An RNA virus hijacks an incognito function of a DNA repair enzyme

Abstract

A previously described mammalian cell activity, called VPg unlinkase, specifically cleaves a unique protein-RNA covalent linkage generated during the viral genomic RNA replication steps of a picornavirus infection. For over three decades, the identity of this cellular activity and its normal role in the uninfected cell had remained elusive. Here we report the purification and identification of VPg unlinkase as the DNA repair enzyme, 5'-tyrosyl-DNA phosphodiesterase-2 (TDP2). Our data show that VPg unlinkase activity in different mammalian cell lines correlates with their differential expression of TDP2. Furthermore, we show that recombinant TDP2 can cleave the protein-RNA linkage generated by different picornaviruses without impairing the integrity of viral RNA. Our results reveal a unique RNA repair-like function for TDP2 and suggest an unusual role in host-pathogen interactions for this cellular enzyme. On the basis of the identification of TDP2 as a potential antiviral target, our findings may lead to the development of universal therapeutics to treat the millions of individuals afflicted annually with diseases caused by picornaviruses, including myocarditis, aseptic meningitis, encephalitis, hepatitis, and the common cold.

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.

Fig. 2.

Identification of p38 as TDP2. (A) Quantified VPg unlinkase activity profile (Upper, red histogram) and SDS/PAGE analysis (Lower) of fractions generated by purification step F show the coelution of p38 with VPg unlinkase activity. Diagonal line (brown) indicates the linear gradient of increasing NaCl concentration (∼150 to 350 mM) used to elute VPg unlinkase. (B) Mass spectrometry analysis of p38 isolated from lane 7 (p38-F12, Upper) and lane 8 (p38-F13, Lower) of the polyacrylamide gel shown in A, Lower identified several tryptic peptides corresponding to TDP2 (in red; overlapping sequence is underlined). (C) Western blot analysis using anti-TDP2 polyclonal antibody (Santa Cruz Biotechnology) confirms the isolation of TDP2 by our purification protocol (lanes labeled by purification step). (D) Relative TDP2 expression levels in different cellular extracts correlate with the VPg unlinkase activity detected previously (7): HeLa (human cervical carcinoma cell line) > K562 (human myeloid leukemia cell line) > NGP (human neuroblastoma cell line) > SKOV3 (human ovarian carcinoma cell line) > RRL (rabbit reticulocyte lysate).

Fig. 3.

Recombinant GST–TDP2 has authentic VPg unlinkase activity. (A) Equivalent amounts of partially purified VPg unlinkase and GST–TDP2 both unlinked VPg from [³⁵S]VPg-PV RNA (Upper), without any apparent degradation of PV RNA (Lower). (B) Increasing amounts of partially purified VPg unlinkase and GST–TDP2, but not GST, unlinked VPg from [³⁵S]VPg-PV RNA and [³⁵S]VPg HRV-PV RNA. Reactions containing RNase A were included to generate markers for VPg–pUp (lanes 6 and 13).

Fig. 4.

Poliovirus infection relocalizes TDP2. (A) Immunofluorescence of TDP2 and viral RNA replication protein 3A. Mock- (Top) or poliovirus-infected HeLa cells were fixed at specific times postinfection; shown are images generated at 2 h (Middle) or 4 h (Bottom) postinfection. Cells were colabeled with antibodies directed against TDP2 (shown in red) or poliovirus protein 3A (green), and nuclei were stained with DAPI. Dashed arrows indicate regions that were largely devoid of TDP2. Arrows indicate regions of poliovirus 3A found adjacent to regions of TDP2. (B) Immunofluorescence of TDP2 and capsid proteins. Mock- (Top) or poliovirus-infected HeLa cells were prepared as described in A above, except that cells were colabeled with anti-TDP2 or poliovirus anti-capsid antibodies. Dashed arrows indicate regions that were largely devoid of TDP2. Arrows indicate regions of viral capsid proteins found adjacent to regions of TDP2.

Fig. 5.

TDP2 sequestration–progeny vRNA shielding model. (A) VPg–RNA linkage of virion RNA (vRNA) following its release from an infecting virion is cleaved by TDP2 (yellow “pac-man” symbol), which generates viral mRNA and free VPg (green sphere). Following translation, the viral mRNA is used as a template for (−) strand RNA synthesis by the viral polymerase (3D^pol) (orange ovals). The (−) strand RNA is then used by the 3D^pol as a template in (+) strand RNA synthesis to generate vRNA, which is either encapsidated or unlinked to participate in viral translation and RNA replication. (B) During the late stage of the replication cycle, TDP2 is excluded from sites of viral RNA synthesis and encapsidation, allowing for the generation of progeny virions.