Toscana virus non-structural protein NSs acts as E3 ubiquitin ligase promoting RIG-I degradation.

It is known that the non-structural protein (NSs) of Toscana virus (TOSV), an emergent sandfly-borne virus causing meningitis or more severe central nervous system injuries in humans, exerts its function triggering RIG-I for degradation in a proteasome-dependent manner, thus breaking off the IFN-ß production. The non-structural protein of different members of Bunyavirales has recently appeared as a fundamental protagonist in immunity evasion through ubiquitination-mediated protein degradation targets. We showed that TOSV NSs has an E3 ubiquitin ligase activity, mapping at the carboxy-terminal domain and also involving the amino-terminal of the protein. Indeed, neither the amino- (NSsΔN) nor the carboxy- (NSsΔC) terminal-deleted mutants of TOSV NSs were able to cause ubiquitin-mediated proteasome degradation of RIG-I. Moreover, the addition of the C-terminus of TOSV NSs to the homologous protein of the Sandfly Fever Naples Virus, belonging to the same genus and unable to inhibit IFN-ß activity, conferred new properties to this protein, favoring RIG-I ubiquitination and its degradation. NSs lost its antagonistic activity to IFN when one of the terminal residues was missing. Therefore, we showed that NSs could behave as an atypical RING between RING (RBR) E3 ubiquitin ligases. This is the first report which identified the E3 ubiquitin ligase activity in a viral protein among negative strand RNA viruses.

Toscana virus NSs protein inhibits the induction of type I interferon by interacting with RIG-I.

Toscana virus (TOSV) is a phlebovirus, of the Bunyaviridae family, that is responsible for central nervous system (CNS) injury in humans. Previous data have shown that the TOSV NSs protein is a gamma interferon (IFN-ß) antagonist when transiently overexpressed in mammalian cells, inhibiting IRF-3 induction (G. Gori Savellini, F. Weber, C. Terrosi, M. Habjan, B. Martorelli, and M. G. Cusi, J. Gen. Virol. 92:71-79, 2011). In this study, we investigated whether an upstream sensor, which has a role in the signaling cascade leading to the production of type I IFN, was involved. We found a significant decrease in RIG-I protein levels in cells overexpressing TOSV NSs, suggesting that the nonstructural protein interacts with RIG-I and targets it for proteasomal degradation. In fact, the MG-132 proteasome inhibitor was able to restore IFN-ß promoter activation in cells expressing NSs, demonstrating the existence of an evasion mechanism based on inhibition of the RIG-I sensor. Furthermore, a C-terminal truncated NSs protein (ΔNSs), although able to interact with RIG-I, did not affect the RIG-I-mediated IFN-ß promoter activation, suggesting that the NSs domains responsible for RIG-I-mediated signaling and interaction with RIG-I are mapped on different regions. These results contribute to identify a novel mechanism for bunyaviruses by which TOSV NSs counteracts the early IFN response.

Structural flexibility of Toscana virus nucleoprotein in the presence of a single-chain camelid antibody.

Phenuiviridae nucleoprotein is the main structural and functional component of the viral cycle, protecting the viral RNA and mediating the essential replication/transcription processes. The nucleoprotein (N) binds the RNA using its globular core and polymerizes through the N-terminus, which is presented as a highly flexible arm, as demonstrated in this article. The nucleoprotein exists in an `open' or a `closed' conformation. In the case of the closed conformation the flexible N-terminal arm folds over the RNA-binding cleft, preventing RNA adsorption. In the open conformation the arm is extended in such a way that both RNA adsorption and N polymerization are possible. In this article, single-crystal X-ray diffraction and small-angle X-ray scattering were used to study the N protein of Toscana virus complexed with a single-chain camelid antibody (VHH) and it is shown that in the presence of the antibody the nucleoprotein is unable to achieve a functional assembly to form a ribonucleoprotein complex.

Ubiquitin and Not Only Unfolded Domains Drives Toscana Virus Non-Structural NSs Protein Degradation.

The non-structural protein NSs of the Phenuiviridae family members appears to have a role in the host immunity escape. The stability of Toscana virus (TOSV) NSs protein was tested by a cycloheximide (CHX) chase approach on cells transfected with NSs deleted versions fused to a reporter gene. The presence of intrinsically disordered regions (IDRs) both at the C- and N-terminus appeared to affect the protein stability. Indeed, the NSsΔC and NSsΔN proteins were more stable than the wild-type NSs counterpart. Since TOSV NSs exerts its inhibitory function by triggering RIG-I for proteasomal degradation, the interaction of the ubiquitin system and TOSV NSs was further examined. Chase experiments with CHX and the proteasome inhibitor MG-132 demonstrated the involvement of the ubiquitin-proteasome system in controlling NSs protein amount expressed in the cells. The analysis of TOSV NSs by mass spectrometry allowed the direct identification of K104, K109, K154, K180, K244, K294, and K298 residues targeted for ubiquitination. Analysis of NSs K-mutants confirmed the presence and the important role of lysine residues located in the central and the C-terminal parts of the protein in controlling the NSs cellular level. Therefore, we directly demonstrated a new cellular pathway involved in controlling TOSV NSs fate and activity, and this opens the way to new investigations among more pathogenic viruses of the Phenuiviridae family.

Toscana virus nucleoprotein oligomer organization observed in solution.

Toscana virus (TOSV) is an arthropod-borne virus belonging to the Phlebovirus genus within the Bunyaviridae family. As in other bunyaviruses, the genome of TOSV is made up of three RNA segments. They are encapsidated by the nucleoprotein (N), which also plays an essential role in virus replication. To date, crystallographic structures of phlebovirus N have systematically revealed closed-ring organizations which do not fully match the filamentous organization of the ribonucleoprotein (RNP) complex observed by electron microscopy. In order to further bridge the gap between crystallographic data on N and observations of the RNP by electron microscopy, the structural organization of recombinant TOSV N was investigated by an integrative approach combining X-ray diffraction crystallography, transmission electron microscopy, small-angle X-ray scattering, size-exclusion chromatography and multi-angle laser light scattering. It was found that in solution TOSV N forms open oligomers consistent with the encapsidation mechanism of phlebovirus RNA.

Truncation of the C-terminal region of Toscana Virus NSs protein is critical for interferon-ß antagonism and protein stability.

Toscana Virus (TOSV) is a Phlebovirus responsible for central nervous system (CNS) injury in humans. The TOSV non-structural protein (NSs), which interacting with RIG-I leads to its degradation, was analysed in the C terminus fragment in order to identify its functional domains. To this aim, two C-terminal truncated NSs proteins, Δ1C-NSs (aa 1-284) and Δ2C-NSs (aa 1-287) were tested. Only Δ1C-NSs did not present any inhibitory effect on RIG-I and it showed a greater stability than the whole NSs protein. Moreover, the deletion of the TLQ aa sequence interposed between the two ΔC constructs caused a greater accumulation of the protein with a weak inhibitory effect on RIG-I, indicating some involvement of these amino acids in the NSs activity. Nevertheless, all the truncated proteins were still able to interact with RIG-I, suggesting that the domains responsible for RIG-I signaling and RIG-I interaction are mapped on different regions of the protein.