NMR structure of the mengovirus Leader protein zinc‐finger domain

Abstract

The Leader protein is a defining feature of picornaviruses from the Cardiovirus genus. This protein was recently shown to inhibit cellular nucleocytoplasmic transport through an activity mapped to its zinc-binding region. Here we report the three-dimensional solution structure determined by nuclear magnetic resonance (NMR) spectroscopy of this domain (residues 5-28) from mengovirus. The domain forms a CHCC zincfinger with a fold comprising a b-hairpin followed by a short a-helix that can adopt two different conformations. This structure is divergent from those of other eukaryotic zinc-fingers and instead resembles motifs found in a group of DNA-binding proteins from Archaea.

Encephalomyocarditis Virus Leader Is Phosphorylated by CK2 and Syk as a Requirement for Subsequent Phosphorylation of Cellular Nucleoporins

“…Bacterial plasmids encoding active N-terminal glutathione Stransferase (GST)-tagged EMCV-R L protein (GST-L E ) have been previously described (9,16). The EMCV-R sequence (GenBank M81861) was from infectious cDNA pEC 9 (15).…”

“…S1 in the supplemental material). While other picornaviruses (e.g., Aphthovirus and Erbovirus L pro ) can encode alternative proteins at this location, the cardiovirus Ls always display an unusual CHCC zinc-binding domain, a central linker, a short acidic region, and additional Ser/Thr-rich motifs, characteristic of their species (7)(8)(9)(10)(11). After translation, these highly charged (pI of ϳ3.7) proteins are released from an L-P1-2A precursor by the activities of the downstream 3C protease.…”

Solution structures of Mengovirus Leader protein, its phosphorylated derivatives, and in complex with nuclear transport regulatory protein, RanGTPase

“…Recombinant L E -GST (EMCV) and mutated derivatives were expressed and purified from Escherichia coli as previously described (6,15,24). GST-L M (Mengo) has also been previously described (14). The protein includes a thrombin cleavage site for GST-tag removal.…”

“…Complicating a resolution of the full L-dependent antiviral mechanism are observations that L E is itself phosphorylated during EMCV infection, in sequential reactions with casein kinase 2 (CK2) and spleen tyrosine kinase (Syk) at residues T 47 and Y 41 , respectively (16). Although not required for Ran interactions, the L E modifications are clearly important to the virus because mutation at these same sites prevents subsequent Nup phosphorylation (5), suppresses NF-κB activation, and restricts infection-dependent IFN I stimulation (IRF-3 inhibition) (10)(11)(12) (14). The problem was solved by treating samples with EDTA after the removal of the GST tag and then refolding by gradual addition of ZnCl 2 .…”

“…1A). The change is in a conserved, amino-proximal CHCC zinc finger motif (aa [10][11][12][13][14][15][16][17][18][19][20][21][22], the structure of which was determined by NMR for the L M protein (14). Technical difficulties hampered resolution of the complete protein, but a full coordinate set was recently completed [Protein Data Bank (PDB) ID code 2M7Y].…”