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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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