Varicella-Zoster Virus ORF9 Is an Antagonist of the DNA Sensor cGAS (2022)
Figure 2

Figure 2: Screening of all VZV ORFs identifies ORF9 as an antagonist of cGAS-mediated DNA-sensing.

(A) HEK293T cells were transfected with pRL-TK, p125-F-Luc, expression plasmids for human cGAS and human STING, as well as expression plasmids for individual ORFs. The next day luciferase activity was determined. For each ORF a z-value was calculated and ORFs sorted by descending values.

(B) The experiment shown in panel (A) was repeated three times and for each ORF in each experiment a rank was determined according to z-value (highest z-value = rank 1). Ranks are displayed as a heatmap. The first row shows results displayed in panel (A).

(C) HEK293T cells were transfected as in (A,B) with expression constructs for VZV ORF9 or empty vector. In parallel, reporter expression was stimulated by co-transfection of the RIG-I-CARD plasmid instead of hcGAS and hSTING.

(D) Western blot analysis of PMA-differentiated THP1 monocytes stably transduced with either V5-tagged VZV ORF9 or GFP using the indicated antibodies.

(E) THP1 monocytes stably transduced with either VZV ORF9 or GFP were PMA-differentiated and transfected with indicated doses of dsDNA. Expression of IFNB1 and IFNL1 was assessed by RT-qPCR. Graphs show expression relative to GAPDH. (F) THP1 monocytes stably transduced with either VZV ORF9 or GFP were treated with the STING agonist diABZi (0.005 μM). Expression of IFNB1, IFIT1 and IFI44 was assessed by RT-qPCR. Graphs show expression relative to GAPDH. See also Fig EV2A.

(G) Schematic detailing the E.coli DHFR degron system.

(H) Schematic detailing the experimental procedure for (J) and (K). See text for details. (I) PMA-differentiated THP1 cells were co-cultured with VZV-ORF9ecDHFR-infected MeWo cells for 1h in the presence of TMP, washed, and incubated for another 24h in the presence of TMP. Cells were then washed extensively and fresh medium was added that was supplemented or not with TMP. Expression levels of IFNB1, IFIT1, and IFI44 were measured by RT-qPCR after a further 24h incubation. Graphs show expression relative to GAPDH. Mean fold changes are indicated.

(J) Cells infected and treated as in (I) with the addition of uninfected cells were analysed by western blotting using the indicated antibodies.

Data information: Panel (A) is representative of three independent biological experiments, where each data point represents a technical replicate (mean ± SD). The three independent experiments are summarised in panel (B), where the data from (A) forms the first row. Panel (C) is representing two independent biological experiments, where each data point represents a technical replicate (mean ± SD). Panels (D) and (E) are representing two independent biological experiments. In (E) each data point represents a technical replicate. Panel (F) shows pooled data from three and two repeats, respectively, where each data point represents an independent biological experiment (horizontal bars show means). Panel (I) shows pooled data from three repeats, where each data point represents an independent biological experiment. Panel (J) is representative of three independent experiments. Statistical analysis in (F) and (I) were paired t-tests.

See also Fig EV2 and EV3.