The Dengue virus protease NS2B3 cleaves cyclic GMP-AMP synthase to suppress cGAS activation.

Dengue virus (DENV) is one of the most prevalent mosquito-transmitted human viruses that causes significant morbidity and mortality worldwide. To persist in the cell and consequently cause disease, DENV is evolved with mechanisms to suppress the induction of type I interferons by antagonizing cGAS-STING signaling. Using recombinant proteins and in vitro cleavage assays, we have shown that the DENV protease NS2B3 is capable of cleaving cGAS in the N-terminal region without disrupting the C-terminal catalytic center. This generates two major cleavage products: cleavage product N-terminal (CP-N) and cleavage product C-terminal (CP-C). We observed reduction in DNA-binding affinity of CP-C as compared to full-length cGAS. Reduction in DNA-binding affinity is also correlated with the decrease in enzymatic activity of CP-C. CP-N, on the other hand, has almost comparable DNA-binding ability as that of the full-length cGAS. In fact, CP-N competitively inhibits cyclic GMP-AMP production by both full-length cGAS and CP-C. We hypothesize that high DNA-binding affinity of CP-N enables it to sequester the DNA from CP-C and noncleaved full-length cGAS and thus reduces the rate of enzyme activation and cyclic GMP-AMP synthesis. Furthermore, we found that NS2B3 physically interacts with full-length cGAS and CP-C, laying the basis for their shuttling to and eventual degradation in the autophagosome. Overall, our study highlights a multifaceted and effective strategy by which an RNA virus antagonizes cGAS-STING signaling which may be useful for the design of antivirals targeting viral proteases.

Immunobiology and structural biology of AIM2 inflammasome.

Absent in melanoma 2 (AIM2) is a cytoplasmic sensor that upon recognizing double-stranded DNA assembles with apoptosis-associated speck-like protein containing a CARD (ASC) and procaspase-1 to form the multi-protein complex AIM2 inflammasome. Double-stranded DNA from bacterial, viral, or host cellular origins triggers AIM2 inflammasome assembly and activation, ultimately resulting in secretion of proinflammatory cytokines and pyroptotic cell death in order to eliminate microbial infection. Many pathogens therefore evade or suppress AIM2 inflammasome to establish infection. On the other hand, AIM2 activation is tightly controlled by multiple cellular factors to prevent autoinflammation. Extensive structural studies have captured the molecular details of multiple steps in AIM2 inflammasome assembly. The structures collectively revealed a nucleated polymerization mechanism that not only pervades each step of AIM2 inflammasome assembly, but also underlies assembly of other inflammasomes and complexes in immune signaling. In this article, we briefly review the identification of AIM2 as a cytoplasmic DNA sensor, summarize the importance of AIM2 inflammasome in infections and diseases, and discuss the molecular mechanisms of AIM2 assembly, activation, and regulation using recent cellular, biochemical, and structural results.

In Vitro Cleavage Assay to Characterize DENV NS2B3 Antagonism of cGAS.

cGAS is a key cytosolic dsDNA receptor that senses viral infection and elicits interferon production through the cGAS-cGAMP-STING axis. cGAS is activated by dsDNA from viral and bacterial origins as well as dsDNA leaked from damaged mitochondria and nucleus. Eventually, cGAS activation launches the cell into an antiviral state to restrict the replication of both DNA and RNA viruses. Throughout the long co-evolution, viruses devise many strategies to evade cGAS detection or suppress cGAS activation. We recently reported that the Dengue virus protease NS2B3 proteolytically cleaves human cGAS in its N-terminal region, effectively reducing cGAS binding to DNA and consequent production of the second messenger cGAMP. Several other RNA viruses likely adopt the cleavage strategy. Here, we describe a protocol for the purification of recombinant human cGAS and Dengue NS2B3 protease, as well as the in vitro cleavage assay.