Domain Organization of Vaccinia Virus Helicase-Primase D5

Abstract

Poxviridae are viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in the Phycodnaviridae. We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single-and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins. IMPORTANCESince the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design. V iruses of the family Poxviridae have a large linear doublestranded DNA (dsDNA) genome coding for up to 250 open reading frames. The double-stranded DNA genome is covalently circularized at both ends and is fully replicated in the cytoplasm. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) (1) and in the Phycodnaviridae (reviewed in reference 2). Like poxviruses, these viruses are also members of the nucleocytoplasmic large DNA virus (NCLDV) clade (3).We are working on vaccinia virus, which was used for the vaccination campaign that led to the eradication of smallpox. It is a convenient and rather safe system for the study of general poxviru...

Online ion-exchange chromatography for small-angle X-ray scattering

“…7c) shows one peak that is slightly shifted to larger distances. This shape, in addition to the clear minima, hints at a mostly spherical shape with a cavity and matches the expected molecular shape well (Hutin et al, 2016).…”

“…A fragment of the helicase-primase D5R representing the D5N and helicase domains, D5 323-785 , was cloned, expressed and purified as described in Hutin et al (2016). The construct was cloned into the pProEx HTb vector (Life Technologies) using the primers 5 0 -GCGCCATGGGTAATAAACTGTTT-AATATTGCAC-3 0 and 5 0 -ATGCAAGCTTTTACGGAGA TGAAATATCCTCTATGA-3 0 and expressed in Escherichia coli BL21 (DE3) Star cells (Novagen).…”

“…To demonstrate the applicability of this approach to a novel sample, we selected D5 323-785 , the D5N and helicase domains of the Vaccinia virus helicase-primase D5. The protein fragment forms a hexamer (320.88 kDa) that is required for its activity (Hutin et al, 2016). After two nickel columns and a size-exclusion chromatography step, a contaminant still persisted and required an additional purification step via ionexchange chromatography (Fig.…”

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

“…The bead models obtained completely independently with both methods are comparable ( Figure 1H). D5 323-785 shows the expected hollow, hexameric structure 18 .…”

In vitro methods for testing antiviral drugs

“…Helicases are also common in some DNA viruses, including poxviruses. These include the vaccinia virus helicase-primase D5 (Bayliss and Smith, 1996;Hutin et al, 2016), E1 protein of bovine papilloma virus 1 (Yang et al, 1993), and the large tumor antigen of SV40 (Stahl et al, 1986). Helicases appear to be attractive targets for antiviral drugs (Briguglio et al, 2011;Frick, 2003;Reynolds et al, 2015), but development of such compounds is challenged by cytotoxicity and bioavailability issues (Kwong et al, 2005).…”