The E3 Ubiquitin Ligase TRIM25 Inhibits Tembusu Virus Replication in vitro.

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused significant economic losses to the duck industry in China since 2010 due to egg production losses and neurological dysfunction. DTMUV is a public health concern because the infection spreads rapidly among birds. Retinoic acid-inducible gene-I (RIG-I)serves as an innate immune sensor and plays a key role in host antiviral defenses. Tripartite motif-containing protein 25 (TRIM25), an E3 ubiquitin ligase, is pivotal for RIG-I ubiquitination and activation. In addition, TRIM25 acts as an interferon-stimulated gene and mediates the antiviral activity. However, the effect of duck TRIM25 on DTMUV has not been assessed. Herein, we reportthe antiviral function of TRIM25 against DTMUV. First, we constructed the pcDNA3.1-c-myc-duTRIM25 plasmid. TRIM25 has a 2052 bp open reading frame that encodes a predicted 684 amino acid protein consisting of a RING finger domain, a B-box domain, a coiled-coil domain, and a PRY/SPRY domain. The protein sequence identity with chicken, mouse, and human TRIM25 is 69.7, 47.8, and 48.3%, respectively. TRIM25 was upregulated in BHK-21 cells, duck embryo fibroblasts, and 293T cellsupon DTMUV infection. The expression of viral RNA and proteins was significantly lower in cells over expressing TRIM25 than in control cells. Furthermore, siRNA-mediated silencing of TRIM25 increased the production of viral progeny. These results help elucidate the molecular mechanisms underlying the host response to DTMUV infection and suggest potential control measures for DTMUV outbreaks.

Identification of determinants that mediate binding between Tembusu virus and the cellular receptor heat shock protein A9

Heat shock protein A9 (HSPA9), a member of the heat shock protein family, is a putative receptor for Tembusu virus (TMUV). By using Western blot and co-immunoprecipitation assays, E protein domains I and II were identified as the functional domains that facilitate HSPA9 binding. Twenty-five overlapping peptides covering domain I and domain II sequences were synthesized and analyzed by using an HSPA9 binding assay. Two peptides showed the capability of binding to HSPA9. Dot blot assay of truncated peptides indicated that amino acid residues 19 to 22 and 245 to 252 of E protein constitute the minimal motifs required for TMUV binding to HSPA9. Importantly, peptides harboring those two minimal motifs could effectively inhibit TMUV infection. Our results provide insight into TMUV-receptor interaction, thereby creating opportunities for elucidating the mechanism of TMUV entry.