ORF3 86-88A mutant was duplicated and also shown as ORF3 95-97A mutant. In the corrected version of Fig. 5A shown below, we have replaced the duplicate image with the correct original data. After careful reexamination of the original data, we are confident that the error in assembling this figure does not affect the validity of the research or the conclusions that we have drawn. We are grateful to the careful reader who brought this mistake to our attention and apologize for any confusion that this error may have caused." The corrected Fig. 5 and its legend appear below.

Article Interactome and Proteome Dynamics Uncover Immune Modulatory Associations of the Pathogen Sensing Factor cGAS Graphical Abstract Highlights d Defined global network of cytoplasmic cGAS protein interactions upon HSV-1 infection d Mapped the temporal proteomes with IFN-and apoptosisinducing HSV-1 strains d Oligoadenylate synthase-like domain of OASL associates with cGAS in the cytoplasm d OASL negatively regulates cGAS-mediated cytokine induction SUMMARY Viral DNA sensing is an essential component of the mammalian innate immune response. Upon binding viral DNA, the cyclic-GMP-AMP synthase (cGAS) catalyzes the production of cyclic dinucleotides to induce type I interferons. However, little is known about how cGAS is homeostatically maintained or regulated upon infection.Here, we define cytoplasmic cGAS interactions with cellular and viral proteins upon herpes simplex virus type 1 (HSV-1) infection in primary human fibroblasts. We compare several HSV-1 strains (wild-type, d109, d106) that induce cytokine responses and apoptosis and place cGAS interactions in the context of temporal proteome alterations using isobaric-labeling mass spectrometry. Follow-up analyses establish a functional interaction between cGAS and 2ʹ-5ʹ-oligoadenylate synthase-like protein OASL. The OAS-like domain interacts with the cGAS Mab21 domain, while the OASL ubiquitin-like domain further inhibits cGASmediated interferon response. Our findings explain how cGAS may be inactively maintained in cellular homeostasis, with OASL functioning as a negative feedback loop for cytokine induction.

In mammalian cells, early defenses against infection by pathogens are mounted through a complex network of signaling pathways shepherded by immune-modulatory pattern-recognition receptors. As obligate parasites, the survival of viruses is dependent upon the evolutionary acquisition of mechanisms that tactfully dismantle and subvert the cellular intrinsic and innate immune responses. Here, we review the diverse mechanisms by which viruses that accommodate DNA genomes are able to circumvent activation of cellular immunity. We start by discussing viral manipulation of host defense protein levels by either transcriptional regulation or protein degradation. We next review viral strategies used to repurpose or inhibit these cellular immune factors by molecular hijacking or by regulating their post-translational modification status. Additionally, we explore the infection-induced temporal modulation of apoptosis to facilitate viral replication and spread. Lastly, the co-evolution of viruses with their hosts is highlighted by the acquisition of elegant mechanisms for suppressing host defenses via viral mimicry of host factors. In closing, we present a perspective on how characterizing these viral evasion tactics both broadens the understanding of virus-host interactions and reveals essential functions of the immune system at the molecular level. This knowledge is critical in understanding the sources of viral pathogenesis, as well as for the design of antiviral therapeutics and autoimmunity treatments.

Dynamically shifting protein-protein interactions (PPIs) regulate cellular responses to viruses and the resulting immune signaling. Here, we use thermal proximity coaggregation (TPCA) mass spectrometry to characterize the on-off behavior of PPIs during infection with herpes simplex virus 1 (HSV-1), a virus with an ancient history of coevolution with hosts. Advancing the TPCA analysis to infer associations de novo, we build a time-resolved portrait of thousands of host-host, virus-host, and virus-virus PPIs. We demonstrate that, early in infection, the DNA sensor IFI16 recruits the active DNA damage response kinase, DNA-dependent protein kinase (DNA-PK), to incoming viral DNA at the nuclear periphery. We establish IFI16 T149 as a substrate of DNA-PK upon viral infection or DNA damage. This phosphorylation promotes IFI16-driven cytokine responses. Together, we characterize the global dynamics of PPIs during HSV-1 infection, uncovering the co-regulation of IFI16 and DNA-PK functions as a missing link in immunity to herpesvirus infection.

The cyclic GMP-AMP synthase (cGAS) protein is a pattern-recognition receptor of the mammalian innate immune system that is recognized as a main cytosolic sensor of pathogenic or damaged DNA. cGAS DNA binding initiates catalytic production of the second messenger, cyclic GMP-AMP, which activates the STING-TBK1-IRF3 signaling axis to induce cytokine expression. Post-translational modification (PTM) has started to be recognized as a critical component of cGAS regulation, yet the extent of these modifications remains unclear. Here, we report the identification and functional analysis of cGAS phosphorylations and acetylations in several cell types under basal and immune-stimulated conditions. cGAS was enriched by immunoaffinity purification from human primary fibroblasts prior to and after infection with herpes simplex virus type 1 (HSV-1), as well as from immune-stimulated STING-HEK293T cells. Six phosphorylations and eight acetylations were detected, of which eight PTMs were not previously documented. PTMs were validated by parallel reaction monitoring (PRM) mass spectrometry in fibroblasts, HEK293T cells, and THP-1 macrophage-like cells. Primary sequence and structural analysis of cGAS highlighted a subset of PTM sites with elevated surface accessibility and high evolutionary sequence conservation. To assess the functional relevance of each PTM, we generated a series of single-point cGAS mutations. Stable cell lines were constructed to express cGAS with amino acid substitutions that prevented phosphorylation (Ser-to-Ala) and acetylation (Lys-to-Arg) or that mimicked the modification state (Ser-to-Asp and Lys-to-Gln). cGAS-dependent apoptotic and immune signaling activities were then assessed for each mutation. Our results show that acetyl-mimic mutations at Lys384 and Lys414 inhibit the ability of cGAS to induce apoptosis. In contrast, the Lys198 acetyl-mimic mutation increased cGAS-dependent interferon signaling when compared with the unmodified charge-mimic. Moreover, targeted PRM quantification showed that Lys198 acetylation is decreased upon infections with two herpesviruses—HSV-1 and human cytomegalovirus (HCMV), highlighting this residue as a regulatory point during virus infection.