This project aims to illuminate the mechanisms and structures of viral enzymes that cap the 5'end of messenger RNA. The m7GpppN cap is a distinctive feature of eukaryal cellular and viral mRNA that is required for mRNA stability and translation. Capping entails three enzymatic reactions: (i) the 5'triphosphate of the pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase (TPase);(ii) the diphosphate RNA end is capped with GMP by RNA guanylyltransferase (GTase);and (iii) the GpppN cap is methylated by RNA (guanine-N7) methyltransferase (MTase). Whereas the three capping reactions are universal in eukarya and DNA viruses, there is a remarkable diversity in the genetic organization of the cap- forming enzymes in different taxa and in different viruses, as well as a complete divergence in the structure and catalytic mechanism of the TPase enzymes found in fungi, protozoa, and several large DNA viruses versus the human TPase. These differences can be exploited to develop novel anti-infective agents directed against capping of the pathogen's mRNAs. They also provide instructive clues to eukaryal phylogeny and evolution of large DNA viruses. This proposal focuses on the poxvirus mRNA capping enzyme, a heterodimer composed of TPase, GTase and MTase domains fused within a single large polypeptide subunit, plus a smaller subunit that binds and stimulates the MTase domain. We plan to functionally map the active sites and domain interfaces of the poxvirus capping enzyme, guided by a new crystal structure of the complete capping enzyme heterodimer. As an outgrowth of our studies of RNA end-modification by viral enzymes, we've developed a new line of research into end-healing and end-sealing enzymes that repair programmed RNA breaks. Programmed RNA damage is an ancient mechanism of responding to cellular stress and distinguishing self from non-self. RNA damage also figures prominently in host responses to virus infection. tRNA damage inflicted by a latent anticodon nuclease PrrC (which breaks tRNALys) underlies a potent host innate immune response to bacteriophage T4 infection, which is thwarted by a virus-encoded tRNA repair system consisting of T4 Pnkp (polynucleotide kinase/phosphatase) and T4 Rnl1 (RNA ligase 1). We have dissected the mechanism, structure, and specificity of Pnkp and Rnl1, and we have extended our analysis to discover or characterize new RNA repair enzymes from viruses, bacteria and human cells. RNA damage and repair now comprise a significant component of the research effort supported by this grant. The current plan focuses on two aspects of the host-pathogen RNA damage/repair dynamic: (i) genetic and structural analysis of PrrC anticodon nucleases and (ii) characterization of a newly discovered two-component RNA repair system (Hen1-Pnkp) that is distributed widely among bacteria.

Public Health Relevance

Viruses must solve (or circumvent) the mRNA capping problem in order to produce viral proteins and attain a successful virus-host dynamic in the face of cellular surveillance systems that sense uncapped RNA ends. Poxviruses are cytoplasmic DNA viruses that encode their own mRNA capping machinery. Structural and mechanistic differences between the poxvirus and mammalian capping systems recommend capping as a target for antipoxviral drug discovery. Antipoxviral strategies are a national public health priority in light of concern that undeclared stocks of smallpox could be used as a bioterror weapon.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM042498-24
Application #
8638782
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
Project End
Budget Start
Budget End
Support Year
24
Fiscal Year
2014
Total Cost
Indirect Cost
City
New York
State
NY
Country
United States
Zip Code
10065
Schmier, Brad J; Shuman, Stewart (2014) Effects of 3'-OH and 5'-PO4 base mispairs and damaged base lesions on the fidelity of nick sealing by Deinococcus radiodurans RNA ligase. J Bacteriol 196:1704-12
Kyrieleis, Otto J P; Chang, Jonathan; de la Peña, Marcos et al. (2014) Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus. Structure 22:452-65
Das, Ushati; Wang, Li Kai; Smith, Paul et al. (2014) Structures of bacterial polynucleotide kinase in a Michaelis complex with GTPýýýMg2+ and 5'-OH oligonucleotide and a product complex with GDPýýýMg2+ and 5'-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosp Nucleic Acids Res 42:1152-61
Das, Ushati; Wang, Li Kai; Smith, Paul et al. (2014) Structures of bacterial polynucleotide kinase in a michaelis complex with nucleoside triphosphate (NTP)-Mg2+ and 5'-OH RNA and a mixed substrate-product complex with NTP-Mg2+ and a 5'-phosphorylated oligonucleotide. J Bacteriol 196:4285-92
Remus, Barbara S; Jacewicz, Agata; Shuman, Stewart (2014) Structure and mechanism of E. coli RNA 2',3'-cyclic phosphodiesterase. RNA 20:1697-705
Chakravarty, Anupam K; Smith, Paul; Jalan, Radhika et al. (2014) Structure, mechanism, and specificity of a eukaryal tRNA restriction enzyme involved in self-nonself discrimination. Cell Rep 7:339-47
Remus, Barbara S; Shuman, Stewart (2014) Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases. RNA 20:462-73
Gao, Pu; Ascano, Manuel; Zillinger, Thomas et al. (2013) Structure-function analysis of STING activation by c[G(2',5')pA(3',5')p] and targeting by antiviral DMXAA. Cell 154:748-62
Wang, Li Kai; Smith, Paul; Shuman, Stewart (2013) Structure and mechanism of the 2',3' phosphatase component of the bacterial Pnkp-Hen1 RNA repair system. Nucleic Acids Res 41:5864-73
Remus, Barbara S; Shuman, Stewart (2013) A kinetic framework for tRNA ligase and enforcement of a 2'-phosphate requirement for ligation highlights the design logic of an RNA repair machine. RNA 19:659-69

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