IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA

Renata C Fleith; Harriet V Mears; Edward Emmott; Stephen C Graham; Daniel S Mansur; Trevor R Sweeney

Our in vitro reconstitution of the human IFIT heterocomplex enabled us to examine the impact of oligomerisation on the cap0 mRNA binding and translation inhibition activity of IFIT1. We opted to use an RRL based translation inhibition assay system as this was previously used to demonstrate differential inhibitory effects of IFIT1 on mRNAs from parainfluenza virus 5 (22) . We used two reporters, one with a well characterised (41), highly structured 5 UTR (ZV, dG=-33.4 kcal/mol) and one with very little structure (-globin, dG=-10.5 kcal/mol), upstream of a Fluc reporter and with the corresponding 3 UTRs. IFIT1 strongly inhibited translation of both reporters ( Figure 3 ), but had a stronger effect on the less-structured -globin reporter, consistent with an emerging consensus that both the methylation state and RNA structure at the 5 end of an mRNA can influence its susceptibility to IFIT1 inhibition (9, 22, 23) . IFIT2 had no detectable impact on the ability of IFIT1 to inhibit translation in the RRL system ( Figure 3C ). In contrast, IFIT3 and to a lesser extent the IFIT2:IFIT3 heterodimer enhanced the translation inhibition effect of IFIT1 on both reporters ( Figure 3C -F).