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, 199 (3), 467-79

Critical Role for the Kinesin KIF3A in the HIV Life Cycle in Primary Human Macrophages

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Critical Role for the Kinesin KIF3A in the HIV Life Cycle in Primary Human Macrophages

Raphaël Gaudin et al. J Cell Biol.

Abstract

Macrophages are long-lived target cells for HIV infection and are considered viral reservoirs. HIV assembly in macrophages occurs in virus-containing compartments (VCCs) in which virions accumulate and are stored. The regulation of the trafficking and release of these VCCs remains unknown. Using high resolution light and electron microscopy of HIV-1-infected primary human macrophages, we show that the spatial distribution of VCCs depended on the microtubule network and that VCC-limiting membrane was closely associated with KIF3A+ microtubules. Silencing KIF3A strongly decreased virus release from HIV-1-infected macrophages, leading to VCC accumulation intracellularly. Time-lapse microscopy further suggested that VCCs and associated KIF3A move together along microtubules. Importantly, KIF3A does not play a role in HIV release from T cells that do not possess VCCs. These results reveal that HIV-1 requires the molecular motor KIF3 to complete its cycle in primary macrophages. Targeting this step may lead to novel strategies to eliminate this viral reservoir.

Figures

Figure 1.
Figure 1.
The distribution of VCCs is dependent on the integrity of the microtubule network. (A) Ultrathin cryosections of macrophages infected with HIV-1 NLAD8 for 15 d were prepared and labeled with anti-Env antibodies and protein A coupled to gold particles of 10-nm diameter (PAG10) and with anti-Pr55Gag antibodies and PAG15. Arrowheads point to viral particles in the process of budding. Bar, 200 nm. (B) 3D reconstruction of macrophages infected with HIV-1 NLAD8 for 7 d and stained for Gag (see Video 1). (C) Confocal micrographs (one plane) of HIV-1–infected macrophages exposed to DMSO or nocodazole (10 µM) for 1 h. Cells were fixed and stained with antibodies specific for the indicated proteins. Bar, 5 µm. These experiments have been repeated three times. (D) Ultrathin cryosections of macrophages infected with HIV-1 NLAD8 for 15 d were double-immunogold labeled for Pr55Gag with PAG15 and for α-tubulin with PAG10. Three representative profiles are presented. Tubulin staining was present at the limiting membrane of VCCs (see arrowheads). Bars, 200 nm.
Figure 2.
Figure 2.
KIF3A is required for viral production by macrophages. (A) Schematic representation of the experimental design. (B) Immunoblot analysis of KIF3A and Tsg101 expression in macrophages transfected with the indicated siRNA. After 4 d, both siRNA specific for KIF3A significantly reduced its level of expression in primary macrophages. (C) Macrophages were infected by HIV-1 and transfected with siRNA as described in A. Cell viability was measured at d 4 p.i. using the CellTiter Glo kit and normalized to the control (siLuc). (D) Primary macrophages were infected with HIV Gag-iGFP ΔEnv pseudotyped with VSV-G and transfected with siRNA as described in A. This virus has a single cycle in macrophages and does not induce syncitia formation. Percentages of GFP+ macrophages were estimated by flow cytometry at d 4 p.i. (E) Infectivity of the virions produced by the macrophages subjected to the indicated siRNA was evaluated using the same amount of p24 (2 ng; see Materials and methods). (F) KIF3A depletion does not affect the early steps of infection. The reporter cell line TZM-bl was transfected with the indicated siRNA. 2 d later, cells were infected with HIV-1. Cells were washed 8 h later, reincubated for an additional 16 h, and assayed for β-gal activity, whose expression is driven by a Tat-sensitive promoter. (G) Dosage of p24 Gag in the 24-h culture supernatants harvested as indicated in A. *** indicates that the difference between the two histogram bars is statistically significant (P < 0.001). Experiments from D–F have been repeated at least two times and from B, C, and G at least three times in quadruplicate.
