Initiation activity of EMC virus RNA, binding to initiation factor eIF-4B and shut-off of host cell protein synthesis

Abstract

PICORNAVIRUSES infecting susceptible animal cells inhibit synthesis of host proteins and direct the cells to synthesise viral proteins¹. The mechanism for the shut-off of host cell protein synthesis is not understood, although several experiments indicate that initiation of host protein synthesis is specifically inhibited¹. When initiation of protein synthesis is reduced by exposing cells infected by picornaviruses to hypertonic medium, synthesis of viral proteins is greatly favoured over that of host proteins^2,3. This suggests that viral templates are more successful in initiation than host templates³. Competition between picornavirus RNA and host mRNAs has been shown directly by in vitro translation experiments^4,5. When these RNAs are added together in saturating amounts to a cell-free protein-synthesising system, viral RNA is preferentially translated over host mRNA^4,5. This competition can be relieved by addition of initiation factor eIF-4B (previously designated IF-M₃)⁶. This has suggested that eIF-4B may be present in limiting amounts in the translation assays, and that viral and host templates compete for available factor⁶. This observation is somewhat surprising, as the 5′-terminal cap sequence m⁷G⁵′pppXmp... is involved in the binding of mRNA to eIF-4B, and picornavirus RNA is not capped (see refs 7 and 8). It has been found that other nucleotide sequences outside the cap participate in this reaction⁹, but the presence of 5′-terminal m⁷G in mRNA promotes interaction with eIF-4B and the initiation process^7,8. Therefore, templates with a ‘capped’ 5′-terminal nucleotide bind to ribosomes more rapidly than their unmethylated counterparts^10,11. We have shown previously that eIF-4B interacts with the RNA of encephalomyocarditis (EMC) virus, a picornavirus, in a nitrocellulose filter binding assay⁷. It has also been shown that eIF-4B is required for in vitro translation of EMC virus RNA¹². In the present report we have examined the basis for the competition between EMC virus RNA and a cellular mRNA at the molecular level. EMC virus RNA binds to ribosomes at a rate similar to that of capped mRNAs, suggesting that features other than the 5′-terminal m⁷G are responsible for the binding of this viral RNA. By measuring the relative affinity of globin mRNA and EMC virus RNA for eIF-4B, we have found that the viral RNA has a greater affinity for this initiation factor than globin mRNA. This high affinity for eIF-4B could explain the fast rate of initiation of the viral RNA and the shut-off of host cell protein synthesis after picornavirus infection.