Poxvirus attack of antiviral defense pathways unleashes an effector-triggered NF-κB response.

Effector-triggered immunity (ETI) is a form of pathogen sensing that involves detection of pathogen-encoded virulence factors or "effectors." To discover ETI pathways in mammals, we developed a screening approach in which we expressed individual virulence factors in a human monocyte cell line and assessed transcriptional responses by RNA sequencing. We identified a poxvirus effector, myxoma virus M3.1, which elicited an antiviral nuclear factor κB (NF-κB) response. NF-κB was unleashed by an ETI pathway that sensed M3.1 attack of two antiviral complexes: zinc finger antiviral protein and TBK1. NF-κΒ activation occurred because the proteins inhibited by M3.1—N4BP1, ZC3H12A, and TBK1—are negative regulators of NF-κB. Our study established a systematic approach for the discovery of ETI pathways, and the results illustrated how negative regulators of immune responses may function in pathogen sensing. Editor's summary: Virulence factors, or effectors, provide an opportunity for cells to detect pathogens specifically, rather than molecules that might be associated with both beneficial and harmful microbes. Although effector-triggered immunity is well established in plants, less is known about its contribution to mammalian defenses. Remick et al. screened transcriptional responses in human monocytes in response to individual virulence factors from the myxoma virus. One factor triggered a nuclear factor κB (NF-κB) response indirectly, because it targeted proteins with antiviral activity that were also negative regulators of NF-κB. On the basis of these results, the authors suggest that molecules that have apparently contradictory functions in innate immunity could have evolved dual functions to safeguard mammalian cells against viral evasion strategies. —Sarah H. Ross INTRODUCTION: Detection of invading pathogens is required for the initiation of host defense against infection. The innate immune system typically recognizes pathogens via germline-encoded receptors that directly bind conserved microbial structures known as pathogen-associated molecular patterns (PAMPs). This form of pathogen sensing is called PAMP-triggered immunity. Studies in plants have revealed an additional form of pathogen sensing called effector-triggered immunity (ETI). Effectors, also called virulence factors, are produced by pathogens to attack host targets to facilitate infection and inhibit host defenses. During ETI, hosts counteract effectors by detecting their virulence activities and mounting a compensatory immune response. Although ETI is an established arm of the plant immune system, relatively few ETI pathways have been characterized in mammals and so their prevalence and functional importance in mammalian immunity is unclear. RATIONALE: We sought to identify ETI pathways in human cells. To do so, we expressed individual viral effectors in human cell lines and screened for effectors that elicited host immune responses. We chose to express viral effectors individually rather than infecting with an intact virus, because intact viruses often block immune signaling pathways through multiple mechanisms, which could obscure our ability to detect ETI responses. We also reasoned that highly human-adapted viruses might evolve to evade or suppress ETI. We thus decided to screen effectors from myxoma virus (MYXV), a rabbit-adapted poxvirus that is lethal to European rabbits. MYXV can infect some human cells, but it is not a natural human pathogen and does not cause disease in humans. Our rationale was that MYXV effectors could lead us to discover human ETI pathways that pathogens must evolve to evade or inhibit to infect humans. RESULTS: Our screesn revealed that a previously uncharacterized MYXV effector, M3.1, activated nuclear factor κB (NF-κB) signaling in human monocytes. NF-κB responses are known to restrict MYXV replication. We reasoned that M3.1-induced NF-κB signaling represented a host ETI response that occurred in response to M3.1 attack of antiviral defense pathways. We discovered that M3.1 disabled two key antiviral pathways: the ZAP complex and TBK1-mediated type I interferon signaling. We found that NF-κB signaling occurred because the host proteins inhibited by M3.1 to block ZAP and TBK1 (i.e., N4BP1, ZC3H12A, and TBK1 itself) are negative regulators of NF-κB. Thus, by attacking these antiviral pathways, M3.1 inadvertently triggered an NF-κB response. During MYXV infection, we found that the ETI response elicited by M3.1 was suppressed, likely by other virulence factors known to inhibit NF-κB signaling. Diverse poxviruses encode M3.1 homologs, some of which also elicited NF-κB signaling. CONCLUSION: Our data support a model in which certain antiviral proteins in host cells are "self-guarded" by virtue of their ability to suppress immune responses. We propose that during infection, viral effectors such as M3.1 attack these host proteins to disable their antiviral activity. However, because the antiviral protein is also a negative immune regulator, viral attack unleashes a compensatory host response. Thus, our results illustrate that negative regulators of immunity can function in pathogen sensing and highlight the complex layers of viral attack and host counterattack that evolve during host-pathogen arms races. Moreover, we established a systematic screening approach for the discovery of ETI pathways. A host ETI pathway detects myxoma virus.: A screen of MYXV effectors revealed M3.1 induced NF-κB. MYXV produces M3.1 to block the antiviral ZAP complex and TBK1. M3.1-deficient MYXV (∆M003.1) is restricted by ZAP. However, in targeting ZAP/TBK1, M3.1 inhibits negative regulators of NF-κB—N4BP1, ZC3H12A, and TBK1—thereby unleashing NF-κB. MYXV overcomes this ETI response by encoding additional effectors to block NF-κB. [ABSTRACT FROM AUTHOR]