Phleboviruses versus the Type I/III Interferon Response: How Sandfly Fever Sicilian Virus NSs Tackles Interferon Induction and PKR-Mediated Restriction

Phleboviruses (order Bunyavirales, family Phenuiviridae) are arthropod-borne viruses that are emerging globally due to the geographic expansion of long-known members and the identification of numerous novel ones. They span a wide spectrum of virulence, comprising clinically inapparent infection, febrile disease, encephalitis, up to severe haemorrhagic fever and multiorgan failure – with novel isolates including both highly virulent members and abundant others with as yet unknown disease potential. Rift Valley fever virus (RVFV), a long-known member, is highly pathogenic for humans and livestock. Thus, RVFV has been subject to extensive molecular characterization, which established the phleboviral non-structural protein NSs as antagonist of the antiviral interferon (IFN) system and main virulence factor in the mammalian host. Sandfly fever Sicilian virus (SFSV), on the other hand, was identified as causative agent of ‘sandfly fever’, a self-limited febrile disease, during World War II. Nowadays, SFSV is one of the geographically most widespread phleboviruses, causing disease mainly in immunologically naïve military troops and travellers. Although SFSV has been thoroughly characterized with regard to its clinical picture, its interaction with the mammalian host remained almost entirely elusive on the molecular level. In this work, we thus elucidated the molecular mechanisms with which the NSs protein of SFSV counteracts the interferon system. We identified that SFSV NSs dampened the induction of both type I and III interferons by specifically masking the DNA-binding domain of the transcription factor interferonregulatory factor 3 (IRF3). Despite IRF3 inhibition, however, SFSV did not fully abrogate IFN induction, leading to IFN-dependent upregulation of related transcription factor IRF7, which was not affected by SFSV NSs and fostered IFN induction. Additionally, SFSV NSs completely failed to impede IFN signalling, resulting in substantial expression of anti-phleboviral IFN-stimulated genes (ISGs). Thus, SFSV NSs appears to be a modulator rather than a full-blown antagonist of the IFN system. Further, protein kinase R (PKR) possesses a strong restrictive activity towards phleboviruses due to the phosphorylation of its substrate eukaryotic elongation factor 2α (eIF2α) and the ensuing block of protein synthesis. Surprisingly, we found that the NSs protein of SFSV conferred PKR resistance and boosted translation without affecting the activation of PKR or the phosphorylation state of eIF2α. Instead, SFSV NSs targeted eIF2B, the central regulatory hub of the integrated stress response (ISR), further downstream. Of note, as previously characterized viral PKR antagonists all act at the levels of PKR activation and eIF2α phosphorylation, targeting of eIF2B by SFSV NSs represented a novel viral evasion strategy. Interestingly, a common theme emerged during our studies: Highly virulent RVFV, on the one hand, utilizes its NSs to induce the degradation of target host factors via the proteasome, thereby acting in a catalytic mode. Furthermore, it establishes a global block of host gene expression to evade the IFN system. The NSs of mildly virulent SFSV, on the other hand, does not affect the expression levels of its host targets, but rather acts in a very specific and stoichiometric manner for both the inhibition of IFN induction and PKR evasion. Given its importance as exclusive phleboviral IFN antagonist, the NSs protein has been speculated to constitute a correlate of virulence. Our data on SFSV NSs support this hypothesis and argue for the characterization of the NSs proteins of novel phleboviruses with respect to their capacity to inhibit IFN induction, IFN signalling, and PKR activity in order to better estimate their potential to induce disease.Phleboviren (Ordnung Bunyavirales, Familie Phenuiviridae) sind Arboviren, die aufgrund der geographischen Expansion bekannter und der Identifizierung zahlreicher neuer Mitglieder global vermehrt auftreten. Sie umfassen ein breites Spektrum an Virulenz, darunter klinisch inapparente Infektionen, fiebrige Erkrankungen, Enzephalitis, bis hin zu hämorrhagischem Fieber und Multiorganversagen – wobei neue Isolate sowohl hochpathogene Mitglieder als auch zahllose andere mit soweit unbekanntem Krankheitspotential beinhalten. Das Rifttalfieber-Virus (RVFV), ein lange bekanntes Mitglied, ist hochpathogen für Mensch und Vieh. Daher wurde RVFV