Random Mutagenesis Defines a Domain of Theiler's Virus Leader Protein That Is Essential for Antagonism of Nucleocytoplasmic Trafficking and Cytokine Gene Expression

Abstract

The leader protein of cardioviruses, Theiler's murine encephalomyelitis virus (TMEV) and encephalomyocarditis virus (EMCV), is a multifunctional protein known to antagonize type I interferon expression and to interfere with nucleocytoplasmic trafficking of host proteins and mRNA. This protein plays an important role in the capacity of TMEV to establish persistent infection of the central nervous system. Mutant forms of the TMEV leader protein were generated by random mutagenesis and selected after retroviral transduction on the basis of the loss of the highly toxic nature of this protein. Theiler's murine encephalomyelitis virus (TMEV) is a neurotropic picornavirus that belongs to the Cardiovirus genus (reviewed by Brahic et al. [5]). The leader (L) protein of TMEV is a short (76 amino acids), very acidic protein. This protein helps the establishment of persistent TMEV infections in the central nervous system by antagonizing innate host defenses. It inhibits the transcription of type I interferon (IFN) and selected cytokine and chemokine genes, likely through inhibition of IRF-3 dimerization (19,25,28,33,34). It also interferes with nucleocytoplasmic trafficking of cellular proteins and blocks mRNA export from the nucleus (11,28). These activities correlate with the phosphorylation of nucleoporin 98 (Nup98) (28).The sequence of the L protein contains three domains: a zinc finger domain that was shown to bind divalent cations (7), an acidic central domain, and a Ser/Thr-rich domain (see Fig. 2). The L protein encoded by encephalomyocarditis virus (EMCV) shows 35% amino acid identity with the TMEV L protein. In the EMCV L protein, the zinc finger and the acidic domain are conserved but the C-terminal region encompassing the Ser/Thr-rich domain is lacking. In spite of this difference, L proteins of cardioviruses share the abilities to antagonize IFN production, to affect nucleocytoplasmic trafficking of mRNA and proteins, and to promote nucleoporin hyperphosphorylation (3,16,21,(25)(26)(27)36).Likely as a consequence of mRNA nuclear export inhibition, the TMEV L protein mediates shutoff of host protein synthesis and is very toxic when expressed in cells (2,11,28). In this work, we took advantage of this toxicity to select L mutants that lost the ability to shut off host protein synthesis in order to identify critical domains of the L protein and to test whether the multiple activities of the L protein can be uncoupled. MATERIALS AND METHODS Cells and viruses.BHK-21 cells were cultured as previously described (34). BALB/3T3, L929, and Phoenix-Eco cells were cultured in Dulbecco's modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (MP Biologicals), 100 IU of penicillin/ml, and 100 g of streptomycin/ml. Phoenix-Eco cells were kindly provided by G. Nolan via the ATCC (SD-3444).TMEV derivatives were produced by electroporation of BHK-21 cells (23) with genomic RNA transcribed in vitro from plasmids carrying the corresponding cDNAs. Virus DA1 was produced from plasmid pTMDA1 (10,22,23). Virus TM598 i...

The Leader Protein of Cardioviruses Inhibits Stress Granule Assembly

“…L-mutant viruses derived from DA1 were TM564 carrying a deletion encompassing codons 6 to 67 of the L region (L ) (29). KJ6 derivatives expressing the wild-type L protein of mengovirus (SPA24) and its corresponding zinc finger mutant (SPA28) were described previously (23).…”

“…1): (i) an amino-terminal zinc finger, (ii) a glutamate/aspartate-rich domain, which confers a very acidic character to the protein (pI about 3.8), (iii) a serine/threonine-rich domain, and (iv) a carboxy-terminal domain, present in the L proteins of TMEV and SAFV (theilovirus species) but lacking in EMCV L and therefore called the Theilo domain. Mutations introduced either in the zinc finger or in the Theilo domain strongly inhibit all the known activities of the TMEV L protein (29).…”

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

“…Complicating this picture are observations (10,21) and predictions (7,22) that L E , as well as L T (from TMEV), is itself phosphorylated during infection. L E exposed to eukaryotic cells or cytosol reacts with antibodies (Ab) specific to phosphotyrosine, predicted as Tyr 41 (10), but other studies suggest that Thr 47 is the target site (21).…”

“…Thr 63 of L T (DA) was suggested as such, because Thr 63 Ala substitution reduced Nup98 phosphorylation in L929 cells. Virus with this mutation had reduced toxicity to BALB/3T3 cells, while an analogous phosphomimetic, Thr 63 Asp, retained the wild-type phenotype (7). The L T Ser 57 (DA, BeAn) locale was also proposed as a putative phosphorylation site, because as one of the few known sequence discontinuities in the Ser/Thr-rich domain, this amino acid (Pro 57 , GDVII) correlates with virus growth kinetics in BHK cells (22).…”

“…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.…”