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

Rong Zhanga,b,1, Babal K. Jhac,1, Kristen M. Ogdend, Beihua Dongc, Ling Zhaoa,2, Ruth Elliotta, John T. Pattond, Robert H. Silvermanc,3, and Susan R. Weissa,3

aDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; bDepartment of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China; cDepartment of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195; and dRotavirus Molecular Biology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

Edited by Ralph S. Baric, University of North Carolina, Chapel Hill, NC, and accepted by the Editorial Board June 18, 2013 (received for review April 16, 2013) Efficient and productive infection often requires viral coun- induction of hepatitis (5–7). ns2 is a member of group II termeasures that block innate immunity. The IFN-inducible 2′,5′- (eukaryotic LigT) of the 2H-phosphoesterase superfamily, which oligoadenylate (2-5A) synthetases (OASs) and ribonuclease (RNase) contain two HxT/S catalytic motifs (8). ns2 has 2′,5′-phosphodi- L are components of a potent host antiviral pathway. We previ- esterase (PDE) activity that cleaves and inhibits the accumulation ously showed that murine (MHV) accessory protein of 2-5A, preventing activation of RNase L while enhancing viral ns2, a 2H phosphoesterase superfamily member, is a phosphodies- growth and pathogenesis (Fig. 1A)(9).Usingmicedeficient in − − terase (PDE) that cleaves 2-5A, thereby preventing activation of RNase L expression (RNase L / ), we demonstrated that ns2 PDE RNase L. The PDE activity of ns2 is required for MHV replication activity allows MHV to replicate to similar titer in the presence or in macrophages and for hepatitis. Here, we show that group A absence of RNase L in vitro in bone marrow–derived macro- (RVA), an important cause of acute gastroenteritis in phages (BMMs) and in the livers of infected mice. However, children worldwide, encodes a similar PDE. The RVA PDE forms mutant viruses expressing PDE-inactive ns2 were able to replicate − − − − the carboxy-terminal domain of the minor core protein VP3 (VP3- only in RNase L / BMM or in the livers of RNase L / mice. CTD) and shares sequence and predicted structural homology with Group II 2H phosphoesterases contain cellular proteins, in-

ns2, including two catalytic HxT/S motifs. Bacterially expressed cluding A-kinase-anchoring protein-7 (AKAP7), and other viral MICROBIOLOGY ′ ′ VP3-CTD exhibited 2 ,5 -PDE activity, which cleaved 2-5A in proteins, including VP3 (C-terminal domain) of group A rotavirus vitro. In addition, VP3-CTD expressed transiently in mammalian (RVA) and polypeptide 1a of (C-terminal protein), cells depleted 2-5A levels induced by OAS activation with poly each of which contains two HxT/S catalytic motifs separated by (rI):poly(rC), preventing RNase L activation. In the context of similar distances (8). Based on this homology, and on evidence recombinant chimeric MHV expressing inactive ns2, VP3-CTD re- that RVA VP3 is a virulence factor (10, 11), we hypothesized stored the ability of the virus to replicate efficiently in macro- that RVA VP3 contains an antagonist of RNase L activation, phages or in the livers of infected mice, whereas mutant viruses similar to MHV ns2. expressing inactive VP3-CTD (H718A or H798R) were attenuated. , nonenveloped viruses with dsRNA , are In addition, chimeric viruses expressing either active ns2 or VP3- a common cause of severe diarrhea in infants and young children. CTD, but not nonfunctional equivalents, were able to protect ribo- somal RNA from RNase L–mediated degradation. Thus, VP3-CTD is Despite using a strategy of gene expression and replication very a2′,5′-PDE able to functionally substitute for ns2 in MHV infection. different from that of coronavirus, we show that RVA uses a sim- Remarkably, therefore, two disparate RNA viruses encode proteins ilar mechanism as MHV to antagonize the activation of RNase L. ′ ′ We found that the C-terminal domain (CTD; ∼143 amino acids) of with homologous 2 ,5 -PDEs that antagonize activation of innate fl immunity. RVA VP3, a minor protein component of the virion core chie y involved in RNA capping (12, 13), has a predicted structure ho- B C ′ ′ | Nidovirales | RNA capping enzyme | mologous to ns2 (Fig. 1 and ). Accordingly, VP3-CTD has 2 ,5 interferon-stiumulated gene PDE activity that cleaves 2-5A in vitro and in intact cells, thereby antagonizing RNase L activation. Furthermore, in chimeric re- combinant MHV strains, VP3-CTD is able to effectively sub- he 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–ribonucle- stitute for a mutant ns2 gene, permitting viral replication in ase (RNase) L pathway is among the most potent IFN- T BMMs and in the livers of infected mice, even in the presence of induced antiviral effectors, blocking viral infections by several RNase L. Our results reveal a previously unknown function for mechanisms, including directly cleaving viral single-stranded RVA VP3, one that has potential importance in counteracting RNA genomes, depleting viral and host mRNA available for antiviral innate immunity. translation, and enhancing type I IFN induction (1). Type I IFNs bind to the cell surface receptor IFNAR (interferon-α/β receptor),

