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

Having successfully formed a stable IFIT2:IFIT3 complex, we attempted to reconstitute the full IFIT1:IFIT2:IFIT3 heterotrimeric complex. An equimolar ratio of IFIT1 was incubated with the purified (preformed) IFIT2:IFIT3 heterodimeric complex. Most of the IFIT1 was incorporated into a larger complex that eluted with a molecular mass of 165 kDa corresponding to a trimer ( Figure 2E ). Analysis of this complex by SDS-PAGE (gel inset) reveals that it contains equimolar amounts of IFIT1, IFIT2 and IFIT3. The remainder of the IFIT1 complexed with free IFIT3, a minor contaminant of the purified IFIT2:IFIT3 heterodimer, and eluted slightly earlier as a hetero-tetrameric complex with a molecular mass of 218 kDa. Finally, we incubated the IFIT2:IFIT3 heterodimer with a two-fold molar excess of IFIT1 at 30 ï‚°C for 1 hour. Analysis by SEC-MALS reveals a clear shift of the heterotrimeric complex to a heterotetrameric complex with a molecular mass of 236 kDa ( Figure 2F ). SDS-PAGE analysis of this complex (gel inset) reveals a molar excess of IFIT1 over IFIT2 and IFIT3 indicating that the heterotetrameric complex consists of two IFIT1 molecules to one molecule each of IFIT2 and 3 (compare gel insets in Figures 2 E and F) . The IFIT1:IFIT2:IFIT3 heterotetramer was less stable than the trimer, precipitating when incubated at 4 ï‚°C. Subsequently, only the IFIT1:IFIT2:IFIT3 heterotrimer was used for further experiments.