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A Conserved Tripeptide Sequence at the C Terminus of the Poxvirus DNA Processivity Factor D4 Is Essential for Protein Integrity and Function.

ABSTRACT: Vaccinia virus (VACV) is a poxvirus, and the VACV D4 protein serves both as a uracil-DNA glycosylase and as an essential component required for processive DNA synthesis. The VACV A20 protein has no known catalytic function itself but associates with D4 to form the D4-A20 heterodimer that functions as the poxvirus DNA processivity factor. The heterodimer enables the DNA polymerase to efficiently synthesize extended strands of DNA. Upon characterizing the interaction between D4 and A20, we observed that the C terminus of D4 is susceptible to perturbation. Further analysis demonstrated that a conserved hexapeptide stretch at the extreme C terminus of D4 is essential for maintaining protein integrity, as assessed by its requirement for the production of soluble recombinant protein that is functional in processive DNA synthesis. From the known crystal structures of D4, the C-terminal hexapeptide is shown to make intramolecular contact with residues spanning the inner core of the protein. Our mutational analysis revealed that a tripeptide motif (215GFI217) within the hexapeptide comprises apparent residues necessary for the contact. Prediction of protein disorder identified the hexapeptide and several regions upstream of Gly215 that comprise residues of the interface surfaces of the D4-A20 heterodimer. Our study suggests that 215GFI217 anchors these potentially dynamic upstream regions of the protein to maintain protein integrity. Unlike uracil-DNA glycosylases from diverse sources, where the C termini are disordered and do not form comparable intramolecular contacts, this feature may be unique to orthopoxviruses.

SUBMITTER: Nuth M

PROVIDER: S-EPMC5207139 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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A Conserved Tripeptide Sequence at the C Terminus of the Poxvirus DNA Processivity Factor D4 Is Essential for Protein Integrity and Function.

Nuth Manunya M Guan Hancheng H Ricciardi Robert P RP

The Journal of biological chemistry 20161111 53

Vaccinia virus (VACV) is a poxvirus, and the VACV D4 protein serves both as a uracil-DNA glycosylase and as an essential component required for processive DNA synthesis. The VACV A20 protein has no known catalytic function itself but associates with D4 to form the D4-A20 heterodimer that functions as the poxvirus DNA processivity factor. The heterodimer enables the DNA polymerase to efficiently synthesize extended strands of DNA. Upon characterizing the interaction between D4 and A20, we observe ...[more]

PMID: 27836975

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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus and the causative agent of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. During lytic reactivation, there is a temporal cascade of viral gene expression that results in the production of new virions. One of the viral factors that is expressed during reactivation is open reading frame 59 (ORF59), the viral DNA polymerase processivity factor. ORF59 plays an essential role for DNA synthesis and is required for the nuclear localization of the viral DNA polymerase (ORF9) to the origin of lytic replication (oriLyt). In addition to its functions in viral DNA synthesis, ORF59 has been shown to interact with chromatin complexes, including histones and cellular methyltransferases. In this study, a series of KSHV BACmids containing 50-amino acid (aa) deletions within ORF59 were generated to determine the interaction domains between ORF59 and histones, as well as to assess the effects on replication fitness as a result of these interactions. These studies show that in the context of infection, ORF59 51 to 100 and 151 to 200 amino acids (aa) are required for interaction with histones, and ORF59 301 to 396 aa are not required for DNA synthesis. Since full-length ORF59 is known to localize to the nucleus, we performed an immunofluorescent assay (IFA) with the ORF59 deletion mutants and showed that all deletions are localized to the nucleus; this includes the ORF59 deletion without the previously identified nuclear localization signal (NLS). These studies further characterize ORF59 and demonstrate its essential role during lytic replication.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the causative agent of potentially fatal malignancies. Lytic replication of KSHV is an essential part of the viral life cycle, allowing for virus dissemination within the infected host and shedding to infect naive hosts. Viral DNA synthesis is a critical step in the production of new infectious virions. One of the proteins that is vital to this process is open reading frame 59 (ORF59), the viral encoded polymerase processivity factor. Previous work has demonstrated that the function of ORF59 is closely connected to its association with other viral and cellular factors. The studies presented here extend that work to include the interaction between ORF59 and histones. This interaction offers an additional level of regulation of the chromatinized viral genome, ultimately influencing DNA synthesis and transcription dynamics.

Dataset Information

A Conserved Tripeptide Sequence at the C Terminus of the Poxvirus DNA Processivity Factor D4 Is Essential for Protein Integrity and Function.

Publications

A Conserved Tripeptide Sequence at the C Terminus of the Poxvirus DNA Processivity Factor D4 Is Essential for Protein Integrity and Function.

Similar Datasets

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