ausgiebig molekular charakterisiert, was das Nichtstrukturprotein NSs als Antagonist des antiviralen Interferon (IFN)-Systems und Hauptvirulenzfaktor im Säugetierwirt etabliert hat. Das sizilianische Sandfliegenfieber-Virus (SFSV) dagegen wurde während des Zweiten Weltkriegs als Erreger des „Sandfliegenfiebers”, einer selbstlimitierten fiebrigen Erkrankung, identifiziert. Heute ist es bekannt als eines der Phleboviren mit der weitesten geographischen Verbreitung und verursacht Symptome hauptsächlich in immunologisch naiven Soldaten und Reisenden. Obwohl SFSV im Hinblick auf das klinische Bild ausführlich charakterisiert wurde, ist seine Interaktion mit dem Säugetierwirt auf der molekularen Ebene fast komplett unbekannt. In dieser Arbeit haben wir daher die molekularen Mechanismen aufgeklärt, mit denen das NSs-Protein von SFSV dem IFN-System entgegenwirkt. Wir konnten zeigen, dass SFSV NSs die Induktion von Typ-I- und III IFN dämpft, indem es gezielt die DNA-Bindedomäne des Transkriptionsfaktors IRF3 verdeckt. Trotz der Inhibition von IRF3 unterband SFSV die IFN-Induktion dennoch nicht vollständig, was zu einer IFNabhängigen Hochregulation des Transkriptionsfaktors IRF7 führt, der nicht von SFSV NSs beeinträchtigt wird und die IFN-Induktion fördert. Zusätzlich versagte SFSV NSs darin, die IFN-Signaltransduktion zu behindern, woraus eine erhebliche Expression anti-phleboviraler IFN-stimulierter Gene (ISGs) resultierte. Folglich scheint SFSV NSs eher ein Modulator als ein starker Antagonist des IFN-Systems zu sein. Daneben besitzt die Proteinkinase R (PKR) aufgrund der Phosphorylierung des eukaryotischen Initiationsfaktors 2α (eIF2α), und der resultierenden Blockade der Proteinbiosynthese eine stark restriktive Aktivität gegenüber Phleboviren. Überraschenderweise fanden wir, dass das SFSV NSs eine PKR-Resistenz vermittelt und die Translation steigert, ohne jedoch die PKR-Aktivierung oder die eIF2α-Phosphorylierung zu beeinträchtigen. Vielmehr wirkt SFSV NSs auf den nachgeschalteten eIF2B-Komplex, den regulatorischen Knotenpunkt der integrierten Stressantwort. Bemerkenswerterweise hemmen alle bisher charakterisierten viralen PKR-Antagonisten die PKR-Aktivierung oder die eIF2α-Phosphorylierung, sodass die Manipulation von eIF2B durch SFSV NSs eine neue virale Evasionsstrategie darstellt. Interessanterweise zeichnete sich bei unseren Untersuchungen ein gemeinsames Muster ab: Das hochvirulente RVFV setzt sein NSs-Protein für den proteasomalen Abbau seiner Zielfaktoren ein, agiert also sozusagen katalytisch. Daneben verursacht es eine globale Blockade der Genexpresssion des Wirtes, um dem IFN-System zu entgehen. Das NSs des weniger virulenten SFSV dagegen beeinträchtigt nicht die Expressionslevel von Zielfaktoren, sondern scheint sowohl die Hemmung der IFN-Induktion als auch die PKR-Evasion auf stöchiometrische Weise zu vermitteln. In Anbetracht seiner Bedeutung als alleiniger phleboviraler IFN-Antagonist wurde spekuliert, dass das NSs-Protein ein Korrelat für die Virulenz darstellt. Unsere Daten zu SFSV NSs unterstützen diese Hypothese und sprechen für die Charakterisierung der NSs-Proteine neuartiger Phleboviren bezüglich ihrer Fähigkeit, die IFN-Induktion, die IFN-Signaltransduktion und die PKR-Aktivität zu hemmen, um ihr Krankheitspotential besser einzuschätzen

Phleboviruses and the Type I Interferon Response

The genus Phlebovirus of the family Bunyaviridae contains a number of emerging virus species which pose a threat to both human and animal health. Most prominent members include Rift Valley fever virus (RVFV), sandfly fever Naples virus (SFNV), sandfly fever Sicilian virus (SFSV), Toscana virus (TOSV), Punta Toro virus (PTV), and the two new members severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV). The nonstructural protein NSs is well established as the main phleboviral virulence factor in the mammalian host. NSs acts as antagonist of the antiviral type I interferon (IFN) system. Recent progress in the elucidation of the molecular functions of a growing list of NSs proteins highlights the astonishing variety of strategies employed by phleboviruses to evade the IFN system

Viral evasion of the integrated stress response through antagonism of eIF2-P binding to eIF2B.

Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2's dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five 'aromatic fingers'. NSs binding preserves eIF2B activity by maintaining eIF2B's conformation in its active A-State

eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition

RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α