, whereas in contrast, the next two genes ORF-K and ORF-L differ by just 1.6 and 2.3% (Table 3).…”

“…Highlevel protein differences are also encountered all across this entire CD-II region whereby these five proteins diverge by 32,20,38,33, and 16% between the EEHV1A and EEHV1B versions (Table 3). Overall, the EEHV1A and EEHV1B subgroups differ here by a total of 32% at the DNA level (1,210 bp out of 3,798 bp), whereas Kiba and Haji themselves differ by just 10 nucleotides (0.3%).…”

Human Cytomegalovirus DNA Replication Requires Transcriptional Activation via an IE2- and UL84-Responsive Bidirectional Promoter Element within ori Lyt

“…For the binding reaction, IE2-FLAG-purified protein (500 ng) was preincubated in 20 l of binding buffer (10 mM Tris-Cl, 0.5 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 50 mM KCl, 8 mM MgCl 2 ; pH 7.5) containing 40 g of poly(dA-dT) ⅐ poly(dA-dT)/ml for 10 min at 20°C. 32 P-labeled probe DNA (20,000 cpm) was then added for 20 min at 20°C. The mixtures were loaded onto 4% nondenaturing acrylamide gels and run at 100 V in 0.5ϫ Tris-borate-EDTA buffer.…”

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.…”