initiating JAK-STAT signaling to the OAS genes, which results in Author contributions: R.Z., B.K.J., K.M.O., B.D., L.Z., J.T.P., R.H.S., and S.R.W. designed elevated levels of OAS proteins. When activated by viral double- research; R.Z., B.K.J., K.M.O., B.D., and R.E. performed research; R.Z., B.K.J., K.M.O., stranded (ds)RNA, certain OAS isoforms use ATP to synthesize B.D., R.E., J.T.P., R.H.S., and S.R.W. analyzed data; and R.Z., B.K.J., K.M.O., B.D., J.T.P., 5′-triphosphorylated 2-5A. Trimer and longer species of 2-5A R.H.S., and S.R.W. wrote the paper. bind with high specificity and affinity to the inactive monomeric The authors declare no conflict of interest. RNase L, causing it to dimerize and become active (2) (Fig. 1A). This article is a PNAS Direct Submission. R.S.B. is a guest editor invited by the Not surprisingly, viruses have evolved multiple mechanisms to Editorial Board. 1 antagonize or prevent activation of RNase L (1). One such virus R.Z. and B.K.J. contributed equally to this work. 2 is mouse hepatitis virus (MHV) (3), a member of the enveloped, Present address: State Key Laboratory of Agricultural Microbiology, Huazhong Agricul- tural University, Wuhan 430070, China. positive-stranded (+)RNA coronavirus species. MHV and re- 3To whom correspondence may be addressed. E-mail: [email protected] or lated 2a-betacoronaviruses (4) encode ns2, a cytoplasmic 30-kDa [email protected]. protein that is dispensable for virus replication in transformed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cell lines but serves as an IFN antagonist and is required for the 1073/pnas.1306917110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1306917110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 residues were located in homologous locations in each structure, suggesting a similar PDE function (Fig. 1C; Fig. S2).

Purified Rotavirus VP3-CTD has 2′,5′-PDE Activity Against 2-5A. Amino acid homology and predicted structural similarity suggested that VP3-CTD, like MHV ns2, was capable of degrading 2-5A. To determine whether VP3-CTD is a 2′,5′-PDE, the protein was expressed in Escherichia coli, purified, and incubated with the 2-5A trimer, (2′-5′)p3A3. Following incubation, the 2-5A sub- strate was separated from the reaction products by HPLC (Fig. 2A). VP3-CTD produced a profile of reaction products with the expected degradation of (2′-5′)p3A3 to (2′-5′)p3A2,ATP,and AMP as a function of incubation time (Fig. 2A). However, mu- tation of the two predicted catalytic His residues of VP3-CTD (H718A and H797R) produced protein that was deficient in 2-5A degradation (Fig. 2B). MHV ns2 generated a profile of reaction products like that of WT VP3-CTD (Fig. 2 A and C) (9). The similar activities of MHV ns2 and rotavirus VP3-CTD were apparent from the kinetics of 2-5A cleavage (Fig. 2C).

