Infection by enteroviruses such as poliovirus (PV), Coxsackie B virus (CVB) and human rhinovirus (HRV), members of the family Picornaviridae
, leads to inhibition of host cell protein synthesis. During PV infection, shutoff begins within 2h, is complete with 4h, and is followed by a phase during which viral translation is also progressively inhibited (Etchison et al., 1982
). The biphasic nature of these changes suggests that shutoff occurs by a multifactorial mechanism, so that factors required for translation of cellular mRNAs (predominantly following cap-dependent initiation) are targeted during the first phase of shutoff and factors that are also required for translation of viral mRNAs may be inactivated later. Enterovirus mRNAs all contain an internal ribosomal entry site (IRES) that promotes initiation by cap-independent ribosomal binding to the mRNA (Hellen and Sarnow, 2001
), unlike most cellular mRNAs, which are translated following cap-dependent initiation
Whereas the mechanism of initiation on enterovirus IRESs is incompletely characterized, many details of the cap-dependent initiation mechanism have been elucidated (Pestova et al., 2007
). First, eIF1, eIF1A and eIF3 promote binding of eIF2•GTP•Met-tRNAMeti
to a 40S ribosomal subunit to form a 43S preinitiation complex. Its attachment to the capped 5′ end of mRNA is mediated by eIF4F (which consists of the eIF4E cap-binding subunit, the eIF4A RNA helicase and eIF4G), and is enhanced by eIF4B and the poly(A) binding protein (PABP). eIF4G coordinates ribosomal recruitment to mRNAs by binding PABP, mRNA and eIFs 3, 4E and 4A. 43S complexes scan to the initiation codon, forming 48S complexes in which the Met-tRNAMeti
anticodon is base-paired to the initiation codon in the ribosomal peptidyl (P) site. In the final subunit-joining stage, eIF5 induces hydrolysis of eIF2-bound GTP, releasing Met-tRNAMeti
into the P site, and eIF5B mediates displacement of eIF2•GDP and other factors from the 48S complex and joining of a 60S subunit to yield an 80S ribosome.
The earliest changes in the translation apparatus during enterovirus infection involve cleavage by the virus-encoded 2A protease (2Apro
) of eIF4GI (which precedes the shutoff of host protein synthesis), and with slightly slower kinetics, of the less abundant eIF4GII (Etchison et al., 1982
; Gradi et al., 1998
). eIF4GI/eIF4GII are cleaved at a single site, splitting domains that bind PABP and eIF4E from those that bind eIF3 and eIF4A (Lamphear et al., 1995), thereby abrogating eIF4G's function in bridging factors that bind capped mRNA, that unwind mRNA and that bind the ribosome. PABP is also cleaved in enterovirus-infected cells, predominantly by the 3C protease (3Cpro
), which separates PABP into an N-terminal fragment that binds eIF4G and the 3′ poly(A) tail of mRNA, and a C-terminal fragment that binds eIF4B and the termination factor eRF3 (Joachims et al., 1999
; Kerekatte et al., 1999
; Kuyumcu-Martinez et al., 2002
). In addition, the translation regulator 4E-BP1 is dephosphorylated during infection. This event sequesters eIF4E and abrogates eIF4F's cap-binding function, which contributes to the shutoff of host protein synthesis and activation of IRES-mediated translation (Gingras et al., 1996
; Svitkin et al., 2005
), but cannot fully account for shutoff because it lags behind this process.
To obtain a more complete overview of the shut-off process, the status in PV and CVB type 3 (CVB3)-infected cells of factors involved in subunit joining was analyzed. eIF5B was found to be cleaved at a single site during enterovirus infection. Cleavage in vitro was mediated by 3Cpro alone and was specific, because eIF1, eIF1A, eIF4B, eIF4F and eIF5 were not substrates for 3Cpro. The time course of eIF5B proteolysis suggest that its cleavage could contribute to the shutoff of host and viral translation observed in enterovirus-infected cells.