Journal:

Article Title: Homo-Oligomerization of Marburgvirus VP35 Is Essential for Its Function in Replication and Transcription

doi: 10.1128/JVI.79.23.14876-14886.2005

Figure Lengend Snippet: Homo-oligomerization of VP35Flag and VP35. (A) VP35Flag and VP35 were coexpressed by using the TNT T7 Quick Coupled Reticulocyte Lysate System and metabolically labeled with [35S]methionine. In vitro translation was followed by coimmunoprecipitation with an anti-Flag or an anti-VP35 antibody. One microliter of the translation reaction mixture and the immunocomplexes was separated by SDS-PAGE and visualized by Bio-Imager analysis. To exclude antibody cross-reactivities, VP35Flag and VP35 were singly expressed in vitro and precipitated with the indicated antibodies. The positions of the proteins are indicated. As a result of internal translation initiation in addition to the full-length products, smaller proteins are synthesized from the templates (*). (B) Huh-T7 cells were transfected with plasmids encoding VP35Flag (500 ng) and VP35 (500 ng) by using Lipofectamine Plus (Invitrogen) according the manufacturer's protocol. At 12 h posttransfection, cells were labeled with [35S]Promix and the proteins were immunoprecipitated with an anti-Flag antibody or anti-NC serum directed against the nucleocapsid proteins NP, VP35, and VP30. Immunocomplexes were separated by SDS-PAGE and visualized by Bio-Imager analysis. To exclude antibody cross-reactivities, VP35Flag and VP35 were singly expressed in vitro and precipitated with the indicated antibodies. *, unspecific cellular proteins.

Article Snippet: The DNA plasmids described were used to in vitro translate VP35 Flag or L Flag simultaneously with VP35 or with VP35 mutants (final volume, 50 μl) and metabolically labeled with [ 35 S]methionine with the TNT T7 Quick Coupled Transcription/Translation System (Promega) according to the supplier's prescription.

Techniques: Metabolic Labelling, Labeling, In Vitro, SDS Page, Synthesized, Transfection, Immunoprecipitation

Journal:

Article Title: Homo-Oligomerization of Marburgvirus VP35 Is Essential for Its Function in Replication and Transcription

doi: 10.1128/JVI.79.23.14876-14886.2005

Figure Lengend Snippet: Localization of the homo-oligomerization domain with VP35 mutants. (A) Coimmunoprecipitation of VP35Flag and VP35 mutants. Flag-tagged VP35 was cotranslated with VP35 mutants with the TNT T7 Quick Coupled Reticulocyte Lysate System and metabolically labeled with [35S]methionine. In vitro translation was followed by coimmunoprecipitation with an anti-Flag or an anti-VP35 antibody. Immunocomplexes were separated by SDS-PAGE and visualized by Bio-Imager analysis. The positions of Flag-tagged VP35 and VP35 mutants are indicated. As a result of internal translation initiation in addition to the full-length product, smaller proteins were synthesized from the templates (*). A schematic representation of the MARV VP35 deletion mutants used for coimmunoprecipitation analyses with VP35Flag is shown at the upper right. The subscript numbers refer to the amino acids of VP35 in the respective mutants. A plus sign indicates an interaction with VP35Flag; a minus sign indicates no interaction with VP35Flag. (B) In silico analysis of the amino acid sequence of VP35 with the COILS 2.2 program. The graph shows a high probability of a coiled-coil structure between amino acids 70 and 120. (Insert) VP35Flag and the VP35 mutant lacking the potential coiled-coil domain (VP35Δ71-119) were cotranslated and analyzed for interaction by coimmunoprecipitation with a Flag-specific and an anti-VP35 antibody. (C) Amino acid sequence of the presumed coiled-coil domain. Note the occurrence of hydrophobic amino acid residues at the first (a) and fourth (d) positions of the heptad repeat (bold and underlined). Results of an in silico analysis of the VP35 substitution mutants (leucine 90 and/or 104 changed to alanine) with the COILS 2.2 program is shown at the bottom. (D) Flag-tagged VP35 substitution mutants were cotranslated with untagged VP35 substitution mutants and analyzed for interaction by coimmunoprecipitation as described above. The positions of the proteins are shown. As a result of internal translation initiation in addition to the full-length products, smaller proteins were synthesized from the templates (*).

Article Snippet: The DNA plasmids described were used to in vitro translate VP35 Flag or L Flag simultaneously with VP35 or with VP35 mutants (final volume, 50 μl) and metabolically labeled with [ 35 S]methionine with the TNT T7 Quick Coupled Transcription/Translation System (Promega) according to the supplier's prescription.

Techniques: Metabolic Labelling, Labeling, In Vitro, SDS Page, Synthesized, In Silico, Sequencing, Mutagenesis

Journal:

Article Title: Homo-Oligomerization of Marburgvirus VP35 Is Essential for Its Function in Replication and Transcription

doi: 10.1128/JVI.79.23.14876-14886.2005

Figure Lengend Snippet: Influence of homo-oligomerization of VP35 on interaction with L. (A) LFlag and VP35 were coexpressed with the TNT T7 Quick Coupled Reticulocyte Lysate System and metabolically labeled with [35S]methionine. In vitro translation was followed by coimmunoprecipitation with an anti-Flag and/or an anti-VP35 antibody. Immunocomplexes were separated by SDS-PAGE and visualized by Bio-Imager analysis. To exclude antibody cross-reactivities, LFlag and VP35 were singly expressed in vitro and precipitated with the indicated antibodies. The positions of the proteins are indicated. (*) In addition to the full-length products, smaller proteins are synthesized from the templates as a result of internal translation initiation. (B) The untagged VP35 substitution mutants (leucine 90 and/or 104 changed to alanine) were cotranslated with LFlag and analyzed for interaction by coimmunoprecipitation as described above. The positions of the proteins are shown. (*) In addition to the full-length product, smaller proteins are synthesized from the templates as a result of internal translation initiation. (C) Schematic representation of the MARV VP35 deletion mutants used for coimmunoprecipitation analyses with LFlag. The subscript numbers refer to the amino acids of VP35 in the respective mutants. A plus sign indicates an interaction with LFlag; a minus sign indicates no interaction with LFlag.

Article Snippet: The DNA plasmids described were used to in vitro translate VP35 Flag or L Flag simultaneously with VP35 or with VP35 mutants (final volume, 50 μl) and metabolically labeled with [ 35 S]methionine with the TNT T7 Quick Coupled Transcription/Translation System (Promega) according to the supplier's prescription.

Techniques: Metabolic Labelling, Labeling, In Vitro, SDS Page, Synthesized