VP3-CTD Degrades 2-5A in Intact Cells. To determine whether VP3- CTD was capable of cleaving 2-5A in intact cells, vectors encoding WT VP3-CTD, mutant VP3-CTD (H718A and H797R), and ns2 were individually transfected into human Hey1b ovarian carci- noma cells. Afterward, cells were treated with poly(rI):poly(rC) to activate OAS. The presence of functional forms of 2-5A in the intact cells was determined by adding nucleotide pools isolated from the treated cells to a specific FRET-based RNase L acti- vation assay (17) (SI Materials and Methods). Protein expression was monitored by Western blot (Fig. 3A). A standard RNase L activation profile with (2′-5′)p3A3 produced a 50% effective concentration (EC50) of about 0.3 nM (2′-5′)p3A3 (Fig. 3B). Fig. 1. Involvement of MHV ns2 and rotavirus VP3 in antagonizing RNase L Although no 2-5A was detected in untreated cells transfected during the IFN antiviral response. (A) Viral–host interactions between MHV with empty vector, cells transfected with empty vector and poly ns2, rotavirus VP3, and the OAS-RNase L system determine the success or (rI):poly(rC) accumulated 2-5A as determined by RNase L ac- failure of viral infections. (B) Location of PDE (phosphodiestase) domains in tivation (Fig. 3C). Remarkably, there was no accumulation of MHV ns2 and rotavirus VP3. GTase, guanylyltransferase; MTase, methyl- transferase. (C) Comparative structural analysis of AKAP7 central domain, 2-5A in cells transfected with VP3-CTD vector and poly(rI):poly VP3-CTD, and ns2. Alignments with AKAP7 were used to predict a structural (rC). In contrast, 2-5A accumulated in cells transfected with the model for SA11 VP3-CTD (amino acids 695–835) and MHV A59 ns2 (Results). mutant VP3-CTD (H718A and H797R) vector and poly(rI):poly PyMol (http://pymol.org) was used for molecular graphics. Ribbon repre- (rC), resulting in RNase L activation. MHV ns2 prevented 2-5A sentation of rat AKAP7 amino acids 89–291 (PDB ID code 2VFY) were com- accumulation in poly(rI):poly(rC)-treated cells to a similar extent pared with the predicted models of the C-terminal region of SA11 VP3 as VP3-CTD. These data indicate that VP3-CTD can degrade – protein and MHV A59 ns2 protein, amino acids 6 195. Colors highlight 2-5A in intact cells. 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. VP3-CTD Rescues Replication of ns2 Mutant Viruses in B6 BMMs. After observing that rotavirus VP3-CTD cleaves 2-5A in vitro and in intact cells, we hypothesized that VP3-CTD could compensate Results for the loss of ns2 function in mutant MHV-infected B6 BMMs. Homology Between MHV ns2 and Rotavirus VP3-CTD Suggests To test this hypothesis, we constructed a series of recombinant a Common Function in Antagonizing the OAS-RNase L Antiviral viruses using an infectious cDNA clone of MHV strain A59 (18) SI Materials and Methods A Pathway. MHV ns2 evades the antiviral activity of IFN in BMMs ( ; Fig. 4 ). The recombinant viruses included WT A59 and A59 expressing ns2 in which the catalytic by degrading 2-5A, the activator of RNase L (9). Sequence H46A H126R alignments between ns2 and VP3-CTD revealed a pair of HxT/S His residues were individually mutated (ns2 or ns2 ). In addition, we constructed isogenic chimeric A59-VP3 viruses ex- motifs, a characteristic feature of the catalytic ligand-binding cleft pressing ns2H126R and either WT VP3-CTD or VP3-CTD in which of 2H phosphoesterases (8) (Fig. S1). To determine whether these the catalytic His residues were individually mutated (ns2H126R- motifs were likely to serve the same function for ns2 and VP3- VP3, ns2H126R-VP3H718A, and ns2H126R-VP3H797R)inplaceof CTD, the amino acid sequences of these molecules were aligned the nonessential ns4 protein (19) (Fig. 4A). against the nearest template of known crystal structure for the The replication kinetics of these viruses was evaluated in central domain of the rat AKAP7 (PDB ID code 2VFY) (14). mouse L2 fibroblasts, a cell type in which MHV does not induce AKAP7 contains a 2H phosphoesterase domain from amino acids IFN-β expression (20). All mutant and chimeric viruses repli- 88–292 that binds AMP. The aligned template was then used to cated with similar kinetics and to similar final titers as A59, in- predict a structural model for VP3-CTD and ns2 using Mod- dicating that neither the mutations in ns2 nor the insertion of eller9V5, which implements comparative protein structure model- VP3-CTD in place of ns4 had any detectable effect on virus ing by satisfaction of spatial restraints (15). The highest confidence replication (Fig. S3A). Western blot analysis demonstrated that models were generated for each molecule, and accuracy of the WT and mutant ns2 and VP3-CTD proteins were expressed in models was assessed using Verify3D (16). The two catalytic His the virus-infected L2 cells (Fig. S3B). Immunofluoresent staining

