Cellular DNA ligase I is recruited to cytoplasmic vaccinia virus factories and masks the role of the vaccinia ligase in viral DNA replication

Abstract

Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. RNA silencing of human DNA ligase I expression and a small molecule inhibitor of human DNA ligase I [corrected] severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding its own ligase might allow VACV to "jump-start" DNA synthesis.

Figure 1. Knock-Down of Cellular Lig1 Inhibits Replication of VACV Ligase Deletion Mutant

(A) Knock-down of cellular DNA ligases. HeLa cells were transfected with siRNAs to Lig1, 3 or 4 or a control non-targeting RNA. After 72 h, cell lysates were analyzed by Western blotting with antibodies to Lig1, 3, 4 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as a loading control. Antibody binding was detected by chemiluminescence. (B) Effects of siRNAs on replication of mutant and wild type VACV. HeLa cells were transfected with siRNAs to Lig1, 3, or 4 or with a control non-targeting siRNA. After 72 h, the cells were infected with 4 PFU per cell of vΔA50gfp or vA50Rev. At indicated times, virus titers were determined by plaque assay (upper panels) or viral DNA was quantified by real-time PCR (lower panels). Experiments were in triplicate and bars represent standard error of the mean. (C) Effect of Lig1 knock-down on late gene expression. HeLa cells were transfected with ligase-specific or control siRNA. After 72 h, the cells were infected with 4 PFU per cell of vΔA50gfp and visualized by fluorescence microscopy at indicated times.

Figure 2. Effects of Ligase Inhibitors

HeLa cells were treated with 50 µM L82 or L67 or with dimethylsulfoxide (DMSO) carrier for 48 h and then infected with 4 PFU per cell of vΔA50gfp or vA50Rev. At 24 h after infection, the cells were harvested and the virus titers determined by plaque assay. Bars represent standard errors of the mean.

Figure 3. Analysis of Cellular Ligases and Replication of vΔA50gfp Ligase Deletion Mutant in Resting and Proliferating HFF

(A) Cellular ligases in resting and proliferating cells. HFF were maintained in medium containing 0.2% FBS for four days to induce the resting state or were passaged in medium containing 10% FBS to allow proliferation. The cells were lysed and the proteins analyzed by Western blotting with antibodies to Lig1, 3, or 4 or to β-actin and detected by chemiluminescence. (B) VACV late gene expression. Resting and proliferating HFF were infected with 0.02 PFU per cell of vΔA50gfp. After 48 h, the cells were visualized by fluorescence microscopy (green) and after 72 h by light microscopy after crystal violet staining (black and white). (C, D) Virus replication and DNA synthesis. Resting and proliferating HFF were infected with 0.02 PFU per cell of vΔA50gfp or vA50Rev. At indicated times, virus titers were determined by plaque assay or viral DNA was quantified by real-time PCR. Experiments were in duplicate and bars indicate standard error. (E) Induction of Lig1 in resting cells by VACV. Lig1 and GAPDH were analyzed by Western blotting of extracts from resting and proliferating HFF that were uninfected or infected for 48 h.

Figure 4. Detection of Lig1 in Cytoplasmic VACV Factories

Uninfected HeLa cells (A) or HeLa cells infected with vΔA50gfp for 12 h (B) were stained with mouse MAb to Lig1 and rabbit polyclonal antibody to the VACV A11 protein followed by secondary fluorescent antibodies and DAPI. Resting HFF were uninfected (C) or infected with vΔA50gfp for 48 h (D) or vA50Rev for 24 h (E) and stained as for panels A and B. Arrowheads point to cytoplasmic factories.