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.