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1306917110 Zhang et al. Downloaded by guest on October 1, 2021 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 VP3H718A;H797R,or(C) MHV A59 ns2. HPLC analyses of the substrate and reaction products at different MICROBIOLOGY times of incubation are indicated. (D) Quantitation of intact 2-5A remaining at different times of in- cubation 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].

− − of virus-infected murine 17Cl-1 cells further demonstrated that no effect on virus replication in RNase L / cells. As expected, both ns2 and VP3-CTD localized to the cytoplasm (Fig. S3C). all tested mutant and chimeric viruses replicated efficiently, with − − We next assessed viral replication in BMMs from B6 mice, similar kinetics and final titer, in RNase L / BMMs (Fig. 4B) a cell type in which MHV induces IFN expression, and ns2 PDE (9). These results demonstrate that the expression of VP3-CTD activity is needed to ensure efficient replication (21). Replication can functionally replace A59 ns2 during infection of B6 BMMs. of ns2 mutant viruses (ns2H46A and ns2H126R) was severely im- A consequence of activation of the OAS–RNase L pathway is paired in B6 BMMs in comparison with that of A59 (Fig. 4B). RNase L–mediated RNA degradation, usually assessed by analysis Intriguingly, the chimeric virus ns2H126R-VP3, expressing VP3- of rRNA integrity (22). To further verify that restoration of ns2 CTD and a mutant ns2, replicated to nearly the same level as mutant virus replication results from blocking the OAS-RNase L A59, suggesting that VP3-CTD was able to replace the function pathway, we compared the state of rRNA in B6 BMMs infected with of ns2 within the A59 . In contrast, chimeric viruses each recombinant virus by RNA chip analysis using an Agilent an- expressing an inactive VP3 PDE in addition to mutant ns2, alyzer. By 12 h postinfection, degradation of rRNA by RNase L was ns2H126R-VP3H718A, and ns2H126R-VP3H797R replicated only to apparent in cells infected with viruses expressing ns2 H46A, the same extent as ns2 mutant viruses, with a final titer almost ns2H126R,ns2H126R-VP3H797R,orns2H126R-VP3H797R (Fig. 4C). In 100-fold less than ns2H126R-VP3. Because 2-5A is responsible for contrast, rRNA remained intact in cells infected with A59 viruses triggering RNase L activation, accumulation of 2-5A should have expressing a functional PDE, either ns2 or VP3-CTD.

Fig. 3. Cleavage of 2-5A in intact cells by VP3 and ns2asdeterminedinRNaseLactivationFRETassays. (A) Lysates of Hey1B cells transfected with plasmid expressing ns2, FLAG-tagged VP3, or FLAG-tagged VP3H718A,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 VP3H718A;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.

Zhang et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 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, per- formed 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 extrac- − − ted 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).

