Relative Contributions of the cGAS-STING and TLR3 Signaling Pathways to Attenuation of Herpes Simplex Virus 1 Replication

Abstract

The innate immune response is crucial for defense against viral infections. Cells recognize virus infection through pattern recognition receptors and induce type I interferons as well as proinflammatory cytokines to orchestrate an innate immune response. Herpes simplex virus 1 (HSV-1) triggers both the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) and Toll-like receptor 3 (TLR3) pathways. It is well known that TLR3 uses the adaptor protein Toll/interleukin-1 receptor (IL-1R) domain-containing adaptor-inducing beta interferon (TRIF) for signaling, but we recently reported that STING signaling also requires TRIF. Because STING directly binds to TRIF, we identified the STING-interacting domain of TRIF and generated STING-noninteracting mutants of human and mouse TRIFs. The mutant TRIFs were unable to support STING signaling, although they were fully functional in the TLR3 pathway. These mutants were used to assess the relative contributions of the TLR3 and STING pathways to the attenuation of HSV-1 replication in mouse and human cell lines. For this purpose, the mouse L929 and NB41A3 cell lines and the human HT1080 and HeLa-M cell lines, in which both the TLR3 and the STING pathways are operational, were used. The TRIF gene was disrupted in these lines by CRISPR/Cas9, before reconstituting them with mutant and wild-type TRIF expression vectors. Infection of the reconstituted cells with HSV-1 revealed that both the cGAS-STING and the TLR3 signaling pathways are required for the attenuation of virus replication, but their relative contributions in attenuating HSV-1 replication were found to be different in mouse versus human cell lines. Thus, our study suggests that the relative contributions of the cGAS-STING and the TLR3 pathways in the attenuation of viral infection may be species specific. IMPORTANCE The magnitude of fatal infections caused by all different viruses in human and animal populations justifies a better understanding of the host innate immune response process that attenuates virus replication. In particular, the relative contributions of different signaling pathways which are responsible for the generation of the innate immune response are still largely unknown. In this study, we used STING-noninteracting TRIF mutants to decipher the relative contributions of the TLR3 and cGAS-STING signaling pathways to the attenuation of HSV-1 infection. We show that the relative contributions of the two pathways to the attenuation of viral infection are different in mouse versus human cell lines. Together, our results provide new insights into the relative contributions of two different signaling pathways in the attenuation of viral infection and may lead to the development of new antiviral strategies aimed at blocking viral infection at very early stages.

Herpes Simplex Virus Type 1 Interactions with the Interferon System

“…cGAS is also upregulated in astrocytes, but not microglia following HSV-1 infections. This suggests that cGAS may also play a role in controlling HSV-1 infections in the central nervous system, but may be species specific [ 83 , 84 , 85 ]. This is further supported in mice with cGAS or STING deficiencies having a higher susceptibility to HSE due to uncontrolled HSV-1 replication in neurons [ 86 ].…”

“…TLR3 deficient astrocytes are susceptible to HSV-2 infections and TLR3 deficient DCs are inefficient in priming CD8+ T cells in response to HSV-1 [ 115 , 116 ]. Neurons and epithelial cell lines that are TLR3 deficient have increased susceptibility to HSV-1 infection [ 84 , 117 ]. However, murine macrophages can compensate for a loss of TLR3 and amount a normal type I IFN response to control HSV-2 infections [ 70 ].…”

“…Interestingly, TLR3 offers no protection to sensory neurons; the major neuronal cell type infected by HSV-1, unless activated prior to infection [ 117 ]. Most recently, it has been hypothesized that the significance of TLR3 may also be species specific [ 84 ]. While human fibroblasts are susceptible to HSV-1 infection in the absence of TLR3, murine fibroblasts are able to compensate for this loss [ 84 , 111 , 112 , 114 ].…”

“…Most recently, it has been hypothesized that the significance of TLR3 may also be species specific [ 84 ]. While human fibroblasts are susceptible to HSV-1 infection in the absence of TLR3, murine fibroblasts are able to compensate for this loss [ 84 , 111 , 112 , 114 ]. While TLR3 loss can be compensated for in vitro, TLR3 deficiencies and/or mutations in humans are associated with increased susceptibility to HSE [ 111 ].…”

The crosstalk between viral RNA- and DNA-sensing mechanisms

“…Recent findings showed that the function of TLR3 on sensing HSV-1 dsRNA to elicit antiviral immunity remains disputed in various cell types and species [72]. Strikingly, the Sen's group recently revealed that the TRIF could combine cGAS-STING and TLR3 pathways by engaging with both STING and TLR3 to establish a powerfully antiviral state to suppress HSV-1 replication [73]. The contribution of these two pathways in controlling HSV-1 infection is also cell types and species specific.…”