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HIV-1 capsids bind and exploit the kinesin-1 adaptor FEZ1 for inward movement to the nucleus.

Malikov V, da Silva ES, Jovasevic V, Bennett G, de Souza Aranha Vieira DA, Schulte B, Diaz-Griffero F, Walsh D, Naghavi MH - Nat Commun (2015)

Bottom Line: Furthermore, both dynein and kinesin-1 motors are required for HIV-1 trafficking to the nucleus.Finally, the ability of exogenously expressed FEZ1 to promote early HIV-1 infection requires binding to kinesin-1.Our findings demonstrate that opposing motors both contribute to early HIV-1 movement and identify the kinesin-1 adaptor, FEZ1 as a capsid-associated host regulator of this process usurped by HIV-1 to accomplish net inward movement towards the nucleus.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA [2] Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.

ABSTRACT
Intracellular transport of cargos, including many viruses, involves directed movement on microtubules mediated by motor proteins. Although a number of viruses bind motors of opposing directionality, how they associate with and control these motors to accomplish directed movement remains poorly understood. Here we show that human immunodeficiency virus type 1 (HIV-1) associates with the kinesin-1 adaptor protein, Fasiculation and Elongation Factor zeta 1 (FEZ1). RNAi-mediated FEZ1 depletion blocks early infection, with virus particles exhibiting bi-directional motility but no net movement to the nucleus. Furthermore, both dynein and kinesin-1 motors are required for HIV-1 trafficking to the nucleus. Finally, the ability of exogenously expressed FEZ1 to promote early HIV-1 infection requires binding to kinesin-1. Our findings demonstrate that opposing motors both contribute to early HIV-1 movement and identify the kinesin-1 adaptor, FEZ1 as a capsid-associated host regulator of this process usurped by HIV-1 to accomplish net inward movement towards the nucleus.

No MeSH data available.


Related in: MedlinePlus

Depletion of FEZ1 inhibits HIV-1 trafficking to the nucleusNHDFs were treated with control or FEZ1-C siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-GFP-Vpr followed by live imaging using a spinning-disc confocal microscope. (a) Still images from movies (Supplementary Movies 1 and 2) taken at the indicated times are shown. Green arrows highlight viral particles entering and traversing the cytoplasm in control siRNA-treated cells. Red arrows highlight representative examples of particles that approach the nucleus but then move long distances back out to the cell periphery in FEZ1-depleted cells. (b) Quantification of the average distance (μm per 2.5 min) traveled by viral particles towards the nucleus (Retrograde motility) or away from the nucleus (Antrograde motility). n≥5 cells and an average of 7–30 viral particles per cell. (c) Quantification of the percentage of virions within 2μm of the nucleus in infected siRNA-treated cells at the indicated time points. n≥20 cells and an average of 80–99 viral particles per cell. (d–f) Depletion of FEZ1 affects HIV-1 particles that have productively fused into the cytoplasm. (d) NHDF cells were treated with control or FEZ1-C siRNAs. 48h after transfection cells were infected with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed in formaldehyde and GFP and Tomato signals were acquired using a spinning-disc confocal microscope. Arrows highlight fused (green, S15-negative) particles proximal to the nucleus in each sample. Representative confocal planes are shown. (e) Quantification of the % fused (green, S15-negative) viral particles within 2μm of the nucleus in samples as described and processed in d. n≥29 cells and an average of 53–55 viral particles per cell. (f) Control siRNA-treated NHDF cells were treated with Bafilomycin A1 for 2h during spinoculation followed by infection with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed and the total number of fused (green, S15-negative) viral particles were quantified and presented as a % of the total number of viral particles. n≥29 cells and an average of 55–80 viral particles per cell. Error bars represent standard deviation. Scale bars represent 10 μm.
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Figure 3: Depletion of FEZ1 inhibits HIV-1 trafficking to the nucleusNHDFs were treated with control or FEZ1-C siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-GFP-Vpr followed by live imaging using a spinning-disc confocal microscope. (a) Still images from movies (Supplementary Movies 1 and 2) taken at the indicated times are shown. Green arrows highlight viral particles entering and traversing the cytoplasm in control siRNA-treated cells. Red arrows highlight representative examples of particles that approach the nucleus but then move long distances back out to the cell periphery in FEZ1-depleted cells. (b) Quantification of the average distance (μm per 2.5 min) traveled by viral particles towards the nucleus (Retrograde motility) or away from the nucleus (Antrograde motility). n≥5 cells and an average of 7–30 viral particles per cell. (c) Quantification of the percentage of virions within 2μm of the nucleus in infected siRNA-treated cells at the indicated time points. n≥20 cells and an average of 80–99 viral particles per cell. (d–f) Depletion of FEZ1 affects HIV-1 particles that have productively fused into the cytoplasm. (d) NHDF cells were treated with control or FEZ1-C siRNAs. 48h after transfection cells were infected with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed in formaldehyde and GFP and Tomato signals were acquired using a spinning-disc confocal microscope. Arrows highlight fused (green, S15-negative) particles proximal to the nucleus in each sample. Representative confocal planes are shown. (e) Quantification of the % fused (green, S15-negative) viral particles within 2μm of the nucleus in samples as described and processed in d. n≥29 cells and an average of 53–55 viral particles per cell. (f) Control siRNA-treated NHDF cells were treated with Bafilomycin A1 for 2h during spinoculation followed by infection with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed and the total number of fused (green, S15-negative) viral particles were quantified and presented as a % of the total number of viral particles. n≥29 cells and an average of 55–80 viral particles per cell. Error bars represent standard deviation. Scale bars represent 10 μm.

