| Summary: | 碩士 === 國立成功大學 === 分子醫學研究所 === 95 === A novel Tandem Affinity Purification (TAP) method combined with mass spectrometry (MALDI-TOF, MS) and database search algorithms are adapted to allow identification of HIV NC protein interacting partners. Our preliminary goal is to identify and characterize cellular proteins associated with HIV nucleocapsid (NC) or during precursor Gag trafficking. NC participates in many steps in virus lifecycle, including (1) viral RNA binding and chaperone activity during reverse transcription and cDNA synthesis in the early stages of viral infection and (2) precursor Gag assembly and HIV RNA packaging process in the late stage of viral particle biosynthesis. The packaging process is highly selective in vivo; however, the affinity and specificity of NC for Ψ (packaging signal) containing viral genomic RNA sequences in vitro is modest at best, suggesting that other viral or cellular factors contribute to the selectivity process. We constructed plasmids and viral vectors harboring HIV NC gene sequences (including mutants with 10 mutations in the NC basic region) with aligned TAP sequence to allow sufficient expression in mammalian cell cultures. In addition to the confirmed in-frame sequences, transfection of these constructs into 293 T cells resulted in the expression of the NC and mNC fusion proteins in the cytosol and can be efficiently identified by specific antibodies against the Tag proteins CBP and proteinA. An optimized Tandem affinity purification (TAP) protocol has been established to compare and isolate candidate proteins that are physically associated with NC. Many NC but not mNC interacting proteins in the final affinity elutents were evident on one-dimension SDS-PAGE gradient gel via comassive blue staining and western blot analysis. Repetitive comparison of the staining patterns and analysis of the protein identities through LC/MS/MS and data mining revealed 30 consistent NC-specific binding proteins. Topoisomersase I, Nuclear DNA helicase II, tubulin, heterogeneous nuclear ribonuclear protein U(HNRPU), suppressor of cyclic-AMP receptor (SCAR), and ribosomal protein subunits are predominant species that may play important roles for NC during cDNA synthesis or precursor Gag trafficking and genomic viral RNA incorporation. Further verification of these NC interaction proteins may require the comparison between the presence and absence of the packaging signal (Ψ) containing viral genomic RNA. Inhibition of the NC binding proteins would assist to reveal their identities and functional relevance in HIV life cycle. This study established a preliminary purification and identification protocols for the proteomic analysis of HIV NC binding proteins and may provide in the future significant insights into the mechanisms of HIV NC-cellular protein interaction networks during initial HIV life cycle, precursor Gag trafficking, and RNA packaging process.
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