The cellular protein Alix interacts with the HPIV1 C proteins.

<p><b>A.</b> Schematic diagram of HPIV1 C expression constructs with a Myc tag attached at the c-terminus. The cDNAs were constructed to contain the coding sequence for C’, C, and Y2 (the cDNA for the Y1 coding sequence was also constructed but not used in this study). The numbers denote the nucleotide position of the start codon relative to the start codon for C’. The location of the F170S point mutation is indicated by the dotted line. The C’^WT_GTG and C’^F170S_GTG constructs bear the native GTG codon as the translational start codon for C’. In contrast, for constructs C’^WT and C’^F170S, an ATG start codon was placed immediately before the native GTG start codon to increase the expression of the C’ isoform at the expense of the smaller C isoforms. The native ACG start codon in the Y2 construct was replaced with an ATG codon to increase the expression of the Y2 isoform. <b>B.</b> Detection of a cellular species that co-immunoprecipitated with the HPIV1 C proteins. The above Myc-tagged C expression constructs were transfected into duplicate cultures of 293 T cells. One set was also transfected with MDA5 and, 42 h post-transfection, transfected again with poly(I:C). 48 h after the initial transfection, both sets of cultures were lysed and subjected to immunoprecipitation with anti-Myc antibodies to isolate the C proteins and possible binding partners, and the precipitated proteins were separated by SDS-PAGE and visualized by silver staining. A portion of the gel is magnified in the inset. The arrow indicates a representative band that co-immunoprecipitated with the Myc-tagged C proteins, regardless of transfection with MDA5 and poly(I:C). This band was excised from a duplicate gel that had been run in parallel and stained with Coomassie blue, and the band was analyzed by mass spectrometry. <b>C.</b> Identification of the co-immunoprecipitated species as the cellular protein Alix. The amino acid sequence of human Alix protein is shown, and peptide sequences that were identified by mass spectrometry are indicated in bold. <b>D.</b> Testing the interaction between endogenous Alix and the HPIV1 C’, C, and Y2 proteins. 293 T cells were transfected with plasmids encoding the indicated Myc-labeled C proteins. Similar to that described in panel B, a subset of cells were also co-transfected with MDA5 and, 42 h post-transfection, transfected again with poly(I:C). The cells were lysed and analyzed by immunoprecipitation with anti-Myc antibodies to isolate C proteins followed by SDS-PAGE and Western blot analysis using an Alix-specific antibody. Endogenous Alix co-immunoprecipitated with the WT C’, C, and Y2 proteins regardless of the presence of MDA5 and poly (I:C). <b>E.</b> Testing the interaction between endogenous Alix and the HPIV1 F170S mutation-containing C protein. As described in panel D, cellular lysates were immunoprecipitated with anti-Myc antibodies to isolate the C proteins. Endogenous Alix co-immunoprecipitated with the WT C proteins but not with the F170S mutation-containing C proteins.