Mentions: The association of FEZ1 with HIV-1 might also affect the movement of incoming virus to the nucleus, rather than playing a direct role in the nuclear entry process. Indeed, FEZ1 is known to act as a kinesin-1 adaptor protein20. To test this, the potential effects of FEZ1 depletion on the movement of HIV-1 particles carrying GFP-tagged Vpr4 were examined. Live imaging of virus in cells treated with either control or FEZ1 siRNAs revealed that in both cases viral particles exhibited long-range bi-directional motility (Supplementary Movies 1, 2 and 3, respectively). Similar bi-directional movement was observed in microglia (Supplementary Movie 4), and has been reported in other cell types4,5, demonstrating that this was not unique to NHDFs. Movies and still images generated from these movies suggested that the majority of particles observed in control siRNA-treated NHDFs gradually moved toward the nucleus (Supplementary Movie 1 and Fig. 3a, upper panels). However, viral particles in FEZ1-depleted cells frequently appeared to make progress towards the nucleus before suddenly making long runs back toward the cell periphery (Supplementary Movies 2 and 3 and Fig. 3a, lower panels). To confirm this, cells were treated and infected as above but imaged at a higher frame rate of 2 frames per second (fps) over a 150s period. Measuring distances traveled by viral particles in either retrograde or anterograde directions revealed that in control siRNA-treated cells particles on average traveled longer retrograde than anterograde distances resulting in a net ~1μm movement toward the nucleus in this time (Fig. 3b). By contrast, in FEZ1-depleted cells viral particles on average traveled longer anterograde distances, resulting in net ~1.5μm movement toward the cell periphery (Fig. 3b). In line with these observations, measuring the distance of viral particles from the nucleus revealed that while the number of viral particles reaching within 2μm of the nucleus increased over time in control siRNA-treated cells (particle numbers gradually decline from their peak, likely due to uncoating, nuclear entry and/or photobleaching at later stages), the majority of viral particles failed to reach within 2μm of the nucleus in FEZ1-depleted cells (Fig. 3c). Finally, to distinguish viral particles that had fused into the cytoplasm from those that might have been non-productively endocytosed, control or FEZ1 siRNA-treated cells were infected with VSV-G-pseudotyped double-labeled virus composed of Vpr-GFP (labeling the viral core) and S15-Tomato (labeling the viral membrane)21. Unfused viral particles appear yellow while fused particles shed the S15-Tomato labeled viral membrane and appear green. As shown in figures 3d and 3e, FEZ1 depletion did not affect the number of green particles in the cell but decreased the number of fused (green) viral particles within 2μm of the nucleus compared with controls. Confirming that these green particles represented fused virus, their number as a percentage of total viral particles in the cell was significantly reduced by treatment of cells with the fusion inhibitor, Bafilomycin (Fig. 3f) similar to previous reports22. These data collectively demonstrated that FEZ1 associated with in vitro assembled HIV-1 CA-NC complexes and was required for efficient movement of HIV-1 across the cytoplasm to the nucleus.


