Vanegas M, Llano M, Poeschla E; Conference on Retroviruses and Opportunistic Infections.
Abstr 10th Conf Retrovir Oppor Infect Feb 10 14 2003 Hynes Conv Cent Boston Mass USA Conf Retrovir Oppor Infect 10th 2003 Boston Mass. 2003 Feb 10-14; 10: abstract no. 239.
Mayo Med Sch, Rochester, MN
BACKGROUND: HIV-1 Integrase (IN) has been reported to localize predominantly in cell nuclei, and to play a role in nuclear import of the HIV-1 pre-integration complex. Nuclear localization signals (NLS) at several locations in IN have been reported. The subcellular localization of other lentiviral INs have not been determined.METHODS: We expressed C-terminally myc-tagged versions of feline immunodeficiency virus (FIV) and HIV-1 IN proteins, referred to below as hIN and fIN, respectively. The amino termini of hIN and fIN were also fused to Myc-tagged chicken pyruvate kinase (PK, ~60 kDa), and expressed from pcDNA3 (pMyc-PK-fIN and pMyc-PK-hIN) or fused to ~27 kDa GFP (pGFP-hIN and pGFP-fIN) and also expressed from pcDNA3. Plasmids were transfected into 293T and HeLa cells and sub-cellular distributions were evaluated by immunofluorescence (IF). DNA sequencing and Western blotting (WB) were used to ensure expression of predicted, single, correctly-sized proteins.RESULTS: The fusion proteins generated by our pMyc-PK-fIN and pMyc-PK-hIN plasmids were exclusively localized in the cytoplasm. In WB, single bands corresponding to the predicted 93 kDa species for the PK fusions were observed. As a positive control, Myc-PK was localized to the nucleus when fused to M9, the nuclear localization domain of the hnRNP A1 protein. We compared these constructs to previously described hIN fusions. IF confirmed nuclear localization of Myc-tagged products of a pcDNA1 construct expressing Myc-PK fused to hIN or Myc-PK fused to residues 161-173 of hIN. WB revealed that these generated fusion proteins of predicted size (~93 kDa and ~60 kDa, respectively), but also a lower molecular weight protein (~55 kDa). When hIN or fIN were fused to the C-terminus of a smaller fusion partner, GFP, each was located predominantly in the nucleus. When expressed without fusion to another protein, Myc-hIN localized predominantly in the nucleus, while Myc-fIN localized variably depending on the location and nature of the epitope tag.CONCLUSIONS: Subcellular localization of FIV and HIV-1 integrase fusions depends on the size of the fusion partner. In agreement with other studies, unfused myc-tagged HIV-1 integrase localizes to the nucleus. Our studies contrast with previously reported results that HIV-1 integrase contains a transferable NLS that confers nuclear localization to large fusion proteins. The results suggest that FIV integrase also does not contain a transferable NLS.
Publication Types:
Keywords:
- Active Transport, Cell Nucleus
- Animals
- Cats
- Cell Nucleus
- Cytoplasm
- Green Fluorescent Proteins
- HIV Integrase
- HIV-1
- Hela Cells
- Humans
- Immunodeficiency Virus, Feline
- Integrases
- Nuclear Localization Signals
- Proto-Oncogene Proteins c-myc
- Recombinant Fusion Proteins
Other ID:
UI: 102260286
From Meeting Abstracts