Cleavage of DAP5 by coxsackievirus B3 2A protease facilitates viral replication and enhances apoptosis by altering translation of IRES-containing genes

Abstract

Cleavage of eukaryotic translation initiation factor 4G (eIF4G) by enterovirus proteases during infection leads to the shutoff of cellular cap-dependent translation, but does not affect the initiation of cap-independent translation of mRNAs containing an internal ribosome entry site (IRES). Death-associated protein 5 (DAP5), a structural homolog of eIF4G, is a translation initiation factor specific for IRES-containing mRNAs. Coxsackievirus B3 (CVB3) is a positive single-stranded RNA virus and a primary causal agent of human myocarditis. Its RNA genome harbors an IRES within the 5′-untranslated region and is translated by a cap-independent, IRES-driven mechanism. Previously, we have shown that DAP5 is cleaved during CVB3 infection. However, the protease responsible for cleavage, cleavage site and effects on the translation of target genes during CVB3 infection have not been investigated. In the present study, we demonstrated that viral protease 2A but not 3C is responsible for DAP5 cleavage, generating 45-and 52-kDa N-(DAP5-N) and C-terminal (DAP5-C) fragments, respectively. By site-directed mutagenesis, we found that DAP5 is cleaved at amino acid G434. Upon cleavage, DAP5-N largely translocated to the nucleus at the later time points of infection, whereas the DAP5-C largely remained in the cytoplasm. Overexpression of these DAP5 truncates demonstrated that DAP5-N retained the capability of initiating IRES-driven translation of apoptosis-associated p53, but not the prosurvival Bcl-2 (B-cell lymphoma 2) when compared with the full-length DAP5. Similarly, DAP5-N expression promoted CVB3 replication and progeny release; on the other hand, DAP5-C exerted a dominant-negative effect on cap-dependent translation. Taken together, viral protease 2A-mediated cleavage of DAP5 results in the production of two truncates that exert differential effects on protein translation of the IRES-containing genes, leading to enhanced host cell death. Coxsackievirus B3 (CVB3), a primary cause of viral myocarditis, is associated with sudden, unexpected death, 1 dilated cardiomyopathy and heart failure. 2 The CVB3 genome consists of a single open reading frame, which is translated to a polyprotein, and subsequently processed by viral proteases 2A and 3C. Similar to other picornaviruses, the CVB3 genome is linked to a small viral peptide, Vpg, rather than a 7-methyl guanosine cap structure at its 5′ terminus. Thus, CVB3 RNA is translated by a cap-independent mechanism and is driven by an internal ribosome entry site (IRES) harbored in the 5′ untranslated region (5′-UTR). 3,4 Viral proteases actively suppress multiple host cell activities that help the virus evade host defense mechanisms, promote viral replication and promote host cell apoptosis. For example, enterovirus proteases can cleave eukaryotic translation initiation factors 4GI (eIF4GI) 5-7 and eIF4GII. 8,9 Picornavirus proteases have also been reported to cleave other canonical translation initiation factors in the cap-dependent translation initiation complex, such as eIF5B 10 and ...

Protein 2B of Coxsackievirus B3 Induces Autophagy Relying on Its Transmembrane Hydrophobic Sequences

“…For instance, 2A and 3C protease cleave the polypeptide translated from viral genome, a single ORF, into viral structural and non-structural proteins [ 1 ]. Viral protease 2A and 3C have also been demonstrated to cleave a plethora of cellular proteins to facilitate viral replication or to inhibit innate immunity [ 33 , 34 , 35 ].…”

Regulation of Apoptosis by Enteroviruses

“…In addition, DAP5-N increases caspase-3 activation and can reduce Bcl-2 expression and enhance p53 translation. Thus, 2A protein signaling leads to increased apoptosis, thereby enhancing viral release (Hanson et al, 2015). Moreover, the 2A protein of EV71 may be a key inducer of apoptosis mediated by the apoptosis-related thioredoxininteracting protein (TXNIP) (Yao et al, 2019; Table 2).…”

Heat shock protein 70 promotes coxsackievirus B3 translation initiation and elongation via Akt-mTORC1 pathway depending on activation of p70S6K and Cdc2

“…Western blotting was conducted by standard protocols as described previously (Hanson et al . , ). The blots were probed with one of the following primary antibodies: monoclonal anti‐mouse β‐actin (Sigma‐Aldrich); monoclonal anti‐VP1 (Leica Microsystem); monoclonal anti‐human Hsp70, polyclonal anti‐human La and monoclonal mouse anti‐HA (Covance); polyclonal anti‐human 4EBP1 (Santa Cruz); polyclonal anti‐human Akt, polyclonal anti‐human c‐myc, polyclonal anti‐human mTORC1, polyclonal anti‐Flag, monoclonal anti‐eGFP, polyclonal anti‐human p‐eEF2K, polyclonal anti‐human p‐eEF2, polyclonal anti‐human p70S6K monoclonal anti‐human Cdc2 and polyclone anti‐human PRAS40 (Cell Signaling).…”