Antiviral protein Y3

Combination therapies consisting of immune checkpoint inhibitors plus anti-VEGF therapy show enhanced antitumor activity and are approved treatments for patients with renal cell carcinoma (RCC). The immunosuppressive roles of VEGF in the tumor microenvironment are well studied, but those of FGF/FGFR signaling remain largely unknown. Lenvatinib is a receptor tyrosine kinase inhibitor that targets both VEGFR and FGFR. Here, we examine the antitumor activity of anti-PD-1 mAb combined with either lenvatinib or axitinib, a VEGFR-selective inhibitor, in RCC. Both combination treatments showed greater antitumor activity and longer survival in mouse models versus either single agent treatment, whereas anti-PD-1 mAb plus lenvatinib had enhanced antitumor activity compared with anti-PD-1 mAb plus axitinib. Flow cytometry analysis showed that lenvatinib decreased the population of tumor-associated macrophages and increased that of IFNγ-positive CD8+ T cells. Activation of FGFR signaling inhibited the IFNγ-stimulated JAK/STAT signaling pathway and decreased expression of its target genes, including B2M, CXCL10, and PD-L1. Furthermore, inhibition of FGFR signaling by lenvatinib restored the tumor response to IFNγ stimulation in mouse and human RCC cell lines. These preclinical results reveal novel roles of tumor FGFR signaling in the regulation of cancer immunity through inhibition of the IFNγ pathway, and the inhibitory activity of lenvatinib against FGFRs likely contributes to the enhanced antitumor activity of combination treatment comprising lenvatinib plus anti-PD-1 mAb. SIGNIFICANCE: FGFR pathway activation inhibits IFNγ signaling in tumor cells, and FGFR inhibition with lenvatinib enhances antitumor immunity and the activity of anti-PD-1 antibodies.

The antiviral protein Y3 produced by the edible mushroom Coprinus comatus disrupts the tobacco mosaic virus (TMV) and inhibits the multiplication of TMV in Nicotiana tabacum; however, the detailed mechanism of its activity remains unclear. In this study, Y3 was demonstrated to interact with TMV coat protein (TMV-CP) in vitro as well as in tobacco plants by using a yeast two-hybrid system and bimolecular fluorescence complementation (BiFC). To explore the interaction site between Y3 and TMV-CP, the phenylalanine (Phe) at the 43rd and arginine (Arg) at the 55th amino acid of Y3 were individually converted to cysteine (Cys) and serine (Ser), named Y3T1 and Y3T2, respectively, and were then used in BiFC assays. Based on the information obtained about disulfide bonds in the protein structure, the two mutations were predicted to change the protein's disulfide bonds. The results showed Y3T1 lost the ability to interact with TMV-CP, suggesting that a specific Phe of Y3 is critical for the interaction between Y3 and CP in plants. Furthermore, a prokaryotic expression system was used to test the antiviral activities of protein Y3 (PY3) and two other mutated proteins (P-Y3T1, P-Y3T2). The results showed that recombinant protein P-Y3 had a slightly lower inhibitory effect against TMV than Y3 extracted directly from mushrooms; further, P-Y3T1 decreased antiviral activity in the tobacco plant significantly compared with P-Y3, suggesting that the anti-TMV effect of Y3 was directly related to the Y3-CP interaction. In contrast, P-Y3T2 was able to still interact with TMV-CP in the tobacco plant, and it increased the ability of the plant to resist TMV compared with PY3, indicating that PY3-T2 could be a candidate peptide for plant protection against TMV and that Y3 may have other inhibitory mechanisms against TMV in addition to its interaction with TMV-CP.

Sendai virus (SeV), belonging to the Respirovirus genus of the family Paramyxoviridae, harbors an accessory protein, named C protein, which facilitates viral pathogenicity in mice. In addition, the C protein is known to stimulate the budding of virus-like particles by binding to the host ALG-2 interacting protein X (Alix), a component of the endosomal sorting complexes required for transport (ESCRT) machinery. However, small interfering RNA (siRNA)-mediated gene knockdown studies suggested that neither Alix nor C protein is related to SeV budding. In the present study, we determined the crystal structure of a complex comprising the C-terminal half of the C protein (Y3) and the Bro1 domain of Alix at a resolution of 2.2 Å to investigate the role of the complex in SeV budding. The structure revealed that a novel consensus sequence, LXXW, which is conserved among Respirovirus C proteins, is important for Alix binding. SeV possessing a mutated C protein with reduced Alix-binding affinity showed impaired virus production, which correlated with the binding affinity. Infectivity analysis showed a 160-fold reduction at 12 h postinfection compared with nonmutated virus, while C protein competes with CHMP4, one subunit of the ESCRT-III complex, for binding to Alix. All together, these results highlight the critical role of C protein in SeV budding. IMPORTANCE Human parainfluenza virus type I (hPIV1) is a respiratory pathogen affecting young children, immunocompromised patients, and the elderly, with no available vaccines or antiviral drugs. Sendai virus (SeV), a murine counterpart of hPIV1, has been studied extensively to determine the molecular and biological properties of hPIV1. These viruses possess a multifunctional accessory protein, C protein, which is essential for stimulating viral reproduction, but its role in budding remains controversial. In the present study, the crystal structure of the C-terminal half of the SeV C protein associated with the Bro1 domain of Alix, a component of cell membrane modulating machinery ESCRT, was elucidated. Based on the structure, we designed mutant C proteins with different binding affinities to Alix and showed that the interaction between C and Alix is vital for viral budding. These findings provide new insights into the development of new antiviral drugs against hPIV1.