Browsing by Author "Lång, Mika"

Alphaviruses are single-stranded positive-sense RNA viruses that have been the cause of numerous epidemics in the past decades. The viral genome codes four nonstructural proteins that are associated with viral RNA replication. The translation product is a polyprotein from which the individual nonstructural proteins are cleaved. The genome is transcribed by the nonstructural proteins to produce a negative strand, which acts as a template in the synthesis of positive strands. The subgenomic RNA, which codes the structural proteins, is transcribed from the negative template strand. The replication of genomic and subgenomic RNA strands is associated with replication complexes composed of the nonstructural proteins. The replication complexes are housed in membrane invaginations called spherules on the plasma membrane and endolysosomal vesicles. Transfection of plasmids expressing viral replicase proteins and template RNA can also induce formation of replication complexes in addition to live virus infection. Purification of the replication complexes is necessary for detailed functional and structural analysis. In this work, two methods were used in the purification of replication complexes. In the first method, mammalian cells were infected with viruses and replication complexes were blocked on the plasma membrane by drug treatment to prevent transport to endolysosomal vesicles. Cells were lysed and nuclei were removed by centrifugation. The postnuclear supernatant was sedimented by ultracentrifugation in a discontinuous gradient. The gradient was fractionated and the fraction containing the replication complexes was subjected to equilibrium ultracentrifugation to separate particles by density. The fraction containing the replication complexes was studied using electron microscope, and protein and lipid analysis. In the second method, mammalian cells were transfected with plasmids expressing a template RNA and the viral polyprotein to induce formation of replication complexes. A sequence coding hemagglutinin peptide had been inserted into the polyprotein sequence coding for the nonstructural protein with polymerase activity. Cells were lysed and nuclei were removed. Rreplication complexes were immunocaptured using the hemagglutinin tag by subjecting the supernatant to anti-HA agarose beads. Captured replication complexes were eluted with Laemmli sample buffer and purification was examined using Western blotting. Spherules associated with replication complexes were observed using electron microscope and the presence of nonstructural proteins was confirmed with antibodies. Spherules were observed in low numbers and Western blotting revealed that samples contained cellular contaminants. Purification of replication complexes using immunocapture was very low, but more than that of untagged samples. Although significant purification of replication complexes was not achieved, progress was made in the optimization of the methods.