Jordan T. Becker

Avian influenza A virus (IAV) is a major global health threat. Not only does it cause significant economic losses to the agricultural industry, but it also presents a risk of spillover into the human population. However, several obstacles must be overcome for a successful cross-species transmission event to occur. One of these obstacles is the innate immunity of the host. While the innate immune response to IAV in humans has been well-studied, the innate immune response to IAV in birds is not as well understood. Here, we explore the interferon-induced immune response by identifying interferon-stimulated genes (ISGs) in chicken, quail, and duck cells, and find that they are largely species-specific. Using this data, we performed a CRISPR knockout ISG screen against two strains of IAV, the mouse-adapted PR8, and the low pathogenicity avian IAV isolate WF10. We find that for PR8, IFITM3 is the dominant restrictor of viral replication with several additional hits with modest effects, consistent with previous work in mammals. For avian WF10, IFITM3 was also a hit however there were two additional hits absent for PR8, the HSP70 co-chaperone protein BAG5 and deubiquitinase OTUD4. Overall, these findings confirm the divergence of interferon-mediated immunity between species and establish a screening system that can be used to identify ISGs important for viral restriction in avian species. IMPORTANCE: Wild birds, especially waterfowl and shore birds, are the primary natural reservoir for influenza A viruses. These birds can transmit IAV to domestic poultry, where the spillover risk to humans increases. Chickens are the most common species of domestic poultry world-wide and are frequently infected with avian influenza viruses. Despite this, relatively little is known about how their immune system responds to influenza infection. This lack of knowledge creates a barrier to finding ways of preventing or mitigating IAV infection in chicken flocks. In this study, we characterized the interferon-induced immune response to IAV in chickens and found that only a small subset of ISGs were responsible for restricting IAV in chicken cells.

Zoonotic viruses are an omnipresent threat to global health. Influenza A virus (IAV) transmits between birds, livestock, and humans. Proviral host factors involved in the cross-species interface are well known. Less is known about antiviral mechanisms that suppress IAV zoonoses. We observed CpG dinucleotide depletion in human IAV relative to avian IAV. Notably, human ZAP selectively depletes CpG-enriched viral RNAs with its cofactor KHNYN. ZAP is conserved in tetrapods, but we uncovered that avian species lack KHNYN. We found that chicken ZAP may not affect IAV (PR8) or CpG-enriched IAV (PR8 _CG ). Human ZAP or KHNYN independently restricted CpG-enriched IAV PR8 _CG by overexpression in chicken cells and by combined knockout in human cells. Additionally, mammalian ZAP-L and KHNYN also independently restricted an avian retrovirus (ROSV). Curiously, platypus KHNYN, the most divergent from eutherian mammals, was also capable of independent restriction of multiple diverse viruses. We suggest that some mammalian KHNYN can act as a bona fide restriction factor with cell-autonomous activity. Furthermore, we speculate that through repeated contact between avian viruses and mammalian hosts, protein changes may accompany CpG-biased mutations or reassortment to evade mammalian ZAP and KHNYN.

Zoonotic viruses are an omnipresent threat to global health. Influenza A virus (IAV) transmits between birds, livestock, and humans. Proviral host factors involved in the cross-species interface are well known. Less is known about antiviral mechanisms that suppress IAV zoonoses. We observed CpG dinucleotide depletion in human IAV relative to avian IAV. Notably, human ZAP selectively depletes CpG-enriched viral RNAs with its cofactor KHNYN. ZAP is conserved in tetrapods but we uncovered that avian species lack KHNYN. We found that chicken ZAP may not affect IAV (PR8) or CpG enriched IAV (PR8 _CG ). Human ZAP or KHNYN independently restricted CpG-enriched IAV PR8 _CG by overexpression in chicken cells and by combined knockout in human cells. Additionally, mammalian ZAP-L and KHNYN also independently restricted an avian retrovirus (ROSV). Curiously, platypus KHNYN, the most divergent from eutherian mammals, was also capable of independent restriction of multiple diverse viruses. We suggest that some mammalian KHNYN can act as a bona fide restriction factor with cell-autonomous activity. Furthermore, we speculate that through repeated contact between avian viruses and mammalian hosts, protein changes may accompany CpG-biased mutations or reassortment to evade mammalian ZAP and KHNYN.

