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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 kinesin-1 inhibits trafficking of HIV-1 particles to the nucleusNHDFs were treated with control siRNA or siRNAs targeting either of two different isoforms of kinesin-1 heavy chain (Kif5A or Kif5B). 48h post-transfection cells were infected with HIV-1-VSV-GFP-Vpr followed by live imaging of particle movement. (a) Still images from movies (Supplementary Movies 5–7) at the indicated time points are shown. Scale bars represent 10 μm. (b) Quantification of the percentage of virions within 2μm of the nucleus in infected cells at the indicated time points. n≥15 cells and an average of 53–91 viral particles per cell. Results are representative of 3 or more independent experiments, and error bars represent standard deviation.
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Figure 6: Depletion of kinesin-1 inhibits trafficking of HIV-1 particles to the nucleusNHDFs were treated with control siRNA or siRNAs targeting either of two different isoforms of kinesin-1 heavy chain (Kif5A or Kif5B). 48h post-transfection cells were infected with HIV-1-VSV-GFP-Vpr followed by live imaging of particle movement. (a) Still images from movies (Supplementary Movies 5–7) at the indicated time points are shown. Scale bars represent 10 μm. (b) Quantification of the percentage of virions within 2μm of the nucleus in infected cells at the indicated time points. n≥15 cells and an average of 53–91 viral particles per cell. Results are representative of 3 or more independent experiments, and error bars represent standard deviation.

Mentions: To determine whether kinesin-1 affected the movement of HIV-1 particles toward the nucleus, NHDFs were treated with control, Kif5A or Kif5B siRNAs and then infected with HIV-1-GFP-Vpr. Live imaging revealed that depletion of either isoform of kinesin-1 potently inhibited the movement of virus towards the nucleus compared with control siRNA-treated samples (Fig. 6a and Supplementary Movies 5–7). These effects were further confirmed by quantification of the total number of particles within 2μm of the nucleus over time (Fig. 6b), which showed that the vast majority of viral particles failed to traffic toward the nucleus in kinesin-1-depleted cells. Furthermore, in kinesin-1 depleted cells virus particles exhibited movements characteristic of either free diffusion in the cytosol or actin-mediated movement. These observations were in line with results from fusion and viral DNA synthesis assays (Fig. 4i–4l), and suggested that in kinesin-1 depleted cells virus particles entered the cytosol but were only capable of short-ranged movements largely at the cell periphery. Furthermore, the requirement for kinesin-1 to facilitate trafficking of HIV-1 to the nucleus suggested that FEZ1’s function as a kinesin-1 adaptor protein might underlie FEZ1’s role in early infection. To test this, we examined the effects on HIV-1 infection of exogenous expression of FEZ1 or a recently described FEZ1 mutant, FEZ1-S58A28. This mutant, where serine 58 is replaced with alanine, fails to bind kinesin-1 in Caenorhabditis elegans28. To confirm this for their human counterparts, GFP control or a GFP-tagged form of human kinesin-1 heavy chain tail24 (containing the FEZ1-binding domain28) was expressed in cells along with FEZ1-Flag or FEZ1-S58A-Flag. GFP was then recovered from cell extracts using sepharose-conjugated GFP-binding peptide29. Binding assays showed that FEZ1-Flag did not bind GFP but specifically bound Kif5B, while the S58A mutation abolished this binding (Fig. 7a). Stable pools of NHDFs expressing Flag control, FEZ1-Flag or FEZ1-S58A-Flag were then generated, and equal expression of exogenous forms of FEZ1 was confirmed by WB analysis (Fig. 7b). Infection of these pools with HIV-1-VSV-luc reporter revealed that exogenous expression of FEZ1-Flag enhanced HIV-1 infection, while FEZ1-S58A-Flag did not increase infection above that of Flag control cells (Fig. 7c). The same results were observed when pools were infected with HIV-1-luc reporter virus pseudotyped with MuLV amphotropic envelope (Fig. 7d), demonstrating that these effects were again independent of the route of viral entry. Importantly, both FEZ1-Flag and FEZ1-S58A-Flag bound in vitro assembled HIV-1 CA-NC equally (Fig. 7e). These interactions were also confirmed by co-transfection of 293A cells with either FEZ1-Flag or FEZ1-S58A-Flag along with a HIV-1 Gag expressing vector. Similar to CA-NC assays both forms of FEZ1 were found to co-IP with HIV-1 Gag (Fig. 7f). Overall, these findings demonstrated that kinesin-1 was required for viral movement to the nucleus and that FEZ1’s ability to bind kinesin-1 was required for this HIV-1-associated adaptor protein to regulate early stages of HIV-1 infection.