Expression of VP3 Enhances Replication of ns2 Mutant Virus in Liver. are produced by two very different RNA viruses; one virus Because VP3 was able to functionally replace ns2 function in (MHV) is pathogenic for mice, whereas the other virus (RVA) BMMs, we hypothesized that replication of ns2 mutant virus in is an important gastrointestinal pathogen in young children. As the liver would be restored by expression of VP3-CTD from the there are relatively few similarities between the enveloped, positive- − − viral genome. To test this idea, B6 and RNase L / mice were stranded RNA coronavirus MHV and the nonenveloped, seg- infected intrahepatically with A59 and mutant viruses, and the mented, dsRNA RVA, it is remarkable that both viruses encode virus titers were determined in the livers of mice killed at day 5 homologous 2H PDE domains. after infection, which is the peak of A59 replication in this organ ns2 is a nonstructural protein translated from a monocistronic (23, 24). As expected, ns2H126R replicated minimally in B6 ′ ′ − − mRNA, whereas the rotavirus 2 ,5 -PDE is found as the C-ter- mice but robustly and to the same level as A59 in RNase L / minal domain of a larger structural protein that also contains mice (Fig. 4D). We compared replication of chimeric viruses rotavirus capping activities. ns2 resides in the cytoplasm (7), the H126R expressing either WT VP3-CTD (ns2 -VP3)ormutant site of replication for most RNA viruses. This localization agrees H126R H797R H126R VP3-CTD (ns2 -VP3 ). Although the titers of ns2 - with ns2 function in 2-5A cleavage and RNase L antagonism. In VP3 virus reached 104 plaque forming units (PFU)/g tissue in contrast, VP3 is a structural protein that is encapsidated in RVA B6 mice, ns2H126R-VP3H797R, expressing inactive PDEs, was un- virions and caps nascent transcripts synthesized within tran- detectable. All of the chimeric viruses replicated equally well in scriptionally active subviral particles. Following translation, VP3 − − RNase L / mice, regardless of the presence or absence of a accumulates in , sites of new particle assembly, RNA functional PDE (Fig. 4D). These results show that expression of the packaging, dsRNA synthesis, and secondary rounds of tran- active VP3 PDE confers increased replication of ns2 mutant virus scription (25). Although it is not known whether VP3 is also in livers of WT B6 animals. Therefore, our results identify a pre- localized diffusely within the cytoplasm, we hypothesize that viously unknown PDE activity of rotavirus VP3-CTD that func- anon–particle-associated form of VP3 mediates its RNase L tions in countering the OAS–RNase L pathway in vivo and in antagonistic activities. Because different isoforms of OAS func- BMMs in vitro. tion at different subcellular locations and may respond to dif- ferent dsRNA triggers to synthesize different forms of 2-5A (1), Discussion future studies to determine the localization of these viral PDEs 2-5A degradation by virus-encoded 2′,5′-PDEs to prevent RNase might enhance an understanding of their function. L activation is a newly recognized viral strategy for evading in- Due to the essential role of VP3 in RNA capping (12, 13) and nate immunity, discovered during studies on the coronavirus lack of a robust reverse genetics system for rotaviruses, it is not MHV (9). Here, we have shown that two highly similar 2′,5′- currently possible to directly assess the role of VP3 PDE activity PDEs capable of cleaving 2-5A and antagonizing RNase L in the context of rotavirus infection. Nonetheless, several lines of

