Solubilization and immunoprecipitation of alphavirus replication complexes.
Alphavirus replication complexes that are located in the mitochondrial fraction of infected cells which pellets at 15,000 x g (P15 fraction) were used for the in vitro synthesis of viral 49S genome RNA, subgenomic 26S mRNA, and replicative intermediates (RIs). Comparison of the polymerase activity in P15 fractions from Sindbis virus (SIN)- and Semliki Forest virus (SFV)-infected cells indicated that both had similar kinetics of viral RNA synthesis in vitro but the SFV fraction was twice as active and produced more labeled RIs than SIN. When assayed in vitro under conditions of high specific activity, which limits incorporation into RIs, at least 70% of the polymerase activity was recovered after detergent treatment. Treatment with Triton X-100 or with Triton X-100 plus deoxycholate (DOC) solubilized some prelabeled SFV RIs but little if any SFV or SIN RNA polymerase activity from large structures that also contained cytoskeletal components. Treatment with concentrations of DOC greater than 0.25% or with 1% Triton X-100-0.5% DOC in the presence of 0.5 M NaCl released the polymerase activity in a soluble form, i.e., it no longer pelleted at 15,000 x g. The DOC-solubilized replication complexes, identified by their polymerase activity in vitro and by the presence of prelabeled RI RNA, had a density of 1.25 g/ml, were 20S to 100S in size, and contained viral nsP1, nsP2, phosphorylated nsP3, nsP4, and possibly nsP34 proteins. Immunoprecipitation of the solubilized structures indicated that the nonstructural proteins were complexed together and that a presumed cellular protein of approximately 120 kDa may be part of the complex. Antibodies specific for nsP3, and to a lesser extent antibodies to nsP1, precipitated native replication complexes that retained prelabeled RIs and were active in vitro in viral RNA synthesis. Thus, antibodies to nsP3 bound but did not disrupt or inhibit the polymerase activity of replication complexes in vitro.
Barton
D J
DJ
Department of Microbiology, Medical College of Ohio, Toledo 43699.
Sawicki
S G
SG
Sawicki
D L
DL
D000524:Alphavirus / Q000235:genetics / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003840:Deoxycholic Acid; D003902:Detergents; D005814:Genes, Viral; D017830:Octoxynol; D011092:Polyethylene Glycols; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012672:Semliki forest virus / Q000235:genetics / Q000502:physiology*; D012845:Sindbis Virus / Q000235:genetics / Q000502:physiology*; D012995:Solubility; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014779:Virus Replication
D003902:Detergents; D012367:RNA, Viral; D014764:Viral Proteins; D003840:Deoxycholic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol