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Human herpesvirus 6B origin-binding protein: DNA-binding domain and consensus binding sequence.

ABSTRACT: We previously demonstrated by a DNA-binding assay that the human herpesvirus 6B (HHV-6B) replication origin has a structure similar to those of alphaherpesviruses, although the HHV-6B and herpes simplex virus type 1 (HSV-1) origin-binding proteins (OBPs) and origins are not interchangeable. Here we describe additional properties of the interaction between HHV-6B OBP and the HHV-6B origin. Competitive electrophoretic mobility shift assays (EMSAs) with DNA duplexes containing single-base alterations allowed deduction of a consensus DNA sequence for HHV-6B-specific OBP binding, YGWYCWCCY, where Y is T or C and W is T or A, while that for HSV-1-specific binding was reported to be YGYTCGCACT. By EMSA, the HHV-6B OBP DNA-binding domain was mapped to a segment containing amino acids 482 to 770. However, in Southwestern (protein-DNA) blotting, the region sufficient for the DNA binding encompassed only amino acids 657 to 770. Similarly, Southwestern blotting showed that amino acids 689 to 851 of HSV-1 OBP had HSV-1 origin-binding activity, although this region was insufficient for origin binding in the EMSA. Although the longer DNA-binding domains identified by EMSA have marginal overall homology among HHV-6B and alphaherpesvirus OBP homologs, the smaller regions sufficient for the binding observed by Southwestern blotting have significant similarity. From these results, we propose a hypothesis that the DNA-binding domain of herpesvirus OBPs consists of two subdomains, one containing a conserved motif that contacts DNA directly, and another, less well conserved, that may modulate either the conformation or accessibility of the binding domain.

SUBMITTER: Inoue N

PROVIDER: S-EPMC189261 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Human herpesvirus 6B origin-binding protein: DNA-binding domain and consensus binding sequence.

Inoue N N Pellett P E PE

Journal of virology 19950801 8

We previously demonstrated by a DNA-binding assay that the human herpesvirus 6B (HHV-6B) replication origin has a structure similar to those of alphaherpesviruses, although the HHV-6B and herpes simplex virus type 1 (HSV-1) origin-binding proteins (OBPs) and origins are not interchangeable. Here we describe additional properties of the interaction between HHV-6B OBP and the HHV-6B origin. Competitive electrophoretic mobility shift assays (EMSAs) with DNA duplexes containing single-base alteratio ...[more]

PMID: 7609026

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Project description:Immediate early proteins of human herpesvirus 6A (HHV-6A) are expressed at the outset of lytic infection and thereby regulate viral gene expression. Immediate early protein 2 (IE2) of HHV-6A is a transactivator that drives a variety of promoters. The C-terminal region of HHV-6A IE2 is shared among IE2 homologs in betaherpesviruses and is involved in dimerization, DNA binding, and transcription factor binding. In this study, the structure of the IE2 C-terminal domain (IE2-CTD) was determined by X-ray crystallography at a resolution of 2.5 Å. IE2-CTD forms a homodimer stabilized by a ?-barrel core with two interchanging long loops. Unexpectedly, the core structure resembles those of the gammaherpesvirus factors EBNA1 of Epstein-Barr virus and LANA of Kaposi sarcoma-associated herpesvirus, but the interchanging loops are longer in IE2-CTD and form helix-turn-helix (HTH)-like motifs at their tips. The HTH and surrounding ?-helices form a structural feature specific to the IE2 group. The apparent DNA-binding site (based on structural similarity with EBNA1 and LANA) resides on the opposite side of the HTH-like motifs, surrounded by positive electrostatic potential. Mapping analysis of conserved residues on the three-dimensional structure delineated a potential factor-binding site adjacent to the expected DNA-binding site. The predicted bi- or tripartite functional sites indicate a role for IE2-CTD as an adapter connecting the promoter and transcriptional factors that drive gene expression.IMPORTANCE Human herpesvirus 6A (HHV-6A) and HHV-6B belong to betaherpesvirus subfamily. Both viruses establish lifelong latency after primary infection, and their reactivation poses a significant risk to immunocompromised patients. Immediate early protein 2 (IE2) of HHV-6A and HHV-6B is a transactivator that triggers viral replication and contains a DNA-binding domain shared with other betaherpesviruses such as human herpesvirus 7 and human cytomegalovirus. In this study, an atomic structure of the DNA-binding domain of HHV-6A IE2 was determined and analyzed, enabling a structure-based understanding of the functions of IE2, specifically DNA recognition and interaction with transcription factors. Unexpectedly, the dimeric core resembles the DNA-binding domain of transcription regulators from gammaherpesviruses, showing structural conservation as a DNA-binding domain but with its own unique structural features. These findings facilitate further characterization of this key viral transactivator.

Project description:The "omics" revolution of recent years has simplified the study of RNA transcripts produced during viral infection and under specific defined conditions. In the quest to find new and differentially expressed transcripts during the course of human Herpesvirus 6B (HHV-6B) infection, we made use of large-scale RNA sequencing to analyze the HHV-6B transcriptome during productive infection of human Molt-3 T-cells. Analyses were performed at different time points following infection and specific inhibitors were used to classify the kinetic class of each open reading frame (ORF) reported in the annotated genome of HHV-6B Z29 strain. The initial search focussed on HHV-6B-specific reads matching new HHV-6B transcripts. Differential expression of new HHV-6B transcripts were observed in all samples analyzed. The presence of many of these new HHV-6B transcripts were confirmed by RT-PCR and Sanger sequencing. Many of these transcripts represented new splice variants of previously reported ORFs, including some transcripts that have yet to be defined. Overall, our work demonstrates the diversity and the complexity of the HHV-6B transcriptome.IMPORTANCERNA sequencing (RNA-seq) is an important tool for studying RNA transcripts, particularly during active viral infection. We made use of RNA-seq to study human Herpesvirus 6B (HHV-6B) infection. Using six different time points, we were able to identify the presence of differentially spliced genes at 6, 9, 12, 24, 48 and 72 hours post-infection. Determination of the RNA profiles in the presence of cycloheximide (CHX) or phosphonoacetic acid (PAA) also permitted identification of the kinetic class of each ORF described in the annotated GenBank file. We also identified new spliced transcripts for certain genes and evaluated their relative expression over time. These data and next-generation sequencing (NGS) of the viral DNA have led us to propose a new version of the HHV-6B Z29 GenBank annotated file, without changing ORF names in order to facilitate trace back and correlate our work with previous studies on HHV-6B.

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Dataset Information

Human herpesvirus 6B origin-binding protein: DNA-binding domain and consensus binding sequence.

Publications

Human herpesvirus 6B origin-binding protein: DNA-binding domain and consensus binding sequence.

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