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. 2017 May 30;114(24):6310–6315. doi: 10.1073/pnas.1610417114

Fig. S1.

Fig. S1.

ICP27 associates with PABP in vivo, and PABP interacts directly with ICP27 in vitro. (A and B) PABP specifically coimmunoprecipitates with ICP27 from HSV-1–infected BHK cell extracts, prepared 5 h postinfection. (A) HSV-1 (KOS 1.1)–infected cell extracts were incubated with normal mouse serum (NMS) (lane 2) or anti-ICP27 monoclonal antibodies 1113 and 1119 (lanes 3–6) in the absence (lane 3) or presence of RNase A, 1, or T1 (lanes 4–6). IP, immunoprecipitation. ICP27 (Upper) or PABP (Lower) was detected by immunoblotting. (Upper) Lower band in the upper panel is IgG heavy chain. (B) Wild type (WT; KOS 1.1) HSV-1, ICP27-null (27lacZ) HSV-1 (where the gene encoding ICP27 is replaced by lacZ), or mock-infected (MI) BHK cell extracts were incubated with a mixture of anti-ICP27 monoclonal antibodies 1113 and 1119 (lanes 1–4) or NMS (lane 5). ICP27 (Upper) and coimmunoprecipitated PABP (Lower) were detected by immunoblotting. RNase A treatment is indicated by a + symbol. The lower protein band in each panel is the IgG heavy chain. PABP is only coimmunoprecipitated from infected cells expressing ICP27. (C) ICP27 and PABP interact directly on a subset of HSV-1 transcripts. The multifunctional ICP27 protein (red) binds to most viral mRNAs, which are polyadenylated and may therefore also bind PABP (green). This property allows for their fortuitous mRNA-mediated coisolation, which is RNase-sensitive. However, our data suggest that ICP27 stimulates the translation of a subset of transcripts (e.g., VP16) by directly recruiting additional PABP to these mRNAs (Fig. 5A). Consequently, on these mRNAs, ICP27 and PABP are present in a functional complex (mediated by protein–protein interactions) that is RNase-insensitive. (D–G) Expression and purification of recombinant proteins. Recombinant proteins were expressed in and purified from E. coli BL21 (DE3) pLysS by affinity chromatography, resolved by SDS/PAGE, and visualized with Gelcode Blue. Molecular masses (kilodaltons) are indicated. (D) GST and GST-PABP. Full-length His-ICP27 (E) and GST-ICP27 (F) are indicated. The lower doublet in E and F was identified by immunoblotting as characteristic N-terminal ICP27 breakdown products. (G) His-PABP. (H) Immobilized purified GST-ICP27 or GST was incubated with purified His-PABP. Coisolated PABP was detected by immunoblotting; input represents 150%.