Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site

Abstract

Cardioviruses have a unique 2A protein (143 aa). During genome translation, the encephalomyocarditis virus (EMCV) 2A is released through a ribosome skipping event mitigated through C-terminal 2A sequences and by subsequent N-terminal reaction with viral 3Cpro. Although viral replication is cytoplasmic, mature 2A accumulates in nucleoli shortly after infection. Some protein also transiently associates with cytoplasmic 40S ribosomal subunits, an activity contributing to inhibition of cellular cap-dependent translation. Cardiovirus sequences predict an eIF4E binding site (aa 126–134) and a nuclear localization signal (NLS, aa 91–102), within 2A, both of which are functional during EMCV infection. Point mutations preventing eIF4E:2A interactions gave small-plaque phenotype viruses, but still inhibited cellular cap-dependent translation. Deletions within the NLS motif relocalized 2A to the cytoplasm and abrogated the inhibition of cap-dependent translation. A fusion protein linking the 2A NLS to eGFP was sufficient to redirect the reporter to the nucleus but not into nucleoli.

Encephalomyocarditis Virus 2A Protein Is Required for Viral Pathogenesis and Inhibition of Apoptosis

“…Inhibition of cap-dependent translation could be due to the binding of 2A to eIF4E, which might impede the interaction of eIF4E with eIF4G (eIF4E-eIF4G-eIF4A interactions are required for initiation of cap-dependent translation). An eIF4E binding site between amino acids 126 to 134 has been located (20). Another possibility is the activation of an inhibitor of translation: when 2A was expressed in BHK-21 cells, 4E-BP1 was observed to be hypophosphorylated (47).…”

Binding Interactions between the Encephalomyocarditis Virus Leader and Protein 2A

“…For example, mutants with deletions in L E (27) or 2A (17) or chimeric viruses exchanging EMCV and theilovirus L X or their 2A (25) typically have incomplete or improperly processed L-P1-2A regions. Presumably, the L-2A interaction, even in this precursor stage, could facilitate proper P1 folding, creating the requisite conformational substrates for sequential reactions with 3C…”

“…No 2A structure is available for any cardiovirus. For all of these sequences though, the C-terminal third of the protein maintains characteristics of an extended alpha helix (17). The first and second portions are not responsive to structure predictions.…”

“…The protein has a very basic pI of 9.67, which presumably allows it to remain nucleolar through rRNA binding contacts. Mutagenic mapping has identified a ribosome protein-like nuclear localization signal (NLS) and a C-proximal eIF4E binding site, which partially overlaps the scission cassette sequences and are common to all known cardioviruses (17). Similar mutations, tested during infection, link the activities of 2A (EMCV) to virus-induced shutdown of cap-dependent translation (4,18).…”

“…Cardiovirus L and 2A interactions with various cellular partners have been the subject of much study and speculation (13,14,17,18). As part of this process, we employed yeast two-hybrid systems to fish out unknown, potential reaction candidates (unpublished).…”