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Journal of Virology, February 2003, p. 1691-1702, Vol. 77, No. 3
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.3.1691-1702.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Properly Folded Nonstructural Polyprotein Directs the Semliki Forest Virus Replication Complex to the Endosomal Compartment

Anne Salonen,1 Lidia Vasiljeva,1 Andres Merits,1,{dagger} Julia Magden,1 Eija Jokitalo,2 and Leevi Kääriäinen1*

Program in Cellular Biotechnology,1 Electron Microscopy Unit, Institute of Biotechnology, Biocenter Viikki, University of Helsinki, Helsinki, Finland2

Received 21 June 2002/ Accepted 28 October 2002

The late RNA synthesis in alphavirus-infected cells, generating plus-strand RNAs, takes place on cytoplasmic vacuoles (CPVs), which are modified endosomes and lysosomes. The cytosolic surface of CPVs consists of regular membrane invaginations or spherules, which are the sites of RNA synthesis (P. Kujala, A. Ikäheimonen, N. Ehsani, H. Vihinen, P. Auvinen, and L. Kääriäinen J. Virol. 75:3873-3884, 2001). To understand how CPVs arise, we have expressed the individual Semliki Forest virus (SFV) nonstructural proteins nsP1 to nsP4 in different combinations, as well as their precursor polyprotein P1234 and its cleavage intermediates. A complex of nsPs was obtained from P123 or P1234, indicating that the precursor stage is essential for the assembly of the polymerase complex. To prevent the processing of the polyprotein and its cleavage intermediates, constructs with the mutation C478A (designated with a superscript CA) in the active site of the protease domain of nsP2 were used. Uncleaved polyproteins containing nsP1 were membrane bound and palmitoylated, and those containing nsP3 were phosphorylated, reflecting properties of authentic nsP1 and nsP3, respectively. Similarly, polyproteins containing nsP1 or nsP2 had enzymatic activities specific for the individual proteins, indicating that they were correctly folded in the precursor state. Uncleaved P12CA was localized almost exclusively to the plasma membrane and filopodia, like nsP1 alone, whereas P12CA3 and P12CA34 were found on cytoplasmic vesicles, some of which contained late endosomal markers. In immunoelectron microscopy these vesicles resembled CPVs in SFV-infected cells. Our results indicate that the nsP1 domain alone is responsible for the membrane association of the nonstructural polyprotein, whereas the nsP1 domain together with the nsP3 domain targets it to the intracellular vesicles.

* Corresponding author. Mailing address: Institute of Biotechnology, P.O. Box 56, 00014 University of Helsinki, Finland. Phone: 358 9 19159400. Fax: 358 9 191 59560. E-mail: leevi.kaariainen{at}helsinki.fi.

{dagger} Present address: Institute of Molecular and Cellular Biology, 51010 Tartu, Estonia.

Journal of Virology, February 2003, p. 1691-1702, Vol. 77, No. 3
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.3.1691-1702.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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