Interferon-Stimulated Gene (ISG)-Expression Screening Reveals the Specific Antibunyaviral Activity of ISG20

Abstract

Bunyaviruses pose a significant threat to human health, prosperity, and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon-stimulated genes (ISGs), whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and the Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae, and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to the case against other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional RNase activity. Through use of an infectious virus-like particle (VLP) assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taking all the data together, we report that ISG20 is a broad and potent antibunyaviral factor but that some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance may influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance.IMPORTANCE There are hundreds of bunyaviruses, many of which cause life-threatening acute diseases in humans and livestock. The interferon (IFN) system is a key component of innate immunity, and type I IFNs limit bunyaviral propagation both in vitro and in vivo. Type I IFN signaling results in the upregulation of hundreds of IFN-stimulated genes (ISGs), whose concerted action generates an “antiviral state.” Although IFNs are critical in limiting bunyaviral replication and pathogenesis, much is still unknown about which ISGs inhibit bunyaviruses. Using ISG-expression screening, we examined the ability of ∼500 unique ISGs to inhibit Bunyamwera orthobunyavirus (BUNV), the prototypical bunyavirus. Using this approach, we identified ISG20, an interferon-stimulated exonuclease, as a potent inhibitor of BUNV. Interestingly, ISG20 possesses highly selective antibunyaviral activity, with multiple bunyaviruses being potently inhibited while some largely escape inhibition. We speculate that the ability of some bunyaviruses to escape ISG20 may influence their pathogenesis.

Novel Insights Into Immune Systems of Bats

“…Several unique ISGs and atypical induction of ISGs have been observed in bat cells (73). In human cells, expression of individual ISGs can inhibit virus replication, such as ISG20 that displays remarkable anti-bunyavirus activity (76). The effects of expressing homologs of atypical bat ISGs in human cells have not been studied.…”

TRIM69 inhibits Vesicular Stomatitis Indiana Virus (VSIV)

“…We have previously used arrayed ISG expression screening of human and rhesus macaque ISG libraries to identify antiviral factors targeting a range of viruses (3, 5, 26). Although, VSIV has previously been subjected to a large-scale screen of ~300 interferon induced genes (4), we reasoned that using larger libraries of arrayed ISGs might identify additional anti-VSIV effectors.…”

“…Although, VSIV has previously been subjected to a large-scale screen of ~300 interferon induced genes (4), we reasoned that using larger libraries of arrayed ISGs might identify additional anti-VSIV effectors. We recently expanded our human ISG library to include >500 ISGs, which can be used in conjunction with our existing library of >300 rhesus macaque ISGs (5, 26) (Figure 1A), all of which are encoded by lentiviral vectors (Figure 1B). In addition, we took advantage of a single-cycle VSIV-GFP system (rVSVΔG-GFP, referred to herein as VSIV-GFP) to allow us to identify strong early blocks to VSIV with high fidelity (27).…”

“…The short generation times inherent to the lifecycles of most viruses means that resistance to inhibition can be rapidly selected in vitro (5, 26, 39, 40). These in vitro evolution approaches can rapidly identify antiviral sensitivity/resistance determinants in viruses targeted by antiviral factors (39, 41).…”

“…The ISG screens were executed as described previously (5, 26). Briefly, MT4 cells were seeded in 96-well plates and transduced with ISG-encoding SCRPSY vectors (one ISG per well).…”