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Journal of Virology, October 2002, p. 10473-10484, Vol. 76, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.20.10473-10484.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Rev Inhibition Strongly Affects Intracellular Distribution of Human Immunodeficiency Virus Type 1 RNAs

Dusan Cmarko,¹ Stig-Ove Bøe,² Catia Scassellati,¹ Anne Marie Szilvay,³ Svend Davanger,⁴ Xiang-Dong Fu,⁵ Gunnar Haukenes,² Karl-Henning Kalland,² and Stanislav Fakan^1*

Centre of Electron Microscopy, University of Lausanne, CH-1005 Lausanne, Switzerland,¹ Centre for Research in Virology, Department of Microbiology and Immunology, The Gade Institute,² Department of Molecular Biology, University of Bergen, High Technology Center of Bergen, N-5020 Bergen,³ Department of Anatomy and Cell Biology, University of Bergen, N-5009 Bergen,Norway,⁴ Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093⁵

Received 1 February 2002/ Accepted 27 June 2002

To define the human immunodeficiency virus type 1 (HIV-1) RNA maturation pathways, we analyzed the intracellular distribution of HIV-1 RNA and the viral regulatory proteins Rev and Tat in transfected COS cells and HIV-1-infected lymphoid C8166 cells by means of ultrastructural in situ hybridization using antisense RNA probes and immunoelectron microscopy. The intranuclear viral RNA occurs in ribonucleoprotein fibrils in the perichromatin and interchromatin regions. The simultaneous demonstration of Rev, Tat, Br-labeled RNA, and cellular proteins SC35 and CRM1 in such fibrils reveals the potential of Rev to associate with nascent HIV pre-mRNA and its splicing complex and transport machinery. In a rev-minus system, the env intron-containing, incompletely spliced viral RNAs are revealed only in the nucleus, indicating that Rev is required to initiate the transport to the cytoplasm. Moreover, env intron sequences frequently occur in the periphery of interchromatin granule clusters, while the probe containing the rev exon sequence does not associate with this nucleoplasmic domain. When cells were treated with the CRM1 inhibitor leptomycin B in the presence of Rev protein, the env intron containing HIV RNAs formed clusters throughout the nucleoplasm and accumulated at the nuclear pores. This suggests that Rev is necessary and probably also sufficient for the accumulation of incompletely spliced HIV RNAs at the nuclear pores while CRM1 is needed for translocation across the nuclear pore complex.

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* Corresponding author. Mailing address: Centre of Electron Microscopy, University of Lausanne, 27 Bugnon, CH-1005 Lausanne, Switzerland. Phone: 41 21 692 5052. Fax: 41 21 692 5055. E-mail: sfakan{at}cme.unil.ch.

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Journal of Virology, October 2002, p. 10473-10484, Vol. 76, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.20.10473-10484.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.