Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification.
The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.
Mayer
Daniel
D
Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Germany.
Baginsky
Sacha
S
Schwemmle
Martin
M
D028981:Bornaviridae / Q000737:chemistry* / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D012367:RNA, Viral / Q000302:isolation & purification*; D012261:Ribonucleoproteins / Q000302:isolation & purification*; D014764:Viral Proteins / Q000302:isolation & purification*
D012367:RNA, Viral; D012261:Ribonucleoproteins; D014764:Viral Proteins