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Journal of Virology, April 2001, p. 3873-3884, Vol. 75, No. 8
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.8.3873-3884.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Biogenesis of the Semliki Forest Virus RNA Replication Complex

Pekka Kujala, Anne Ikäheimonen, Neda Ehsani, Helena Vihinen, Petri Auvinen, and Leevi Kääriäinen*

Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, FIN-00014 University of Helsinki, Finland

Received 18 September 2000/Accepted 8 January 2001

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.

* Corresponding author. Mailing address: Institute of Biotechnology, P.O. Box 56, Viikinkaari 9, 00014 University of Helsinki, Finland. Phone: 358-9-191 59400. Fax: 358-9-191 59560. E-mail: leevi.kaariainen{at}helsinki.fi.

Journal of Virology, April 2001, p. 3873-3884, Vol. 75, No. 8
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.8.3873-3884.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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