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Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1
Université Paris 5, Paris, France.
Université Paris VI and Université Paris VII, Paris, France.
Södertörn University, Avdelning Naturvetenskap. Stockholm University.
Université Paris 5, Paris, France.
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2002 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 47, 45091-45098 p.Article in journal (Refereed) Published
Abstract [en]

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

Place, publisher, year, edition, pages
2002. Vol. 277, no 47, 45091-45098 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:sh:diva-15773DOI: 10.1074/jbc.M207439200ISI: 000179404800065PubMedID: 12228227ScopusID: 2-s2.0-0347298811OAI: oai:DiVA.org:sh-15773DiVA: diva2:508454
Available from: 2012-03-08 Created: 2012-03-07 Last updated: 2017-02-13Bibliographically approved
In thesis
1. Dynamic aspects of nucleocytoplasmic trafficking
Open this publication in new window or tab >>Dynamic aspects of nucleocytoplasmic trafficking
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular structures and compartmentalization is the result of a dynamic steady state exchange between its components. This thesis is focused in investigations of dynamic properties of green fluorescent protein (GFP)-labeled proteins in live cells using confocal laser microscopy in combination with bleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP).

Studies of dynamic properties of c-Myc in living cells showed that c-Myc is shuttling between the nucleus and the cytoplasm. c-Myc also enters the nucleoli during certain conditions. Nucleolar c-Myc is dynamically associated to structural component(s) of nucleoli, but can exchange with soluble pools in the nucleoplasm and cytoplasm.

Photobleaching experiments showed that a significant fraction of HIV-1 Vpr is dynamically associated with the NE and rapidly exchanges between the nucleoplasm and the cytoplasm. The yeast two-hybrid system, pull-down experiments and co-immunoprecipitating was used to show that Vpr interacts specifically and directly with a domain in the N-terminal portion of the NPC protein hCG1. The results suggest that the specific interaction of HIV-1 Vpr with the nucleoporin hCG1 results in the dynamic retention of Vpr at the nuclear envelope.

The distribution and dynamic properties of NPC proteins was investigated in NIH/3T3 cells, lacking the pore membrane protein gp210. Confocal laser scanning microscopy and FRAP experiments showed that the absence of gp210 from nuclear pores of NIH/3T3 cells did neither alter the distribution nor dynamic properties of POM121 and NUP107 (two NPC proteins stably integrated in the NPC).

Degradation of the integral nuclear pore membrane protein POM121 during apoptosis was investigated in relation to other apoptotic events. POM121 cleavage, which is the earliest sign of dismantling of the nuclear membrane, is due to caspase-3-dependent cleavage at aspartate-531. Loss of nuclear compartmentalization in live cells undergoing apoptosis was monitored as appearance of GFP-NLS in the cytoplasm. The time of appearance of cytoplasmic GFP-NLS correlated with caspase-3-dependent cleavage of POM121. Both events occured concomitantly with collapsing of chromatin against the nuclear periphery, but preceded the onset of nucleosomal DNA fragmentation.

Translocation ability of the cell-penetrating peptide, transportan, into living cells was investigated. Recombinantly expressed GFP was purified and conjugated to chemically synthesized transportan via a disulfide bond and added to tissues culture cells. Transportan was able to internalize a 27 kDa protein such as GFP in a native folded state into living cells.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, Stockholms universitet, 2004. 41 p.
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-32045 (URN)91-7265-803-7 (ISBN)
Public defence
2004-05-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
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Available from: 2017-02-13 Created: 2017-02-13 Last updated: 2017-02-13Bibliographically approved

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