Vaccinia virus nicking-joining enzyme is encoded by K4L (VACWR035)

Abstract

Vaccinia virus encodes an enzyme with DNA modifying activity that cleaves and inefficiently cross-links cruciformic DNA. This enzyme is contained within the virion, expressed at late times postinfection, and processes DNA in an energy-independent, Mg2+ ion-independent manner. Viral nuclease activity was measured in extracts from cells infected with well-defined viral mutants. Since some viral extracts lacked nuclease activity, the gene encoding the activity was postulated to be one of the open reading frames absent in the viruses lacking activity. Inducible expression of each candidate open reading frame revealed that only the gene VACWR035, or K4L, was required for nuclease activity. A recombinant virus missing only the open reading frame for K4L lacked nuclease activity. Extracts from a recombinant virus expressing K4L linked to a FLAG polypeptide were able to cleave and cross-link cruciformic DNA. There were no significant differences between the virus lacking K4L and wild-type vaccinia virus WR with respect to infectivity, growth characteristics, or processing of viral replicative intermediate DNA, including both telomeric and cross-linked forms. Purification of the K4L FLAG polypeptide expressed in bacteria yielded protein containing nicking-joining activity, implying that K4L is the only vaccinia virus protein required for the nicking-joining enzymatic activity.

FIG. 1.

Reaction products after virion nuclease reaction. Plasmids containing single-stranded regions, such as an extruded cruciform (pECHC), were converted to nicked circular (NC), linear (LIN), or covalently continuous cross-linked (XL) forms after incubation with the nuclease.

FIG. 2.

Nuclease activity is not present in all poxvirus recombinants. Cytoplasmic lysates from BS-C-1 cells infected at a multiplicity of infection of 1 with vaccinia virus WR, MVA, v811, v796, v759, and virion extract from vaccinia virus WR (left to right) were used in a nuclease assay at 55°C for 2 hours, and the DNA sample was resolved by electrophoresis through a 1.4% agarose gel. The rightmost lane contains the plasmid used as a substrate in the nuclease assays, pECHC. The positions of the pECHC nicked circle (NC), linear (LIN), and supercoiled (SC) forms are denoted by arrows.

FIG. 3.

Nuclease activity is present in cells transfected with K4L. Nuclease assays were run using mock-infected extract, virion extract derived from vaccinia virus WR, or cytoplasmic extracts prepared from cells transfected with pPG552 or pT7K4LFLAG and infected with vaccinia virus WR in the presence of cytosine arabinoside (second through fifth lanes). These samples, and the plasmid pECHC (left-most lane) used in the nuclease assays, were resolved by electrophoresis through a 1.4% agarose gel. The positions of the pECHC nicked circle (NC), linear (LIN), and supercoiled (SC) forms are denoted by arrows.

FIG. 4.

Sensitivity of virus recombinants to inhibitors of viral replication. Duplicate monolayers of BS-C-1 cells were infected with the same dilution of vK4LKO, vK4Lrestore, or vaccinia virus WR and incubated with no drug (upper panel), 1 mg/ml G418 (middle panel), or 50 μg/ml bromodeoxyuridine (lower panel).

FIG. 5.

The K4L open reading frame is required for nuclease activity. Cytoplasmic extracts from uninfected BOS cells or cells infected with vaccinia virus WR, vK4Lrestore, and vK4LKO and pECHC plasmid alone (mock lane) were used in a nuclease assay with pECHC plasmid, and the products were resolved by electrophoresis through a 1.4% agarose gel. The position of the pECHC nicked circle (NC), linear (LIN), and supercoiled (SC) forms are denoted by arrows.

FIG. 6.

The K4L open reading frame encodes the viral nicking-joining enzyme. An affinity-purified preparation of the bacterial K4L protein (pT7K4LFLAG) and the virion extract from the recombinant virus vK4Lrestore were used in a nuclease assay with pECHC, and the products were resolved by electrophoresis through a 1.4% agarose gel. The band corresponding to the linear pECHC molecule was purified and used for electrophoresis through a neutral agarose (left panel) or alkaline agarose (right panel) gel with untreated pECHC or pECHC linearized by digestion with the restriction endonuclease EcoRI. The positions of the pECHC cross-linked (XL), linear (LIN), and supercoiled (SC) forms are denoted by arrows. Band intensity was quantitated on a Fuji LAS-1000 after staining the gels with SYBR Gold (Molecular Probes).