The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon

Abstract

Variola virus (VARV) caused smallpox, one of the most devastating human diseases and the first to be eradicated, but its deliberate release represents a dangerous threat. Virulent orthopoxviruses infecting humans, such as monkeypox virus (MPXV), could fill the niche left by smallpox eradication and the cessation of vaccination. However, immunomodulatory activities and virulence determinants of VARV and MPXV remain largely unexplored. We report the molecular characterization of the VARV- and MPXV-secreted type I interferon-binding proteins, which interact with the cell surface after secretion and prevent type I interferon responses. The proteins expressed in the baculovirus system have been purified, and their interferon-binding properties characterized by surface plasmon resonance. The ability of these proteins to inhibit a broad range of interferons was investigated to identify potential adaptation to the human immune system. Furthermore, we demonstrate by Western blot and activity assays the expression of the type I interferon inhibitor during VARV and MPXV infections. These findings are relevant for the design of new vaccines and therapeutics to smallpox and emergent virulent orthopoxviruses because the type I interferon-binding protein is a major virulence factor in animal models, vaccination with this protein induces protective immunity, and its neutralization prevents disease progression.

Figure 1.

Cell surface binding of VACV IFNα/βBP and its VARV and MPXV orthologues. A) Anti-V5 immunofluorescent staining of nonpermeabilized HeLa cells incubated with supernatants containing the indicated V5-tagged recombinant proteins. Cell nuclei were visualized using DAPI. B) Confocal microscopy of cells incubated as before, showing plasmatic membrane localization of the indicated proteins.

Figure 2.

Inhibition of type I but not type III IFN-induced signaling by the VACV B18 protein and its VARV and MPXV orthologues. HeLa or A549 cells were incubated with supernatants containing the indicated V5-tagged recombinant proteins and subsequently exposed to 1000 U of hIFNα (A), hIL-29 (B), or hIL-28A (C) for the indicated times. Samples were analyzed by Western blot using anti-phospho-STAT-1, anti-STAT-1, and anti-V5 antibodies as indicated. Molecular size markers (kDa) are at left.

Figure 3.

Inhibition of type I IFN antiviral activity by VACV IFNα/βBP and its VARV and MPXV orthologues. Supernatants containing the indicated recombinant proteins were preincubated with 50 U of each type I IFN and subsequently added to HeLa cell monolayers. After a 16 h incubation, cells were infected with VSV, and cell viability was determined after 48 h. Controls of nontreated, uninfected (−VSV), and infected (+VSV) cells are at left, separated by a dashed line. Results are means ± sd of triplicate samples from one representative experiment.

Figure 4.

Differential binding of VACV B18 and VARV B17 to human and murine IFNs. Purified recombinant VACV B18 or VARV B17 were coupled to a sensor chip and screened for interactions with the indicated molecules by SPR. Top panels: binding curves of each protein to indicated human and murine type I IFNs. Bottom panels: binding curves of each protein to human and murine type III IFNs and the related human and murine IL-10. Curves correspond to injections of 10 nM hIFNα-A, 10 nM hIFNβ, 10 nM mIFNα, and 100 nM of all other analytes. All curves are normalized to an arbitrary value of 100 RU for comparative purposes; hIFNα-A curve is shown as a reference. Data correspond to one representative experiment.

Figure 5.

Expression and activity of the IFNα/βBP in supernatants from MPXV- or VARV-infected cells. A) Supernatants from VACV-, MPXV-, or VARV-infected cells were analyzed by Western blot using an anti-B18 protein antibody along with negative (media) and purified recombinant B18 protein (VACV B18) as indicated. B, C) Increasing amounts of supernatants from MPXV strain USA- or RCG-infected cells (B) or VARV strain BSH75 X or mock-infected cells (media) (C) were preincubated with either 5 or 50 U of type I hIFNs as indicated and added to HeLa cell monolayers for 16 h. As a control, 10 ng of purified VACV B18 protein was preincubated with IFN and added to HeLa cell monolayers as before. Cells were infected with VSV, and cell viability was determined after 48 h. Controls of nontreated, uninfected (−VSV) and infected (+VSV) cells were included in each case. Results are means ± sd of triplicate samples from one representative experiment.