Fig 2: Model: PPM1G restricts innate immune signaling mediated by STING and MAVS and is hijacked by KSHV ORF33 for immune evasion.STING and MAVS are key adaptors in cytosolic nucleic acid–sensing pathways during virus infection. Their phosphorylation by TBK1 is an essential and conserved mechanism that leads to recruitment of IRF3 and subsequent activation of the type I IFN pathway. However, cells must regulate their IFN production carefully to prevent inflammation and autoimmunity. Therefore, the activities of STING and MAVS must be tightly regulated to ensure a proper innate immune homeostasis in infected host cells. Host protein phosphatase PPM1G, as identified in this study, dephosphorylates p-STING and p-MAVS and tones down the response of the cytosolic DNA– and RNA–sensing pathways, thereby contributing to maintenance of a balanced antiviral innate immune response (left). However, this system is hijacked by KSHV for immune evasion. During KSHV de novo infection, tegument protein ORF33 is released from KSHV virion into the cytoplasm. ORF33 directly interacts with STING/MAVS and recruits PPM1G, resulting in enhanced dephosphorylation of p-STING/p-MAVS, impaired recruitment of IRF3 and suppression of type I IFN pathway. When KSHV is reactivated from latency, newly synthesized ORF33 is also likely to facilitate viral lytic replication by recruiting PPM1G to dephosphorylate p-STING/MAVS, thereby suppressing host antiviral activities (right).

Fig 3: PPM1G knockout or knockdown enhances host defense against both DNA and RNA viruses.(A and B) Effect of PPM1G knockout on SeV replication. WT or PPM1G KO HEK293 cells were infected with SeV (50 HA U/ml) for the indicated time periods. (A) SeV M and NP mRNA levels were measured by RT-qPCR. (B) SeV genome copy number in culture supernatant at 24 hours was measured by RT-qPCR. (C and D) Effect of PPM1G knockout on HSV-1 replication. WT or PPM1G KO HEK293 cells were infected with HSV-1 (MOI = 3) for the indicated time periods. (C) HSV-1 gD and gJ mRNA levels were measured by RT-qPCR. (D) HSV-1 titers in supernatant at 24 hours were measured. (E to I) Influence of PPM1G knockdown on reactivation levels of KSHV. iSLK.219 cells were transfected with the indicated siRNA for 48 hours and then induced for 48 hours. GFP served as a marker for KSHV latent infection, and red fluorescent protein (RFP) served as a marker for KSHV lytic replication. (F) RFP intensity from (E) was quantified using ImageJ and normalized to control. (G) The expression of K8/KbZIP and ORF45 in iSLK.219 cells was examined with the indicated antibodies. (H) KSHV genome copy number in supernatant was measured by qPCR. (I) Supernatants from (E) were used to infect iSLK cells for 24 hours, and GFP expressed from viral genome was measured by fluorescence-activated cell sorting. (A to D and F to H) Data presented are means ± SEM of three independent measurements, representative of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001; Student’s t test, n = 3.

Fig 4: PPM1G negatively regulates the antiviral innate immune response.(A and B) Impact of PPM1G on IFNβ production, as described in the legend to Fig. 1 (B and C). (C) Impact of PPM1G on dimerization and phosphorylation levels of IRF3, as described in the legend to Fig. 1G. (D and E) Influence of PPM1G knockdown on IFNβ mRNA levels. (D) HEK293 cells were transfected with indicated siRNA. After 48 hours, cells were challenged as in Fig. 1J, or with infection of HSV-1 [multiplicity of infection (MOI) = 3] for 12 hours. (E) Cells were transfected with the siRNA for 48 hours. iSLK cells were infected with KSHV for 24 hours (left), or iSLK.219 cells were induced for 48 hours (right), and IFNβ mRNA levels were measured. (F) Effect of PPM1G knockout on IFNβ mRNA levels. WT or PPM1G KO HEK293 cells were challenged for 12 hours as described in the legend to Fig. 1J. (G) Effect of PPM1G knockout on phosphorylation levels of IRF3. WT or PPM1G KO HEK293 cells were challenged for 12 hours as described in the legend to Fig. 3 (A and C). (H) The phenotype exhibited by PPM1G knockout cells was reversed by introducing WT but not the catalytically inactive (D496A) PPM1G mutant. PPM1G KO HEK293 cells were transfected with the indicated expression plasmids for 24 hours. Cells were challenged as described in the legend to Fig. 1C. (A, B, D to F, and H) Data presented are means ± SEM of three independent measurements, representative of three independent experiments. **P < 0.01 and ***P < 0.001; ns, not significant; Student’s t test, n = 3. See also fig. S7.

Fig 5: KSHV ORF33 recruits PPM1G to dephosphorylate STING and MAVS.(A) Coomassie Blue–stained gel of purified His-PPM1G and His-PPM1G D496A from bacteria. (B and C) The effect of His-PPM1G and His-PPM1G D496A on the phosphorylation levels of STING and MAVS was examined by in vitro phosphatase assay. (D and E) Interaction between endogenous PPM1G and exogenously expressed ORF33 (D) or ORF33 expressed from viral genome (E) was examined by coimmunoprecipitation (co-IP). (F to I) Enhancement of interaction between PPM1G and STING (F and G) or MAVS (H and I) by ORF33 was examined by co-IP. See also fig. S6.