Spondweni virus (SPONV) is the closest known relative of Zika virus (ZIKV). SPONV pathogenesis resembles that of ZIKV in pregnant mice, and both viruses are transmitted by Aedes aegypti mosquitoes. We aimed to develop a translational model to further understand SPONV transmission and pathogenesis. We found that cynomolgus macaques ( Macaca fascicularis ) inoculated with ZIKV or SPONV were susceptible to ZIKV but resistant to SPONV infection. In contrast, rhesus macaques ( Macaca mulatta ) supported productive infection with both ZIKV and SPONV and developed robust neutralizing antibody responses. Crossover serial challenge in rhesus macaques revealed that SPONV immunity did not protect against ZIKV infection, whereas ZIKV immunity was fully protective against SPONV infection. These findings establish a viable model for future investigation into SPONV pathogenesis and suggest that the risk of SPONV emergence is low in areas with high ZIKV seroprevalence due to one-way cross-protection between ZIKV and SPONV.

Double-stranded RNA produced during viral replication and transcription activates both protein kinase R (PKR) and ribonuclease L (RNase L), which limits viral gene expression and replication through host shutoff of translation. In this study, we find that APOBEC3B forms a complex with PABPC1 to stimulate PKR and counterbalances the PKR-suppressing activity of ADAR1 in response to infection by many types of viruses. This leads to translational blockage and the formation of stress granules. Furthermore, we show that APOBEC3B localizes to stress granules through the interaction with PABPC1. APOBEC3B facilitates the formation of protein-RNA condensates with stress granule assembly factor (G3BP1) by protecting mRNA associated with stress granules from RNAse L-induced RNA cleavage during viral infection. These results not only reveal that APOBEC3B is a key regulator of different steps of the innate immune response throughout viral infection but also highlight an alternative mechanism by which APOBEC3B can impact virus replication without editing viral genomes.

APOBEC3 enzymes are single-stranded DNA cytosine-to-uracil deaminases with beneficial roles in antiviral immunity and detrimental roles in cancer mutagenesis. Regarding viral infection, all seven human APOBEC3 enzymes have overlapping roles in restricting virus types that require DNA for replication, including EBV, HIV, human papillomavirus (HPV), and human T-cell leukemia virus (HTLV).

Spondweni virus (SPONV) is the closest known relative of Zika virus (ZIKV). SPONV pathogenesis resembles that of ZIKV in pregnant mice, and both viruses are transmitted by Aedes aegypti mosquitoes. We aimed to develop a translational model to further understand SPONV transmission and pathogenesis. We found that cynomolgus macaques ( Macaca fascicularis ) inoculated with ZIKV or SPONV were susceptible to ZIKV, but resistant to SPONV infection. In contrast, rhesus macaques ( Macaca mulatta ) supported productive infection with both ZIKV and SPONV and developed robust neutralizing antibody responses. Crossover serial challenge in rhesus macaques revealed that SPONV immunity did not protect against ZIKV infection, whereas ZIKV immunity was fully protective against SPONV infection. These findings establish a viable model for future investigation into SPONV pathogenesis, and suggest the risk of SPONV emergence is low in areas with high ZIKV seroprevalence due to one-way cross-protection between ZIKV and SPONV. Teaser: Identification of asymmetric immune interactions between Zika and Spondweni viruses in macaque monkeys.

Viruses have evolved diverse mechanisms to antagonize host immunity such as direct inhibition and relocalization of cellular APOBEC3B (A3B) by the ribonucleotide reductase (RNR) of Epstein-Barr virus. Here, we investigate the mechanistic conservation and evolutionary origin of this innate immune counteraction strategy. First, we find that human gamma-herpesvirus RNRs engage A3B via largely distinct surfaces. Second, we show that RNR-mediated enzymatic inhibition and relocalization of A3B depend upon binding to different regions of the catalytic domain. Third, we show that the capability of viral RNRs to antagonize A3B is conserved among gamma-herpesviruses that infect humans and Old World monkeys that encode this enzyme but absent in homologous viruses that infect New World monkeys that naturally lack the A3B gene. Finally, we reconstruct the ancestral primate A3B protein and demonstrate that it is active and similarly engaged by the RNRs from viruses that infect humans and Old World monkeys but not by the RNRs from viruses that infect New World monkeys. These results combine to indicate that the birth of A3B at a critical branchpoint in primate evolution may have been a driving force in selecting for an ancestral gamma-herpesvirus with an expanded RNR functionality through counteraction of this antiviral enzyme.