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. 2007 Aug 3:4:54.
doi: 10.1186/1742-4690-4-54.

Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

Boyan Grigorov  1 , Didier Décimo, Fatima Smagulova, Christine Péchoux, Marylène Mougel, Delphine Muriaux, Jean-Luc Darlix
Affiliations

Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

Boyan Grigorov et al. Retrovirology. .

Abstract

Background: The HIV-1 nucleocapsid protein (NC) is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT), gRNA dimerization and packaging, and virion assembly.

Results: We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired.

Conclusion: These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

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Figures

Figure 1
Figure 1
A. The HIV-1 NC Zinc finger mutants. The sequence of HIV-1 NCp7 (1–55) is shown. Mutations H23C and H44C are indicated. Deletions ΔZF1 and ΔZF2 correspond to a complete deletion of the zinc fingers (ZF). ΔNC was described elsewhere [24]. B. Gag expression and maturation in HeLaP4 cells. HelaP4 cells were transfected with the pNL4.3 DNA (wild-type or either one of the NC mutants) and subsequently harvested and lysed. Viral proteins were analyzed by SDS-PAGE and revealed by immunoblotting with anti-CAp24. Immunodetection of the Gag maturation products. Wild-type HIV-1 and NC mutants are indicated. Lanes "V" and "Cell" representing pelletable virions from culture medium and cell lysates, respectively. Pr55Gag, p41(MA-CA), p49(MA-CA-p2-NC) and CAp24/p25 are indicated by arrows.
Figure 2
Figure 2
Localization of HIV-1 Gag carrying NC zinc finger mutations by immunofluorescence microscopy. Cells were transfected with the indicated viral DNA and then fixed and stained with an anti-CAp24 antibody, as described in material and methods. In addition, cytoplasmic ring-like membranes were found labeled with these two latter Gag mutants (zoomed picture) in less than 1% of the cells. Note that the images obtained for NC(ΔZF1ZF2) was also found for NC(H23H44C).
Figure 3
Figure 3
Plasma membrane accumulation of the HIV-1 NC(ΔZF1ZF2) Gag. HeLa cells were transfected with wild-type HIV-1 (A) or NC(ΔZF1ZF2) (B) DNA, then fixed and stained for the detection of Gag with an anti-MAp17; with an anti-CD63/Lamp3 for late endosomes, and with an anti-Lamp1 for lysosomes, as indicated. Zoomed-1 picture shows wild-type Gag colocalization with CD63/Lamp3 late endosomal marker (26 ± 6%) and zoomed-2 picture with the Lamp1 marker (6 ± 2%). In contrast, zoomed-3 picture shows less colocalization of this mutant with Lamp3 in comparison to wt (6 ± 4%). Zoomed-4 picture shows an accumulation of NC(ΔZF1ZF2)-Gag mutant at the PM, and less or equal with Lamp1(+) intracytoplasmic vesicles (3 ± 1%).
Figure 4
Figure 4
Intracellular localization of the gRNA in cells expressing the NC zinc finger-mutant Gag. HeLa cells were transfected with wild-type HIV-1 (A), or NC(ΔZF1ZF2) (B), or NC(H23H44C) (D) DNA, then fixed and stained for the detection of the gRNA by FISH, as described in material and methods. The fluorescent Cy3-labelled oligonucleotide probe hybridized to the HIV-1gag gene (in red). The nucleus was stained with Dapi in the "mock" HIV-negative cells (C). The arrows indicate the accumulation of wt gRNA at the PM.
Figure 5
Figure 5
Subcellular localization of Gag and the gRNA. Subcellular fractionations of 293T cells expressing wild-type HIV-1 (A, as in (21)) or NC(ΔZF1ZF2) (B) or ΔNC (C) were analyzed by OptiPrep gradient centrifugation. Cells were broken as described in materials and methods and the post-nuclear supernatant (PNS) was fractionated by Optiprep gradient. 20 μl of each fraction were loaded on SDS-PAGE, and Gag and Lamp2 were analyzed by immunoblotting using anti-Cap24 and anti-Lamp2 antibodies. Each fraction of the gradient was tested for the presence of the gRNA by RT-PCR as described in materials and methods. The expected 132 bp DNA fragment was detected on 1% agarose gel. In addition to the gradient analyses, the immunofluorescence (IF) detections are shown, representing the cells stained with an anti-CAp24 (in green) for Gag (A) or mutated Gag (B and C), and the FISH treatment of the 293T cells expressing HIV-1 (A) or the NC Gag mutants (B and C) for the gRNA using the Gag-oligo-Cy3 probe (in red).
Figure 6
Figure 6
Electron microscopy analysis of HIV-1 NC mutant virions. Virions were produced by DNA transfected HeLaP4 cells and further processed as indicated in materials and methods. Bar is 100 nm.
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