(A); HEK293T cells were transfected with plasmids expressing PA, PB1, or PB2 and 24 hours later, cytosolic extracts were prepared and used for immunoprecipitation assays using anti-eIF4GI or a control antibody. The immunoprecipitates were separated by SDS-PAGE, and Western blots were probed with antibodies against the indicated proteins. (B); Scheme of the HA-tagged PB2 proteins used in part C. (C); HEK293T cells were transfected with the corresponding plasmids and immunoprecipitations assays were performed as indicated in (A). The Western blot was probed with anti eIF4GI and anti HA antibodies.

(A); Coomassie staining of His-PB2 and His-VP1 proteins expressed and purified from bacteria. Asterisk represent PB2 degradation products or PB2 fragments generated from premature termination since they are detected with the anti-PB2 antibody (data not shown). (B); A collection of 122 overlapping peptides representing the complete eIF4GI factor was synthesized on a cellulose membrane, which was incubated sequentially in the presence or absence of purified His-protein s and the corresponding primary and secondary antibodies, and assayed in Western blots. After each incubation the membrane was stripped and used for the next detection. The sequence of incubation was as follows: primary and secondary antibodies used to detect PB2 (top, left); incubation with His-PB2 protein followed by its corresponding primary and secondary antibodies (bottom, left); primary and secondary antibodies used to detect VP1 (top, right); and finally His-VP1 followed by its corresponding primary and secondary antibodies (bottom, right). The arrows represent eIF4GI-PB2 interacting peptides. (C); Representation of the PB2 interaction sites in the eIF4GI sequence.

HEK293T cells were processed as described in Fig. 5. (A); Metabolic labeling of treated and untreated cells with rapamicyn. The right panel shows 4E-BP1 phosphorylation on Ser 65. (B); CAT mRNA accumulation measured by RT-PCR. (C); CAT protein accumulation in the different conditions described in Fig. 5. (D); Rates of CAT protein/CAT mRNA accumulation in each condition described in Fig. 5. Standard deviations are indicated by bars, asterisks (*=p<0.05) indicate statistical significance determine d by Student’s T-test. (E); Representation of the relative translation efficiency presented in part (D)

(A); Rabbit reticulocyte extracts were depleted of eIF4E by the addition of purified 4E-BP1 protein and incubation with a 7mGTP resin. After removal of the bound complexes, the eIF4E-depleted lysates were used to assess the translation of in vitro transcribed cap-CAT:EMCV-IRES-Luc RNA or purified cytoplasmic RNA from infected cells (− 4E lanes). Subsequently, purified His-eIF4E recombinant protein was added (+ 4E lanes). The bottom panel shows the amounts of the indicated proteins in the eIF4E-depleted preparations and after the addition of recombinant His-eIF4E protein. The synthesized proteins were metabolically labeled and analyzed by SDS-PAGE. (B); Cytoplasmic RNA from HEK293T infected cells isolated after 6 hpi and dicistronic cap-CAT:EMCV IRES-Luc RNA obtained by in vitro transcription were used for in vitro translation in reticulocyte lysates, with or without increasing concentrations of purified 4EBP1. The bottom panel shows the amounts of 4EBP1 added to the reactions. The synthesized proteins were processed as described in part (A).

A); Recombinant His-eIF4GI (left) and His-PB2 (right) proteins expressed and purified from bacteria were analyzed by Western blotting with specific (Ab-eIF4GI and Ab-PB2) and anti-His antibodies. (B), Interaction of eIF4GI and PB2 proteins. Purified His-eIF4GI and His-PB2 proteins were tested for immunoprecipitation with preimmune or anti-eIF4GI antibodies when they were incubated alone (His-eIF4GI or His-PB2) or when both proteins were incubated together (His-eIF4GI+His-PB2) and the immunocomplexes analyzed by Western blotting with the indicated antibodies.

In Part A and B we evaluated whether the presence in trans of the viral polymerase confers rapamycin resistance to a cellular-like CAT mRNA (pCMV-CAT) or a viral-like CAT mRNA (pCMV-NS-CAT), both transcribed by the cellular RNA polymerase II. In Part C we examined whether the translation of a viral-like CAT mRNA transcribed by the viral polymerase is rapamycin insensitive. In this case, the antisense CAT vRNA was previously used to reconstitute and purify viral RNPs (NS-CAT-vRNPs). These NS-CAT-vRNPs were then transfected in cells expressing the polymerase and the NP proteins and, therefore, the corresponding CAT-mRNA was synthesized by the influenza virus polymerase.