Rev inhibition strongly affects intracellular distribution of human immunodeficiency virus type 1 RNAs.
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.
Cmarko
Dusan
D
Centre of Electron Microscopy, University of Lausanne, 27 Bugnon, CH-1005 Lausanne, Switzerland.
Bøe
Stig-Ove
SO
Scassellati
Catia
C
Szilvay
Anne Marie
AM
Davanger
Svend
S
Fu
Xiang-Dong
XD
Haukenes
Gunnar
G
Kalland
Karl-Henning
KH
Fakan
Stanislav
S
D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D005091:Exons; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism*; D015696:Gene Products, tat / Q000235:genetics; D015752:Genes, env; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007438:Introns; D028884:Karyopherins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010957:Plasmids; D012326:RNA Splicing; D012333:RNA, Messenger; D012367:RNA, Viral / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D014162:Transfection; D014771:Virion; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D000097606:Exportin 1 Protein
D015694:Gene Products, rev; D015696:Gene Products, tat; D028884:Karyopherins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D018160:Receptors, Cytoplasmic and Nuclear; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D000097606:Exportin 1 Protein; C073288:SRSF2 protein, human