Structural features of the single-stranded DNA-binding protein of Epstein–Barr virus

Mumtsidu, Eleni; Makhov, Alexander M.; Konarev, Petr V.; Svergun, Dmitri I.; Griffith, Jack D.; Tucker, Paul A.

doi:10.1016/j.jsb.2007.10.014

Cited by 10 publications

(20 citation statements)

References 67 publications

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“…We previously reported that deletion of the last 60 amino acids of ICP8 abolishes its ability to form double-helical protein filaments (24). Similar results were obtained with the C-terminus deletion mutant of BALF2 (24).…”

Section: Expression and Purification Of The Orf6⌬c Proteinsupporting

confidence: 73%

“…Because fluorescence polarization measures binding in solution as contrasted to a gel matrix, we would place more confidence in those values. Extensive analysis of the binding of ICP8 to short DNA molecules has shown that one monomer covers 15.8 nt (24,28,29). Our estimate of 22 nt bound by a single monomer of ORF6 is greater than the ICP8 value even though ORF6 is slightly smaller than ICP8.…”

Section: Discussionmentioning

confidence: 57%

“…5C). ICP8⌬C, however, had been shown to form filaments with ssDNA, which also had a larger diameter (24). The ORF6⌬C particles did not appear to be tightly stacked as in the case with wild-type ORF6.…”

Section: Expression and Purification Of The Orf6⌬c Proteinmentioning

confidence: 90%

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Interaction of Kaposi's Sarcoma-Associated Herpesvirus ORF6 Protein with Single-Stranded DNA

Özgür

Griffith

2014

J Virol

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Kaposi's sarcoma-associated herpesvirus (KSHV) ORF6 is homologous to the herpes simplex virus 1 (HSV-1) ICP8 and EpsteinBarr virus (EBV) BALF2 proteins. Here, we describe its single-stranded DNA (ssDNA) binding properties. Based on previous findings with ICP8 and BALF2, a 60-amino-acid C-terminal deletion mutant of Orf6 was generated, and the protein was purified to explore the function of the C terminus in ssDNA binding. We showed that full-length ORF6 binds cooperatively to M13 ssDNA, disrupting its secondary structure and extending it to a length equivalent to that of duplex M13 DNA. The width of the ORF6-ssDNA filament is 9 nm, and a 7.3-nm repeat can be distinguished along the filament axis. Fluorescence polarization analysis revealed that the wild-type and C-terminal mutant ORF6 proteins bind equally well to short ssDNA substrates, with dissociation constant (K d ) values of 2.2 ؋ 10 ؊7 M and 1.5 ؋ 10 ؊7 M, respectively. These values were confirmed by electrophoretic mobility shift assay (EMSA) analysis, which also suggested that binding by the full-length protein may involve both monomers and small multimers. While no significant difference in affinities of binding between full-length ORF6 and the C-terminal deletion mutant were observed with the short DNAs, binding of the C-terminal mutant protein to M13 ssDNA showed a clear lack of cooperativity as seen by electron microscopy (EM). Incubation of a duplex DNA containing a long single-stranded tail with doublehelical ORF6 protein filaments revealed that the ssDNA segment can be enveloped within the protein filament without disrupting the filament structure. IMPORTANCEThis work describes the biochemical characterization of the single-stranded DNA binding protein of KSHV, ORF6, central to viral DNA replication in infected cells. A C-terminal deletion mutant protein was generated to aid in understanding the role of the C terminus in DNA binding. Here we analyze the binding of the wild-type and mutant proteins to short oligomeric and longer genomic ssDNA substrates. Although it is capable of interacting with the short substrates, the inability of mutant ORF6 to form oligomers in solution hindered it from fully covering the long genomic substrates. We previously showed that ORF6 forms long filaments in solution, and we showed here that these can absorb ssDNA without disruption of the filament structure. This work will provide an important basis for future studies by us and/or others.

