Viral proteomics: The emerging cutting-edge of virus research

Zhou, ShengTao; Liu, Rui; Zhao, Xia; Huang, CanHua; Wei, YuQuan

In 2004, Dziembowski et al. [40] documented the tandem affinity purification (TAP) procedure, which employs a fusion tag consisting of two sites separated by a protease cleavable spacer. This technology has been applied in a variety of virus research. Jorba et al. [41] purified the intracellular complexes formed by the influenza polymerase in human cells by the TAP approach. The purified complexes contained the heterotrimeric polymerase and a series of associated proteins that were not apparent in purifications of control untagged polymerase. Further identification of influenza polymerase-associated proteins was achieved by MALDI-MS and validated by Western blotting. The roles of these proteins in influenza infection were substantiated by colocalization with virus ribonucleoproteins in infected human cells. The majority of the associated human factors were nuclear proteins involved in cellular RNA synthesis, modification, and nucleocytoplasmic export, except for cytosolic proteins involved in translation and transport. The interactions annotated in this proteomic research suggest a significant role for the influenza polymerase in the infection cycle other than in RNA replication and transcription. In research by Mayer et al. [42] , strep-tagged viral nucleoprotein (NP-Strep) bait and TAP-tagged polymerase subunit bait (TAP tag) were applied to explore the interacting proteins of influenza A viral ribonucleoprotein (vRNP). Forty-one proteins were identified using NP-Strep bait, and four proteins were found using TAP-tagged bait. These included importin-β3 and PARP-1, the two polymerase-associated factors. These studies proved the effectiveness of TAP purification in protein interaction analysis. However, the application of this technology is limited because of its relatively low throughput compared with other methods.