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1306917110 Zhang et al. Downloaded by guest on October 1, 2021 evidence suggest that, like ns2, RVA VP3-CTD functions to promoting proteasome-mediated degradation of IFN regulatory prevent RNase L activation via 2-5A degradation. First, in vitro, factors (IRFs) and β-transducin repeat-containing protein (βTrCP) both VP3-CTD and ns2 degrade 2-5A from the 2′,3′-termini of (33). The interplay of these overlapping innate immune antago- 2-5A molecules, releasing 5′-AMP residues one at a time until nistic activities may enhance viral infection, spread, and patho- the 5′-terminal ATP moiety is released (9) (Fig. 2). The mini- genesis in specific cell types. RVA viremia is more common than mum active species of 2-5A is triadenylate; therefore, once previously thought, with virus detected in the blood and extra- a single 5′-AMP residue is cleaved from trimer 2-5A to yield the intestinal tissues of many infected children (34). Additionally, dimeric species, (2′,5′)p3A2, the ability to activate RNase L is lost RVA is capable of infecting a subpopulation of human plasma- (26). VP3-CTD and ns2 also have a remarkable ability to prevent cytoid dendritic cells (pDCs) (35). pDC contact with rotavirus activation of RNase L in cells transfected with the potent OAS results in activation, maturation, and cytokine production. In- activator, poly(rI):poly(rC) (Fig. 3C). To be effective, 2′,5′-PDE terestingly, IFNα production in infected pDCs is impaired; yet activity needs to deplete 2-5A to levels lower than those neces- levels of IRF7 are similar to those in uninfected cells, which sug- sary to maintain RNase L activation. Our findings emphasize the gests that NSP1 is not solely responsible for reduced IFN pro- ability of VP3-CTD and ns2 to rapidly and efficiently degrade duction. Further research is needed, but it is possible that infection 2-5A at a rate that exceeds the ability of OAS to synthesize 2-5A. of pDCs serves as a mechanism by which rotavirus escapes the A chimeric MHV recombinant expressing both VP3-CTD and an intestine and that antagonism ofRNaseLvia2-5Acleavageby H126R inactive ns2 PDE (ns2 -VP3) replicated to nearly the same VP3-CTD might function to enhance viral spread. level as A59 in B6 BMMs and prevented rRNA cleavage, in- To prevent cellular and tissue damage from excessive activation dicating that the rotavirus PDE has the same function with of RNase L, certain host proteins are able to degrade 2-5A mole- a similar level of activity as ns2 in the context of MHV infection cules. Unlike virally encoded 2′,5′-PDEs, cellular enzymes that H126R (9) (Fig. 4B). Finally, VP3-CTD expressed by ns2 -VP3 was mediate 2-5A decay, most notably the exonuclease-endonuclease- able to confer replication in the livers of B6 mice, whereas phosphatase PDE12 (2′-PDE) (36–38), are often insufficient to a chimeric virus expressing both mutant ns2 and mutant VP3 prevent RNase L activation in response to potent stimulation of H126R H797R (ns2 -VP3 ) failed to replicate at the lower level of de- OAS proteins by viral dsRNA. Host 2-5A degrading enzymes de- H126R tection (Fig. 4D). The failure of ns2 -VP3 to replicate to the scribed to date have amino acid sequences that are unrelated to ns2 same level as A59 in liver is most likely due to perturbation of the and VP3. Thus, it is possible that there may be other viral non–2H- ns4 region of the genome, as previously observed for chimeric PDEs with the same function of ns2 and VP3-CTD that would be viruses in which ns4 was replaced with EGFP (27). Thus, although missed by the searches for proteins with 2H-PDE homology (8). MICROBIOLOGY A59 induces inflammation and necrosis in the liver and the livers H126R The cellular enzyme AKAP7, however, is a 2H phosphoesterase of mice infected with ns2 show no evidence of pathological family member with amino acid and predicted structural homology changes (9), we were unable to measure differences in liver pa- H126R H126R to both ns2 and VP3 (14). The 2H phosphoesterase domain of thology between mice infected with ns2 and ns2 -VP3, AKAP7 could be the host gene originally acquired by an ancestral as neither virus replicated to a high enough titer to produce nidovirus (precursor to both and ) and by fi signi cant hepatitis (28). a rotavirus to evade the antiviral activity of the OAS-RNase L in- Eight species of rotavirus (RVA-RVH) have been proposed, nate immunity pathway. We are currently investigating the possible with RVA causing the majority of human disease (29, 30). Based role of AKAP7 in 2-5A turnover during viral infections. Further- on alignment of VP3 sequences (Fig. S4), all RVA strains, in- more, we predict that inhibitors of viral and host 2′,5′-PDEs may cluding those of both human and animal isolates, encode a sim- have antiviral activities and will be useful research tools for probing ilar PDE domain. The presence of similar 2H-PDE motifs and the roles of these enzymes in innate immunity. numerous other conserved residues suggests that a PDE domain is also present at the VP3 C terminus of RVB and RVG strains. Materials and Methods The absence of comparable PDE-like domains for RVC, RVF, Cell Lines and Mice. Murine L2 fibroblast (L2), murine 17 clone-1 fibroblast and RVH strains accounts for the comparative decreased size of (17Cl-1), and baby hamster kidney cells expressing MHV receptor (BHK- their VP3 proteins. Because VP3 PDE cleaves 2-5A and antag- MHVRs) were cultured as described previously (18, 31). Plaque assays were onizes RNase L and it is absent from some rotavirus species, this performed on L2 cells (39). B6 mice were purchased from Jackson Labora- region likely is the virulence factor to which the VP3-encoding tory. RNase L−/− mice (B6, 10 generations of back-crossing) (40) were bred in gene has previously been linked (10, 11), and its presence may the University of Pennsylvania animal facility. Hey1b human ovarian carci- contribute to the success of the RVA. noma cell lines were cultured as described previously (a gift of A. Marks, University of Toronto, Toronto, Canada) (41) and standardized for studies on Cell type might be an important determinant of viral outcomes – resulting from the antagonistic effects of ns2 and VP3 on RNase L. the OAS RNase L pathway (42). We showed previously that, although dispensable for replication in ′ ′ Plasmids and Recombinant Viruses. The 3′ terminus of VP3 encoding 143 transformed cell lines, the 2 ,5 -PDE activity of ns2 was required for fi fi amino acids was ampli ed from the plasmid pBSmod g3(VP3-SA11) derived ef cient MHV replication in myeloid cells and for pathogenesis in from SA11-4F virus strain (GenBank accession no. DQ838641) and cloned into the liver (21). However, MHV replication in the central nervous mammalian expression vector pCAGGS, resulting in the pC-VP3. The His resi- system, the other major target of infection, and the consequent dues at positions 46 and 126 were both mutated resulting in pC-VP3H46A; H126R. encephalitis was independent of 2′,5′-PDE activity (31). This The ORFs for VP3 and mutant VP3H46A; H126R CTDs were cloned into E. coli phenotype can be attributed at least in part to the low levels of expression vector pMAL-c2, resulting in pMAL-VP3 and pMAL-VP3H46A; H126R. expression of IFN-responsive genes, including OAS, and the muted pC-ns2 and pMAL-ns2 used in this study were constructed previously (9). IFN signaling response in the brain compared with the liver (21). Construction of recombinant viruses, BMM preparation, infection of BMMs, Rotavirus primarily infects mature enterocytes of the villus and viral plaque assays are described in the SI Materials and Methods. small intestine (32). Because rotavirus species that lack the VP3- ′ ′ fi CTD, such as RVC, are capable of producing infections in 2 ,5 -PDE Activity Assays. Expression and puri cation of VP3 are described in SI Materials and Methods. The 2′,5′-PDE activities of ns2 and VP3 were de- humans that result in gastroenteritis (29), we predict that 2′,5′- termined with purified (2′-5′)p3A3 as substrate followed by HPLC to sep- PDE activity is dispensable for successful replication in the arate the substrate from the products (43) (for details, see SI Materials gastrointestinal tract but that this activity might be important for and Methods). spread to distal sites and enhanced virulence. RVAs encode a protein (NSP1) in addition to the VP3-CTD that antagonizes Quantification of 2-5A by FRET Assays. 2-5A present in cell extracts was host innate immune defenses. NSP1 inhibits IFN production by measured by activation of recombinant, human RNase L in FRET assays (44).

