Biogenesis of the Semliki Forest virus RNA replication complex.
The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326-338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed 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 antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.
Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, FIN-00014 University of Helsinki, Finland.
P
P
Kujala
A
A
Ikäheimonen
N
N
Ehsani
H
H
Vihinen
P
P
Auvinen
L
L
Kääriäinen
D000818:Animals; D000914:Antibodies, Viral; D015415:Biomarkers / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure / Q000821:virology; D002467:Cell Nucleus / Q000648:ultrastructure / Q000821:virology; D006224:Cricetinae; D003593:Cytoplasm / Q000648:ultrastructure / Q000821:virology; D005455:Fluorescent Antibody Technique; D046911:Macromolecular Substances; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D011233:Precipitin Tests; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012672:Semliki forest virus / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure*; D019065:Virus Assembly; D014779:Virus Replication
D000914:Antibodies, Viral; D015415:Biomarkers; D046911:Macromolecular Substances; D012367:RNA, Viral