Figure 3.
Figure 3.
KIF3A is dispensable for HIV-1 production by infected T cells. (A) Schematic representation of the experimental design. (B) Immunoblot analysis of KIF3A and α-tubulin expression in Jurkat T cells expressing the indicated shRNA. After 3 d, all shRNA specific for KIF3A reduced its level of expression down to less than 20% in Jurkat T cells. (C) Dosage of p24 Gag in 20-h culture supernatants of NL4-3 ΔEnv-infected Jurkat T cells harvested as indicated in A. Values presented have been corrected to the number of HIV-infected cells present in each sample, as calculated using CellTiter Glo (for cell viability) and anti-Gag staining analyzed by flow cytometry (for percentage of infection). (D) Confocal micrographs of primary T cells infected with VSV-G–pseudotyped HIV-1 NL4-3 for 24 h and stained for the indicated markers. Note that Gag distribution is highly polarized at the plasma membrane, whereas KIF3A distribution is totally different. Bars, 5 µm. (E) Ultrathin cryosection of Jurkat T cells infected with HIV-1 NL4-3 and immunogold labeled for Pr55Gag with PAG10. Bar, 200 nm.
Figure 4.
Figure 4.
The VCCs are associated with tubulin and the kinesin KIF3A. (A) Confocal micrographs of HIV-1–infected macrophages. Cells were fixed and stained with antibodies specific for the indicated proteins. A z-projection of a stack of images is presented on the top row, and the bottom row shows one confocal plane of the magnification of the boxed area. Bar, 10 µm. (B) Structured illumination microscopy of HIV-1–infected macrophages stained for p24 and KIF3A. (C) 3D reconstruction with half opacity of the region boxed in B. Bar, 3 µm. (D) Immuno-EM of ultrathin cryosection of macrophages infected with HIV-1 NLAD8 for 7 d labeled for the indicated proteins. Arrowheads point to KIF3A staining associated with α-tubulin at the limiting membrane of a VCC. Bar, 500 nm. Experiments have been repeated at least twice.
Figure 5.
Figure 5.
KIF3A depletion results in intracellular accumulation of VCCs. (A) Examples of 3D reconstructions obtained after thresholding of image stacks acquired by confocal microscopy of HIV-1–infected macrophages transfected with the indicated siRNA and stained for Gag. (B–E) Effects of KIF3A depletion on the indicated parameters within HIV-1–infected macrophages. Reconstructions as seen in A were segmented and quantified using Imaris software. KIF3A knockdown was estimated in parallel by immunoblot in the eight donors used for these experiments. The average efficiency of silencing was 72.9%, ranging from 55 to 95%. n.s., not statistically significant.
Figure 6.
Figure 6.
KIF3A-iCh is recruited to active VCCs. (A) Schematic representation of the KIF3A-iCh construct. (B and C) Correlative light and electron microscopy. (B) Primary macrophages grown on coverslips with coordinates were coinfected with HIV-1 Gag-iGFP and KIF3A-iCherry lentiviral vector, fixed, and imaged by spinning disk microscopy at d 7 p.i. Bar, 5 µm. (C) The same coverslips were then embedded in Epon and processed for EM. An overview of the macrophage imaged in B is presented in the top left inset. Example of mature and immature virions are presented in the bottom left inset. Bar, 100 nm. The VCCs are in the top third of the cell. (D) Two magnifications of the VCC-rich region, with a viral budding profile shown in the inset (bar, 200 nm).
Figure 7.
Figure 7.
Dynamics of internally tagged forms of KIF3A and Gag in primary macrophages. General view of a macrophage coinfected with HIV-1 Gag-iGFP ΔEnv and KIF3A-iCherry lentiviral vector and cultured for 5 d. The cell was imaged for 5 h by spinning disc microscopy. (A) Z-projection of the overlaid transmission, GFP and mCherry fluorescent intensities at time 0 (left, bar, 5 µm). The red square region is magnified for GFP and mCherry channels and the overlay of the two (panels 2–4; bar, 2 µm). Time 0, 1, 2, and 3 h 40 of a 3D reconstruction of the boxed area are presented with the tracking of the VCCs visible on the 3 h 40 panel (see Videos 3 and 4). Bar, 2 µm.

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