Inhibition of mRNA export and dimerization of interferon regulatory factor 3 by Theiler's virus leader protein

Abstract

Theiler's murine encephalomyelitis virus (TMEV or Theiler's virus) is a neurotropic picornavirus that can persist lifelong in the central nervous system of infected mice, causing a chronic inflammatory demyelinating disease. The leader (L) protein of the virus is an important determinant of viral persistence and has been shown to inhibit transcription of type I interferon (IFN) genes and to cause nucleocytoplasmic redistribution of host proteins. In this study, it was shown that expression of the L protein shuts off synthesis of the reporter proteins green fluorescent protein and firefly luciferase, suggesting that it induces a global shut-off of host protein expression. The L protein did not inhibit transcription or translation of the reporter genes, but blocked cellular mRNA export from the nucleus. This activity correlated with the phosphorylation of nucleoporin 98 (Nup98), an essential component of the nuclear pore complex. In contrast, the data confirmed that the L protein inhibited IFN expression at the transcriptional level, and showed that transcription of other chemokine or cytokine genes was affected by the L protein. This transcriptional inhibition correlated with inhibition of interferon regulatory factor 3 (IRF-3) dimerization. Whether inhibition of IRF-3 dimerization and dysfunction of the nuclear pore complex are related phenomena remains an open question. In vivo, IFN antagonism appears to be an important role of the L protein early in infection, as a virus bearing a mutation in the zinc finger of the L protein replicated as efficiently as the wild-type virus in type I IFN receptor-deficient mice, but had impaired fitness in IFNcompetent mice.

Theiler's Murine Encephalomyelitis Virus Leader Protein Is the Only Nonstructural Protein Tested That Induces Apoptosis When Transfected into Mammalian Cells

“…Unlike the L of aphthoviruses, the cardiovirus L does not have a proteolytic function. Recently, studies have shown that cardiovirus L binds Ran-GTPase and phosphorylates nucleoporins, leading to disruption of the nuclear pore complex that spans the nuclear envelope and the Ran gradient (27,28,30). The inhibition of nuclear import allows measurement of the efflux of nuclear proteins into the cytoplasm after loading the nucleus with a marker protein (pGFP NLS ).…”

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

“…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 .…”

“…Because picornaviruses replicate in the cytoplasm, this inhibition is detrimental only to the cell. Among the measured results, there is antagonism of IFN transcription (9-12), impediment of cellular stress granule formation (13), and retention of cellular mRNA transcripts in the nucleus (12). These cumulative activities allow cardioviruses to negate almost all host antiviral innate immune responses and enhance their pathogenicity during infection.…”

“…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].…”

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

“…The extra common residues ("Theilo domain"), and the extra TMEV-specific residues (serine/threonine [Ser/Thr] domain), make L S (71 aa) and L T (76 aa) longer than the L E , although they all are expected to have reasonably similar biological functions (11). The L T (DA strain) has been linked to disruption in active NPC trafficking (24), induction of Nup phosphorylation (specifically Nup98) (25), and inhibition of interferon responses (26). The L E from EMCV-R can replace L T (DA) in recombinant virus, albeit with reduced viral replication efficiency (11).…”