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Section: Expression and Purification Of The Orf6⌬c Proteinsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 57%

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Interaction of Kaposi's Sarcoma-Associated Herpesvirus ORF6 Protein with Single-Stranded DNA

Özgür

Griffith

2014

J Virol

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“…The HSV ssDNA binding protein ICP8 has been reported to form double-helical protein filaments in vitro in the absence of DNA (24,25,38). ICP8 homologs BALF2 from Epstein-Barr virus (EBV) and ORF6 from Kaposi's sarcoma-associated herpesvirus (KSHV) also form double-helical protein filaments (26,39), suggesting that filament formation is a common property of herpesvirus ssDNA binding proteins. Removal of the 60 C-terminal amino acids from ICP8, BALF, or ORF6 has been reported to eliminate filament formation and to reduce cooperative binding to ssDNA (26,27,40).…”

Section: Resultsmentioning

confidence: 99%

“…The 60 C-terminal residues of ICP8 are required both for filament formation and for cooperative binding to ssDNA (26,27). In 2005, the crystal structure of ICP8 lacking the C-terminal 60 amino acid residues was reported, revealing an N-terminal domain (residues 9 to 1038) that was described as containing head, neck, and shoulder regions (28).…”

mentioning

confidence: 99%

ICP8 Filament Formation Is Essential for Replication Compartment Formation during Herpes Simplex Virus Infection

Darwish

Grady

Bai

et al. 2016

J Virol

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Herpes simplex virus (HSV) dramatically reorganizes the infected-cell nucleus, leading to the formation of prereplicative sites and replication compartments. This process is driven by the essential viral single-stranded DNA (ssDNA) binding protein ICP8, which can form double-helical filaments in the absence of DNA. In this paper, we show that two conserved motifs, FNF (F1142, N1143, and F1144) and FW (F843 and W844), are essential for ICP8 self-interactions, and we propose that the FNF motif docks into the FW region during filament formation. Mammalian expression plasmids bearing mutations in these motifs (FNF and FW) were unable to complement an ICP8-null mutant for growth and replication compartment formation. Furthermore, FNF and FW mutants were able to inhibit wild-type (WT) virus plaque formation and filament formation, whereas a double mutant (FNF-FW) was not. These results suggest that single mutant proteins are incorporated into nonproductive ICP8 filaments, while the double mutant is unable to interact with WT ICP8 and does not interfere with WT growth. Cells transfected with WT ICP8 and the helicase-primase (H/P) complex exhibited punctate nuclear structures that resemble prereplicative sites; however, the FNF and FW mutants failed to do so. Taken together, these results suggest that the FNF and FW motifs are required for ICP8 self-interactions and that these interactions may be important for the formation of prereplicative sites and replication compartments. We propose that filaments or other higher-order structures of ICP8 may provide a scaffold onto which other proteins can be recruited to form prereplicative sites and replication compartments. IMPORTANCEFor nuclear viruses such as HSV, efficient DNA replication requires the formation of discrete compartments within the infectedcell nucleus in which replication proteins are concentrated and assembled into the HSV replisome. In this paper, we characterize the role of filament formation by the single-stranded DNA binding protein ICP8 in the formation of prereplicative sites and replication compartments. We propose that ICP8 protein filaments generate a protein scaffold for other cellular and viral proteins, resulting in a structure that concentrates both viral DNA and replication proteins. Replication compartments may be similar to other types of cellular membraneless compartments thought to be formed by phase separations caused by low-affinity, multivalent interactions involving proteins and nucleic acids within cells. ICP8 scaffolds could facilitate the formation of replication compartments by mediating interactions with other components of the replication machinery. H erpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of an infected cell in large globular domains called replication compartments (1). These compartments are non-membrane-bound structures in the nucleus that serve to concentrate and compartmentalize viral and cellular molecules that are required for gene expression, DNA replication, and ...

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Characterization of the single-stranded DNA binding protein pVVGJΦ of VGJΦ phage from Vibrio cholerae

Falero

Caballero

Trigueros

et al. 2011

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Structural features of the single-stranded DNA-binding protein of Epstein–Barr virus

Cited by 10 publications

References 67 publications

Interaction of Kaposi's Sarcoma-Associated Herpesvirus ORF6 Protein with Single-Stranded DNA

Interaction of Kaposi's Sarcoma-Associated Herpesvirus ORF6 Protein with Single-Stranded DNA

ICP8 Filament Formation Is Essential for Replication Compartment Formation during Herpes Simplex Virus Infection

Characterization of the single-stranded DNA binding protein pVVGJΦ of VGJΦ phage from Vibrio cholerae

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