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An RNA Virus Hijacks an Incognito Function of a DNA Repair Enzyme

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An RNA Virus Hijacks an Incognito Function of a DNA Repair Enzyme An RNA virus hijacks an incognito function of a DNA repair enzyme Richard Virgen-Slanea, Janet M. Rozovicsa, Kerry D. Fitzgeralda, Tuan Ngob, Wayne Choub, Gerbrand J. van der Heden van Noortc, Dmitri V. Filippovc, Paul D. Gershonb, and Bert L. Semlera,1 aDepartment of Microbiology and Molecular Genetics, School of Medicine, and bDepartment of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697; and cLeiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands Edited* by Charles M. Rice, The Rockefeller University, New York, NY, and approved July 26, 2012 (received for review May 14, 2012) A previously described mammalian cell activity, called VPg unlin- generated in bacteria, we showed the in vitro cleavage of the kase, specifically cleaves a unique protein–RNA covalent linkage VPg–RNA linkage for both poliovirus and human rhinovirus. generated during the viral genomic RNA replication steps of a pi- Finally, confocal microscopy of HeLa cells infected with polio- cornavirus infection. For over three decades, the identity of this virus revealed that TDP2 is relocalized in the cell to cytoplasmic cellular activity and its normal role in the uninfected cell had sites distinct from those containing viral proteins associated with remained elusive. Here we report the purification and identifica- RNA replication or encapsidation, suggesting that the virus may tion of VPg unlinkase as the DNA repair enzyme, 5′-tyrosyl–DNA exclude VPg unlinkase/TDP2 from viral complexes that require an intact VPg–RNA linkage during the later stages of infection. We phosphodiesterase-2 (TDP2). Our data show that VPg unlinkase fi activity in different mammalian cell lines correlates with their dif- discuss the implications of our ndings to highlight how a large ferential expression of TDP2. Furthermore, we show that recombi- family of medically important RNA viruses appears to have hijacked a unique phosphodiesterase function from a host cell nant TDP2 can cleave the protein–RNA linkage generated by DNA repair enzyme, providing an extraordinary example of different picornaviruses without impairing the integrity of viral pathogen ingenuity. RNA. Our results reveal a unique RNA repair-like function for TDP2 and suggest an unusual role in host–pathogen interactions Results and Discussion for this cellular enzyme. On the basis of the identification of TDP2 fi fi To identify VPg unlinkase, we developed a multi-step puri cation as a potential antiviral target, our ndings may lead to the de- procedure using our recently described VPg unlinkase activity assay, velopment of universal therapeutics to treat the millions of indi- which resolves [35S]methionine radiolabeled-VPg released from fl viduals af icted annually with diseases caused by picornaviruses, poliovirus vRNA (35S-PV1–RNA) by Tris-tricine SDS/PAGE (7). including myocarditis, aseptic meningitis, encephalitis, hepatitis, Our assay was optimized for rapid detection of VPg unlinkase ac- and the common cold. tivity (Materials and Methods). During the development of this pu- rification scheme, we screened different synthetic compounds 5′-tyrosyl-RNA phosphodiesterase | poliovirus | human rhinovirus | and nucleic acids as competitive inhibitors to be used in the af- translation initiation finity purification of VPg unlinkase. We made a fortuitous ob- servation that single-strand DNA (ssDNA) is ∼100-fold more NA viruses with limited genomic coding capacities require the efficient at inhibiting VPg unlinkase activity than synthetic RNA Rextensive use of host cell functions to carry out their replica- with or without a 5′-tyrosyl–RNA bond (Fig. S1). This key finding tion cycles in mammalian cells. As such, unraveling the mecha- prompted us to include ssDNA cellulose in our purification protocol. nisms of gene expression and replication for these viruses Because VPg unlinkase activity is found in both cytoplasmic and necessitates that we identify the cellular proteins responsible for nuclear extracts (2), we initiated our purification procedure using such functions. For the positive-strand RNA viruses in the pi- total cell homogenate from uninfected HeLa cells. After subject- cornavirus family (including poliovirus, human rhinovirus, and ing the homogenate to high-speed centrifugation to pellet cellular foot-and-mouth disease virus), host cell functions are required for debris and large complexes containing nucleic-acid–binding pro- several steps in their intracellular replication cycles, including teins, the supernatant (S370), which contained ∼75% of the initial viral translation and RNA synthesis. These viruses use a small activity, was fractionated sequentially by heparin-sepharose, viral protein (VPg) as a primer for viral RNA synthesis, which ssDNA-cellulose, anion-exchange, size-exclusion, and cation-ex- results in the linkage of all nascent viral RNAs