Biogenesis of the Semliki Forest virus RNA replication complex

The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Fores t virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individ ual ns proteins. All ns proteins were associated with large cytoplasmic vac uoles (CPV), the inner surfaces of which were covered by small invagination s, or spherules, typical of alphavirus infection. All ns proteins were loca lized by immune-electron microscopy (EM) to the limiting membranes of CPV a nd to the spherules, together with newly labeled viral RNA. Along with earl ier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol, 2:326-338, 1968), these results suggest that ind ividual spherules represent template-associated RNA polymerase complexes. I mmunoprecipitation of radiolabeled ns proteins shelved that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein anti sera showed that CPV were derivatives of late endosomes and lysosomes. Inde ed, CPV frequently contained endocytosed bovine serum albumin-coated gold p articles, introduced into the medium at different times after infection. Wi th time, increasing numbers of spherules were also observed on the cell sur faces; they were occasionally released into the medium, probably by secreto ry lysosomes. We suggest that the spherules arise by primary assembly of th e RNA replication complexes at the plasma membrane, guided there by nsP1, w hich has affinity to lipids specific for the cytoplasmic leaflet of the pla sma membrane. Endosomal recycling and fusion of CPV with the plasma membran e can circulate spherules between the plasma membrane and the endosomal-lys osomal compartment.