HIV-1 capsids bind and exploit the kinesin-1 adaptor FEZ1 for inward movement to the nucleus.

Malikov V, da Silva ES, Jovasevic V, Bennett G, de Souza Aranha Vieira DA, Schulte B, Diaz-Griffero F, Walsh D, Naghavi MH - Nat Commun (2015)

Depletion of FEZ1 inhibits HIV-1 trafficking to the nucleusNHDFs were treated with control or FEZ1-C siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-GFP-Vpr followed by live imaging using a spinning-disc confocal microscope. (a) Still images from movies (Supplementary Movies 1 and 2) taken at the indicated times are shown. Green arrows highlight viral particles entering and traversing the cytoplasm in control siRNA-treated cells. Red arrows highlight representative examples of particles that approach the nucleus but then move long distances back out to the cell periphery in FEZ1-depleted cells. (b) Quantification of the average distance (μm per 2.5 min) traveled by viral particles towards the nucleus (Retrograde motility) or away from the nucleus (Antrograde motility). n≥5 cells and an average of 7–30 viral particles per cell. (c) Quantification of the percentage of virions within 2μm of the nucleus in infected siRNA-treated cells at the indicated time points. n≥20 cells and an average of 80–99 viral particles per cell. (d–f) Depletion of FEZ1 affects HIV-1 particles that have productively fused into the cytoplasm. (d) NHDF cells were treated with control or FEZ1-C siRNAs. 48h after transfection cells were infected with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed in formaldehyde and GFP and Tomato signals were acquired using a spinning-disc confocal microscope. Arrows highlight fused (green, S15-negative) particles proximal to the nucleus in each sample. Representative confocal planes are shown. (e) Quantification of the % fused (green, S15-negative) viral particles within 2μm of the nucleus in samples as described and processed in d. n≥29 cells and an average of 53–55 viral particles per cell. (f) Control siRNA-treated NHDF cells were treated with Bafilomycin A1 for 2h during spinoculation followed by infection with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed and the total number of fused (green, S15-negative) viral particles were quantified and presented as a % of the total number of viral particles. n≥29 cells and an average of 55–80 viral particles per cell. Error bars represent standard deviation. Scale bars represent 10 μm.
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Figure 3: Depletion of FEZ1 inhibits HIV-1 trafficking to the nucleusNHDFs were treated with control or FEZ1-C siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-GFP-Vpr followed by live imaging using a spinning-disc confocal microscope. (a) Still images from movies (Supplementary Movies 1 and 2) taken at the indicated times are shown. Green arrows highlight viral particles entering and traversing the cytoplasm in control siRNA-treated cells. Red arrows highlight representative examples of particles that approach the nucleus but then move long distances back out to the cell periphery in FEZ1-depleted cells. (b) Quantification of the average distance (μm per 2.5 min) traveled by viral particles towards the nucleus (Retrograde motility) or away from the nucleus (Antrograde motility). n≥5 cells and an average of 7–30 viral particles per cell. (c) Quantification of the percentage of virions within 2μm of the nucleus in infected siRNA-treated cells at the indicated time points. n≥20 cells and an average of 80–99 viral particles per cell. (d–f) Depletion of FEZ1 affects HIV-1 particles that have productively fused into the cytoplasm. (d) NHDF cells were treated with control or FEZ1-C siRNAs. 48h after transfection cells were infected with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed in formaldehyde and GFP and Tomato signals were acquired using a spinning-disc confocal microscope. Arrows highlight fused (green, S15-negative) particles proximal to the nucleus in each sample. Representative confocal planes are shown. (e) Quantification of the % fused (green, S15-negative) viral particles within 2μm of the nucleus in samples as described and processed in d. n≥29 cells and an average of 53–55 viral particles per cell. (f) Control siRNA-treated NHDF cells were treated with Bafilomycin A1 for 2h during spinoculation followed by infection with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed and the total number of fused (green, S15-negative) viral particles were quantified and presented as a % of the total number of viral particles. n≥29 cells and an average of 55–80 viral particles per cell. Error bars represent standard deviation. Scale bars represent 10 μm.
Mentions: The association of FEZ1 with HIV-1 might also affect the movement of incoming virus to the nucleus, rather than playing a direct role in the nuclear entry process. Indeed, FEZ1 is known to act as a kinesin-1 adaptor protein20. To test this, the potential effects of FEZ1 depletion on the movement of HIV-1 particles carrying GFP-tagged Vpr4 were examined. Live imaging of virus in cells treated with either control or FEZ1 siRNAs revealed that in both cases viral particles exhibited long-range bi-directional motility (Supplementary Movies 1, 2 and 3, respectively). Similar bi-directional movement was observed in microglia (Supplementary Movie 4), and has been reported in other cell types4,5, demonstrating that this was not unique to NHDFs. Movies and still images generated from these movies suggested that the majority of particles observed in control siRNA-treated NHDFs gradually moved toward the nucleus (Supplementary Movie 1 and Fig. 3a, upper panels). However, viral particles in FEZ1-depleted cells frequently appeared to make progress towards the nucleus before suddenly making long runs back toward the cell periphery (Supplementary Movies 2 and 3 and Fig. 3a, lower panels). To confirm this, cells were treated and infected as above but imaged at a higher frame rate of 2 frames per second (fps) over a 150s period. Measuring distances traveled by viral particles in either retrograde or anterograde directions revealed that in control siRNA-treated cells particles on average traveled longer retrograde than anterograde distances resulting in a net ~1μm movement toward the nucleus in this time (Fig. 3b). By contrast, in FEZ1-depleted cells viral particles on average traveled longer anterograde distances, resulting in net ~1.5μm movement toward the cell periphery (Fig. 3b). In line with these observations, measuring the distance of viral particles from the nucleus revealed that while the number of viral particles reaching within 2μm of the nucleus increased over time in control siRNA-treated cells (particle numbers gradually decline from their peak, likely due to uncoating, nuclear entry and/or photobleaching at later stages), the majority of viral particles failed to reach within 2μm of the nucleus in FEZ1-depleted cells (Fig. 3c). Finally, to distinguish viral particles that had fused into the cytoplasm from those that might have been non-productively endocytosed, control or FEZ1 siRNA-treated cells were infected with VSV-G-pseudotyped double-labeled virus composed of Vpr-GFP (labeling the viral core) and S15-Tomato (labeling the viral membrane)21. Unfused viral particles appear yellow while fused particles shed the S15-Tomato labeled viral membrane and appear green. As shown in figures 3d and 3e, FEZ1 depletion did not affect the number of green particles in the cell but decreased the number of fused (green) viral particles within 2μm of the nucleus compared with controls. Confirming that these green particles represented fused virus, their number as a percentage of total viral particles in the cell was significantly reduced by treatment of cells with the fusion inhibitor, Bafilomycin (Fig. 3f) similar to previous reports22. These data collectively demonstrated that FEZ1 associated with in vitro assembled HIV-1 CA-NC complexes and was required for efficient movement of HIV-1 across the cytoplasm to the nucleus.