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 kinesin-1 inhibits trafficking of HIV-1 particles to the nucleusNHDFs were treated with control siRNA or siRNAs targeting either of two different isoforms of kinesin-1 heavy chain (Kif5A or Kif5B). 48h post-transfection cells were infected with HIV-1-VSV-GFP-Vpr followed by live imaging of particle movement. (a) Still images from movies (Supplementary Movies 5–7) at the indicated time points are shown. Scale bars represent 10 μm. (b) Quantification of the percentage of virions within 2μm of the nucleus in infected cells at the indicated time points. n≥15 cells and an average of 53–91 viral particles per cell. Results are representative of 3 or more independent experiments, and error bars represent standard deviation.
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Related In: Results  -  Collection

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Figure 6: Depletion of kinesin-1 inhibits trafficking of HIV-1 particles to the nucleusNHDFs were treated with control siRNA or siRNAs targeting either of two different isoforms of kinesin-1 heavy chain (Kif5A or Kif5B). 48h post-transfection cells were infected with HIV-1-VSV-GFP-Vpr followed by live imaging of particle movement. (a) Still images from movies (Supplementary Movies 5–7) at the indicated time points are shown. Scale bars represent 10 μm. (b) Quantification of the percentage of virions within 2μm of the nucleus in infected cells at the indicated time points. n≥15 cells and an average of 53–91 viral particles per cell. Results are representative of 3 or more independent experiments, and error bars represent standard deviation.
Mentions: To determine whether kinesin-1 affected the movement of HIV-1 particles toward the nucleus, NHDFs were treated with control, Kif5A or Kif5B siRNAs and then infected with HIV-1-GFP-Vpr. Live imaging revealed that depletion of either isoform of kinesin-1 potently inhibited the movement of virus towards the nucleus compared with control siRNA-treated samples (Fig. 6a and Supplementary Movies 5–7). These effects were further confirmed by quantification of the total number of particles within 2μm of the nucleus over time (Fig. 6b), which showed that the vast majority of viral particles failed to traffic toward the nucleus in kinesin-1-depleted cells. Furthermore, in kinesin-1 depleted cells virus particles exhibited movements characteristic of either free diffusion in the cytosol or actin-mediated movement. These observations were in line with results from fusion and viral DNA synthesis assays (Fig. 4i–4l), and suggested that in kinesin-1 depleted cells virus particles entered the cytosol but were only capable of short-ranged movements largely at the cell periphery. Furthermore, the requirement for kinesin-1 to facilitate trafficking of HIV-1 to the nucleus suggested that FEZ1’s function as a kinesin-1 adaptor protein might underlie FEZ1’s role in early infection. To test this, we examined the effects on HIV-1 infection of exogenous expression of FEZ1 or a recently described FEZ1 mutant, FEZ1-S58A28. This mutant, where serine 58 is replaced with alanine, fails to bind kinesin-1 in Caenorhabditis elegans28. To confirm this for their human counterparts, GFP control or a GFP-tagged form of human kinesin-1 heavy chain tail24 (containing the FEZ1-binding domain28) was expressed in cells along with FEZ1-Flag or FEZ1-S58A-Flag. GFP was then recovered from cell extracts using sepharose-conjugated GFP-binding peptide29. Binding assays showed that FEZ1-Flag did not bind GFP but specifically bound Kif5B, while the S58A mutation abolished this binding (Fig. 7a). Stable pools of NHDFs expressing Flag control, FEZ1-Flag or FEZ1-S58A-Flag were then generated, and equal expression of exogenous forms of FEZ1 was confirmed by WB analysis (Fig. 7b). Infection of these pools with HIV-1-VSV-luc reporter revealed that exogenous expression of FEZ1-Flag enhanced HIV-1 infection, while FEZ1-S58A-Flag did not increase infection above that of Flag control cells (Fig. 7c). The same results were observed when pools were infected with HIV-1-luc reporter virus pseudotyped with MuLV amphotropic envelope (Fig. 7d), demonstrating that these effects were again independent of the route of viral entry. Importantly, both FEZ1-Flag and FEZ1-S58A-Flag bound in vitro assembled HIV-1 CA-NC equally (Fig. 7e). These interactions were also confirmed by co-transfection of 293A cells with either FEZ1-Flag or FEZ1-S58A-Flag along with a HIV-1 Gag expressing vector. Similar to CA-NC assays both forms of FEZ1 were found to co-IP with HIV-1 Gag (Fig. 7f). Overall, these findings demonstrated that kinesin-1 was required for viral movement to the nucleus and that FEZ1’s ability to bind kinesin-1 was required for this HIV-1-associated adaptor protein to regulate early stages of HIV-1 infection.

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