Zhang et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 Briefly, RNase L was incubated with (2′-5′)p3A3 or 2-5A present in isolated, day 5 after infection, the mice were killed. After perfusion with PBS, nucleotide pools from cells (see SI Materials and Methods for extraction livers were removed, homogenized, and titrated by plaque assays on L2 method). The FRET probe [5′-(6-FAM-UUAUC AAAUUCUUAUUUGCCCCAUU- cells as previously described. All mouse experiments were reviewed and ′ UUUUUGGUUUA-BHQ-1)-3 ] was added and incubated at 20 °C for 45 min. approved by the University of Pennsylvania Institutional Animal Care fl Fluorescence was measured with a Wallac 1420 uorimeter (Perkin-Elmer) and Use Committee. (excitation 485 nM/emission 535 nm with a 0.1-s integration time). Statistical Analysis. Two-tailed t tests were performed to determine statistical rRNA Cleavage Assays. 2-5A–induced rRNA cleavage was monitored in virus- significance. P values are shown in the figures. Data were analyzed with infected B6 BMMs. Total cellular RNA was isolated at 6 or 12 h after infection GraphPad Prism (GraphPad Software). with the RNeasy kit (Qiagen). RNA was quantified by a Nanodrop analyzer, and equal quantities of RNA were resolved on RNA chips using an Agilent 2100 BioAnalyzer (45). ACKNOWLEDGMENTS. We thank Sasha Stone and L. Dillon Birdwell for technical help. This work was supported by National Institutes of Health Grants RO1-AI104887 (to S.R.W. and R.H.S.) and R56-AI-095285 (to S.R.W.) −/− Mouse Infections. Four-week-old B6 or RNase L mice were anesthetized and the Intramural Research Program of the National Institute of Allergy with isoflurane (IsoFlo; Abbott Laboratories) and inoculated intrahepatically and Infectious Diseases, National Institutes of Health (J.T.P. and K.M.O.). R.Z. with each virus (2,000 PFU per mouse) in 50 μL PBS containing 0.75% BSA. At was supported in part by the China Scholarship council.

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