Translational control by viral proteinases

Abstract

Most RNA viruses have evolved strategies to regulate cellular translation in order to promote preferential expression of the viral genome. Positive strand RNA viruses express large portions, or all of their proteome via translation of large polyproteins that are processed by embedded viral proteinases or host proteinases. Several of these viral proteinases are known to interact with host proteins, particularly with the host translation machinery, and thus, encompass the dual functions of processing of viral polyproteins and exerting translation control. Picornaviruses are perhaps the best characterized in regards to interaction of their proteinases with the host translation machinery and will be emphasized here. However, new findings have shown that similar paradigms exist in other viral systems which will be discussed.

Fig. 1

Schematic illustrating scaffolding protein eIF4GI (and eIF4GII) (shown in red) as an extended structure bearing a series of binding domains for other translation factors PABP, eIF4E, eIF4A (two sites), eIF3 and mnk-1. The NH2 and COOH-termini of eIF4G are shown. The locations of known protease cleavage sites are depicted with arrows. A central region of eIF4G linking eIF4E- and eIF3-binding domains has been expanded in the box to illustrate locations of individual proteinase cleavage sites.

Fig. 2

Schematic illustrating PABP (shown in green) with its multiple binding partners. The structure of PABP is illustrated with four numbered RRM domains that interact with RNA, a flexible proline-rich linking domain and a structured COOH-terminal domain that contains a binding cleft for eRF3, eIF4B and PAIP2. RRMs 2 and 3 interact with eIF4G and UNR. A region near RRM 4 interacts with 60S ribosomal subunits in yeast and may be involved in PABP oligomerization. The location of viral proteinase cleavage sites is indicated. Other binding sites for PAIP1 and PAIP2 are not shown for clarity.

Fig. 3

A working model for dual roles of 2A^pro and 3C^pro in inhibition of de novo translation and ribosome recycling on capped mRNAs. (A) Model for ribosome recycling. 40S subunit is depicted on the AUG codon after completing scanning. It is unclear if the cap structure is released by eIF4F during scanning (dashed line) or the 5′ UTR is looped out (solid line). Ribosomes that have reached the stop codon bind eRF3 which may interact with PABP-CTD to facilitate recycling of 60S subunits to waiting 40S subunits on initiation codons, or alternatively, both subunits may recycle. The PABP-CTD may dynamically switch back and forth between binding eRF3 and eIF4B (white arrows). This recycling step can function after eIF4G is cleaved. PABP oligomerization involving the CTD (yellow arrows) may prevent other PABP-CTD from interactions with eIF4B and eRF3. (B) Model for translation shutoff in PV-infection. After cleavage of eIF4G by 2A^pro (orange arrows), de novo binding of 40S subunits to mRNA via the cap structure is blocked and recycling of ribosomes is blocked by cleavage of PABP by 3C^pro (green arrows). Cleaved eIF4G may still retain mRNA in closed loop configuration.

Fig. 4

Kinetics experiments reveal severely diminished ability of 2A^pro to inhibit translation after polysomes form. (A) Immunoblot shows rapid cleavage of eIF4GI in HeLa translation lysates incubated with excess CVB3 2A^pro. (B) Schematic depicting ribosome loading of capped/polyadenylated luciferase RNA after addition of RNA to translation lysates at timepoint zero. Luciferase enzymatic activity takes 8 min to appear, marking the time when fully loaded polysomes first appear on Luc RNA. Ribosome recycling cannot occur until after 8 min. (C) Effect of adding 2A^pro to lysates at various times before or after Luc RNA. 2A^pro was preincubated with lysate 5 min, or added at 0, 4 or 8 min after Luc RNA (depicted in panel B by yellow arrows). The graph shows the accumulation of LUC relative light units plotted as percentage of the translation in mock-treated control lysate. Continued efficient polysome translation after eIF4G cleavage is likely due to ribosome recycling since de novo initiation is blocked. (D) Effect of addition of 2A^pro or 3C^pro to translation lysate at 11 min after RNA was added (arrow). 3C^pro inhibited translation more effectively than 2A^pro when added late. Drastic translation inhibition requires both 2A^pro and 3C^pro.