Bottom Line: Furthermore, both dynein and kinesin-1 motors are required for HIV-1 trafficking to the nucleus.Finally, the ability of exogenously expressed FEZ1 to promote early HIV-1 infection requires binding to kinesin-1.Our findings demonstrate that opposing motors both contribute to early HIV-1 movement and identify the kinesin-1 adaptor, FEZ1 as a capsid-associated host regulator of this process usurped by HIV-1 to accomplish net inward movement towards the nucleus.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA [2] Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.

ABSTRACT
Intracellular transport of cargos, including many viruses, involves directed movement on microtubules mediated by motor proteins. Although a number of viruses bind motors of opposing directionality, how they associate with and control these motors to accomplish directed movement remains poorly understood. Here we show that human immunodeficiency virus type 1 (HIV-1) associates with the kinesin-1 adaptor protein, Fasiculation and Elongation Factor zeta 1 (FEZ1). RNAi-mediated FEZ1 depletion blocks early infection, with virus particles exhibiting bi-directional motility but no net movement to the nucleus. Furthermore, both dynein and kinesin-1 motors are required for HIV-1 trafficking to the nucleus. Finally, the ability of exogenously expressed FEZ1 to promote early HIV-1 infection requires binding to kinesin-1. Our findings demonstrate that opposing motors both contribute to early HIV-1 movement and identify the kinesin-1 adaptor, FEZ1 as a capsid-associated host regulator of this process usurped by HIV-1 to accomplish net inward movement towards the nucleus.

No MeSH data available.


Related in: MedlinePlus