Fig 5. UL42 inhibits DNA binding and oligomerization of cGAS.

(A&B) UL42 impairs the binding of cGAS to dsDNA. HEK293T cells (2×10⁶) were transfected with the indicated plasmids (2 μg each, except for UL42 in (B), where increased amounts of 1, 2, 3, and 4 μg were used as indicated). Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. (C) Quantification of cGAS-bound viral DNAs in cells infected with wild-type or UL42-deficient HCMV. HFFs (2x10⁶) were infected with HCMV-WT or HCMV-ΔUL42 (MOI = 1) for 12 hr. The cell lysates were immunoprecipitated with anti-cGAS and cGAS-bound DNAs were extracted and analyzed by qPCR analyses with primers for the indicated viral genes. (D) Effects of UL42 on self-association of cGAS and MITA. HEK293 cells (2×10⁶) were transfected with the indicated plasmids for 20 hr before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. (E) Effects of UL42 on self-association of cGAS. HEK293T cells (2×10⁶) were transfected with the indicated plasmids (2 μg each, except for UL42 was transfected with increased amounts of 1, 2, 3, and 4 μg) for 20 hrs before pull-down assays analysis with the indicated antibodies. Graphs show mean ± SD, n = 3. *p<0.05, **p<0.01 (unpaired t test).