Orthoreovirus outer-fiber proteins are substrates for SUMO-conjugating enzyme Ubc9

Abstract

Reoviruses are potential anticancer agents due to their ability to induce cell death in tumor cells. Grass carp reovirus (GCRV) is one of the best characterized models on reovirus pathogenesis in vitro. However, there is little known about how SUMOylation affects reovirus pathogenesis. The SUMO conjugating enzyme 9 (Ubc9) determines the targets of SUMOylation. Here, the protein interactions between reovirus outer fiber proteins, specifically GCRV-104 VP55, and Ubc9 were probed using a yeast two-hybrid system. The N-terminal coiled-coil domain of VP55, containing a single lysine residue, was responsible for the interaction between VP55 and Ubc9 in yeast. In solid phase binding assays, a single amino acid mutation (K87R) prevented Ubc9 from binding to VP55. Overexpression of Ubc9 enhanced GCRV-104 infection efficiency, and knockdown of Ubc9 in CIK cells inhibited viral replication, which suggested that Ubc9 was a proviral factor. Furthermore, Ubc9 was shown to bind outer fiber proteins from type II GCRV, avian reovirus and mammalian reovirus in yeast. To our knowledge, this is the first study to show that Ubc9 binds to reovirus outer-fiber proteins and likely contributes to efficient orthoreovirus replication. These results suggest that SUMOylation modifications could be targeted to improve the therapeutic efficacy of oncolytic reovirus.

Repression of SUMOylation pathway by grass carp reovirus contributes to the upregulation of PKR in an IFN-independent manner

“…SUMO1/2 and Ubc9 are among the key molecules in host SUMOylation machinery, and we have reported the involvement of Ubc9 in the interaction between grass carp reovirus and host cells [ 8 ]. The transcription levels of Grass carp SUMO1/SUMO2 and Ubc9 in muscle, heart, intestine, kidney, gill, liver, spleen and brain were quantitatively measured by real time RT-PCR (Figure 2 ).…”

“…Grass carp reovirus (GCRV) has served as a protype of aquareoviruses (family Reoviridae, genus Aquareovirus) due to its strong pathogenicity for grass carp Ctenopharyngodon idellus by causing grass carp hemorrhagic disease; it has also been extensively studied for the interaction mechanism between dsRNA virus and dsRNA-initiated antiviral response [ 7 ]. In our previous study, grass carp Ubc9 bound to the N-terminal coiled-coil domain of GCRV-104 fiber protein and promoted viral infection efficiency [ 8 , 9 ]. However, it remains to be clarified whether direct SUMOylation of viral target or SUMOylation-mediated innate immune response is responsible for the pro-viral effect of Ubc9.…”

Grass Carp Reovirus VP56 Allies VP4, Recruits, Blocks, and Degrades RIG-I to More Effectively Attenuate IFN Responses and Facilitate Viral Evasion

“…Several attempts have been made to find out the membrane receptor of aquareovirus, including fibulin-4, Ubc9, LITAF, LamR, TIA1, etc. ( 33 – 37 ). However, the receptor of GCRV-II on the cell surface is still not determined.…”

Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus

“…cDNAs of purified RNAs, from infected cells or supernatants, were synthesized using PrimeScript reverse transcription system (TakaRa, Japan) following its product protocol. Real-time RT-PCR was performed according to our previous methods [ 7 , 23 ] using a CFX96 real-time PCR system (Bio-rad, USA) with the primer pairs of JX01F: CAAGACCATTCAAGACTC; JX01R: TCACTCACTTCGACTAAT and 104F: ATCGTCTTCAACCGCATAG; 104R: GGGCGTTACTTCCCTCAAC.…”