to VPg via an O4- change chromatography, resulting in the generation of a nearly (5′-uridylyl)tyrosine bond (reviewed in ref. 1). Following genome homogeneous enzyme preparation in which activity was enriched release from the infecting virion, however, the VPg–RNA linkage by >10,000-fold (Fig. 1 B and C). The resulting 38-kDa poly- of virion RNA (vRNA) is short lived (refer to the picornavirus peptide (p38) isolated by this purification scheme (Fig. 1B, lane 7, RNA species depicted in Fig. 1A). Upon polysome association, purification step F) corresponded in size with the VPg unlinkase VPg is removed from vRNA by a cellular enzyme (2), which will detected during our initial characterization of this activity (Fig. be referred to as “VPg unlinkase” in this report. Interestingly, all S2). Analysis of fractions from purification step F (cation-ex- viral RNAs packaged into virions maintain VPg at the 5′ ends of change chromatography) verified the coelution of p38 (Fig. 2A, progeny RNAs, suggesting that VPg unlinkase activity may be Lower) with VPg unlinkase activity (Fig. 2A, Upper and Fig. S3). modulated during the later stages of picornavirus infections. Since The protein corresponding to p38 was excised from two dif- its discovery in 1978 (2), several studies have described the partial ferent lanes of a polyacrylamide gel (Fig. 2A, lanes 7 and 8, purification (3, 4) and biochemical characterization (reviewed in ref. 5) of VPg unlinkase and explored its possible role(s) during picornavirus infections (2, 3, 6–9). Despite these efforts, the cellular Author contributions: R.V.-S., K.D.F., and B.L.S. designed research; R.V.-S., J.M.R., K.D.F., T.N., identity of this remarkable enzyme has remained elusive. In this and P.D.G. performed research; R.V.-S., J.M.R., T.N., W.C., G.J.v.d.H.v.N., D.V.F., and P.D.G. report we describe the identification of VPg unlinkase from contributed new reagents/analytic tools; R.V.-S., K.D.F., T.N., P.D.G., and B.L.S. analyzed uninfected HeLa cells as 5′-tyrosyl–DNA phosphodiesterase-2 data; and R.V.-S., K.D.F., and B.L.S. wrote the paper. (TDP2), a DNA repair enzyme with additional proposed functions The authors declare no conflict of interest. in the mammalian cell. Western blot analysis demonstrated the *This Direct Submission article had a prearranged editor. presence of TDP2 in fractionated extracts enriched for VPg 1To whom correspondence should be addressed. E-mail: [email protected]. unlinkase activity and correlated cell-specificdifferencesinVPg This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. unlinkase activity with levels of TDP2. Using recombinant TDP2 1073/pnas.1208096109/-/DCSupplemental. 14634–14639 | PNAS | September 4, 2012 | vol. 109 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1208096109 Downloaded by guest on September 28, 2021 A B ABCDEF VPg RNA 70 kDa 50 kDa VPg 5' NCR 3' NCR poly(A)n 40 kDa p38 Coding region 30 kDa 25 kDa 5' NCR 3' NCR poly(A)n 20 kDa Coding region 15 kDa RNA 1 23 4567 C Activity Protein Specific Activity Yield Fold (units) (mg) (units/mg) (%) Enrichment A S370 532 193 3 100 1 B Heparin-Sepharose 442 13 35 83 13 C ssDNA-Cellulose 427 3 141 80 51 D Mono Q 327 0.2 1341 62 486 E Sephacryl S-200 118 0.09 1279 22 463 F Mono S 43 0.001 34483 8 12489 Fig. 1. Isolation of VPg unlinkase. (A) Illustration depicting the removal of VPg (green orb) from vRNA (Upper) generating viral mRNA (Lower) is shown. Models of the 5′-terminal chemical structure (based on uridylylated foot-and-mouth disease virus VPg; PDB 2F8E) (29) of vRNA (Upper box) or viral mRNA (Lower box) are shown. Red arrow indicates the bond that is cleaved by VPg unlinkase. (B) SDS/PAGE analysis (protein gel stained with SYPRO Ruby) of the purification process (labeled by purification step, C) shows the isolation of p38. A longer exposure of the gel (Right) was required to visualize proteins in E and F.(C) Table summarizing the purification of VPg unlinkase. Activity units were quantified from the relative levels of VPg signal generated in a 20-μL reaction incubated for 3 min at 30 °C. Protein concentrations were determined by Bradford assay and SDS/PAGE analysis. Lower) corresponding to elution fractions F12 and F13 in the labeled virion RNA ([35S]VPg-PV RNA) isolated from purified activity profile (Fig. 2A, Upper) and subjected to trypsin digestion poliovirus was incubated with GST–TDP2 or partially purified followed by nano-liquid chromatography (nano-LC) MS/MS VPg unlinkase, the unlinking of VPg was observed (Fig. 3A, analysis. This analysis unequivocally identified p38 as TDP2 via Upper). Analysis of these reactions by 1% (wt/vol) agarose gel unique peptides highlighted in Fig. 2B, Upper and Lower.TDP2is electrophoresis verified that the release of VPg from vRNA was aMg2+/Mn2+-dependent cellular hydrolase known to cleave the not due to RNA degradation (Fig. 3A, Lower). We previously 5′-tyrosyl–DNA bond generated as a result of topoisomerase- reported that VPg unlinkase can remove VPg from different mediated DNA damage (10).
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