Homologous 2',5'-phosphodiesterases from disparate RNA viruses antagonize antiviral innate immunity

Abstract

Efficient and productive virus infection often requires viral countermeasures that block innate immunity. The IFN-inducible 2',5'-oligoadenylate (2-5A) synthetases (OASs) and ribonuclease (RNase) L are components of a potent host antiviral pathway. We previously showed that murine coronavirus (MHV) accessory protein ns2, a 2H phosphoesterase superfamily member, is a phosphodiesterase (PDE) that cleaves 2-5A, thereby preventing activation of RNase L. The PDE activity of ns2 is required for MHV replication in macrophages and for hepatitis. Here, we show that group A rotavirus (RVA), an important cause of acute gastroenteritis in children worldwide, encodes a similar PDE. The RVA PDE forms the carboxy-terminal domain of the minor core protein VP3 (VP3-CTD) and shares sequence and predicted structural homology with ns2, including two catalytic HxT/S motifs. Bacterially expressed VP3-CTD exhibited 2',5'-PDE activity, which cleaved 2-5A in vitro. In addition, VP3-CTD expressed transiently in mammalian cells depleted 2-5A levels induced by OAS activation with poly(rI):poly(rC), preventing RNase L activation. In the context of recombinant chimeric MHV expressing inactive ns2, VP3-CTD restored the ability of the virus to replicate efficiently in macrophages or in the livers of infected mice, whereas mutant viruses expressing inactive VP3-CTD (H718A or H798R) were attenuated. In addition, chimeric viruses expressing either active ns2 or VP3-CTD, but not nonfunctional equivalents, were able to protect ribosomal RNA from RNase L-mediated degradation. Thus, VP3-CTD is a 2',5'-PDE able to functionally substitute for ns2 in MHV infection. Remarkably, therefore, two disparate RNA viruses encode proteins with homologous 2',5'-PDEs that antagonize activation of innate immunity.

Keywords: Nidovirales; RNA capping enzyme; Reoviridae; interferon-stiumulated gene.

Fig. 1.

Involvement of MHV ns2 and rotavirus VP3 in antagonizing RNase L during the IFN antiviral response. (A) Viral–host interactions between MHV ns2, rotavirus VP3, and the OAS-RNase L system determine the success or failure of viral infections. (B) Location of PDE (phosphodiestase) domains in MHV ns2 and rotavirus VP3. GTase, guanylyltransferase; MTase, methyltransferase. (C) Comparative structural analysis of AKAP7 central domain, VP3-CTD, and ns2. Alignments with AKAP7 were used to predict a structural model for SA11 VP3-CTD (amino acids 695–835) and MHV A59 ns2 (Results). PyMol (http://pymol.org) was used for molecular graphics. Ribbon representation of rat AKAP7 amino acids 89–291 (PDB ID code 2VFY) were compared with the predicted models of the C-terminal region of SA11 VP3 protein and MHV A59 ns2 protein, amino acids 6–195. Colors highlight similar regions of secondary structure. The two conserved His residues (in His-x-S/T motifs) of AKAP7, VP3, and ns2 (mutated in this study) are shown as sticks in red.

Fig. 2.

VP3-CTD is a 2′,5′-PDE that cleaves 5′-AMP from the 2′,3′-termini of 2-5A molecules. The 2-5A substrate, (2′-5′)p₃A₃, was incubated with purified proteins: (A) CTD of SA11 VP3, (B) CTD of SA11 VP3^H718A;H797R, or (C) MHV A59 ns2. HPLC analyses of the substrate and reaction products at different times of incubation are indicated. (D) Quantitation of intact 2-5A remaining at different times of incubation were determined by calculating the area under the peaks from the HPLC profiles [shown as a percentage of the substrate (2′-5′)p₃A₃ that remained intact].

Fig. 3.

Cleavage of 2-5A in intact cells by VP3 and ns2 as determined in RNase L activation FRET assays. (A) Lysates of Hey1B cells transfected with plasmid expressing ns2, FLAG-tagged VP3, or FLAG-tagged VP3^H718A,H797R were analyzed by Western blot using antibodies directed against ns2, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG), or GAPDH. (B) Standard curve of RNase L activated by different concentrations of (2′-5′)p₃A₃ as determined in FRET assays. (C) Detection of 2-5A in Hey1b cells transfected with empty vector (vector) or with plasmids expressing VP3-CTD, mutant VP3^H718A;H797R-CTD, or ns2 (as indicated) and then transfected with poly(rI):poly(rC). FRET assays were performed on nucleotide pools isolated from the treated cells. RFU, relative fluorescence units.

Fig. 4.

Comparison of chimeric MHVs expressing WT or mutant ns2 and VP3. (A) Schematic diagram of recombinant viruses. (B) B6 or RNase L^−/− BMM were infected (1 PFU per cell) and virus titered from the supernatant at the indicated times. Data are from one representative experiment of two, performed in triplicate and shown as the means ± SEM. (C) B6 BMMs were infected (1 PFU per cell), and at 12 h after infection, total cellular RNA was extracted and analyzed on RNA chips. (D) B6 or RNase L^−/− mice were infected intrahepatically (2,000 PFU per mouse). Infectious virus was titered from the livers of mice killed at 5 d after infection (n = 5). The dashed line represents the limit of detection. Data are from one representative experiment of two and are shown as the means ± SEM (***P < 0.001).