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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

FEZ1 is a positive regulator of HIV-1 infection in non-neuronal human cells(a) Endogenous levels of FEZ1 in neuroblastoma cells (Sh-SY5Y), microglia (CHME3) or primary normal human dermal fibroblasts (NHDF). Equal cell numbers were lysed and analyzed by WB using anti-FEZ1 antibody. Equal loading was confirmed using anti-GAPDH antibody. (b–i) FEZ1 is required for early HIV-1 infection regardless of the route of viral entry. NHDF (b–c), CHME3 (d–e) or Thp-1 differentiated into macrophages (f–g) were transfected with control siRNA (control) or independent FEZ1 siRNAs (FEZ1-B or FEZ1-C). 48h post-transfection cells were infected with HIV-1-VSV-luc followed by measurements of luciferase activity to determine levels of infection in NHDF (b), CHME3 (d) or differentiated Thp-1 (f) cells. (c, e and g) WB analysis showing the extent of FEZ1 depletion in samples from b, d and f, respectively. β-actin or eIF4E were used as loading controls. (h and i) Rescue of RNAi-mediated FEZ1 depletion restores HIV-1 infection. (h) Pools of NHDF cells expressing either Flag control or FEZ1-Flag were treated with either control or FEZ1-C siRNAs followed by HIV-1-VSV-luc infection and measurements of infectivity as in b–d. (i) WB analysis showing the levels of FEZ1 in samples from h. eIF4E was used as a loading control. (j and k) NHDF or CHME3 cells were transfected with control or FEZ1-C siRNAs followed by infection with HIV-1-Ampho-luc 48h post-transfection. Luciferase assays were used to determine levels of infection in NHDF (j) or CHME3 (k). Results are representative of 3 or more independent experiments, and error bars represent standard deviation. Molecular weight markers (in kDa) are shown to the right of WBs.
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Figure 1: FEZ1 is a positive regulator of HIV-1 infection in non-neuronal human cells(a) Endogenous levels of FEZ1 in neuroblastoma cells (Sh-SY5Y), microglia (CHME3) or primary normal human dermal fibroblasts (NHDF). Equal cell numbers were lysed and analyzed by WB using anti-FEZ1 antibody. Equal loading was confirmed using anti-GAPDH antibody. (b–i) FEZ1 is required for early HIV-1 infection regardless of the route of viral entry. NHDF (b–c), CHME3 (d–e) or Thp-1 differentiated into macrophages (f–g) were transfected with control siRNA (control) or independent FEZ1 siRNAs (FEZ1-B or FEZ1-C). 48h post-transfection cells were infected with HIV-1-VSV-luc followed by measurements of luciferase activity to determine levels of infection in NHDF (b), CHME3 (d) or differentiated Thp-1 (f) cells. (c, e and g) WB analysis showing the extent of FEZ1 depletion in samples from b, d and f, respectively. β-actin or eIF4E were used as loading controls. (h and i) Rescue of RNAi-mediated FEZ1 depletion restores HIV-1 infection. (h) Pools of NHDF cells expressing either Flag control or FEZ1-Flag were treated with either control or FEZ1-C siRNAs followed by HIV-1-VSV-luc infection and measurements of infectivity as in b–d. (i) WB analysis showing the levels of FEZ1 in samples from h. eIF4E was used as a loading control. (j and k) NHDF or CHME3 cells were transfected with control or FEZ1-C siRNAs followed by infection with HIV-1-Ampho-luc 48h post-transfection. Luciferase assays were used to determine levels of infection in NHDF (j) or CHME3 (k). Results are representative of 3 or more independent experiments, and error bars represent standard deviation. Molecular weight markers (in kDa) are shown to the right of WBs.

Mentions: While naturally high FEZ1 levels contribute to neuronal resistance to HIV-1 infection8,9, the potential contribution of FEZ1 to infection in other cell types remains unknown. To begin exploring this, endogenous FEZ1 levels were measured in different human cell types by western blotting (WB). In line with our previous work, FEZ1 was naturally expressed at high levels in the neuroblastoma line, SH-SY5Y compared to either the human microglia line, CHME3 or primary normal human dermal fibroblasts (NHDFs) (Fig. 1a). FEZ1 levels in a variety of T cell and monocyte lines were also found to be either comparable with or lower than those in NHDFs (Supplementary Fig. 1), demonstrating that FEZ1 expression in various natural target cell types (microglia, T cells and monocytes) and NHDFs is notably lower than in neuronal cells. To test if endogenous FEZ1 levels in non-neuronal cells could influence their susceptibility to infection, FEZ1 was depleted in NHDFs, microglia (CHME3) or macrophages (differentiated Thp-1) using RNAi. siRNA-treated cells were then infected with HIV-1 carrying a luciferase reporter gene and pseudotyped with vesicular stomatitis virus G (VSV-G) envelope glycoprotein, widely used to improve HIV-1 infectivity and which mediates endocytic fusion, one of the entry pathways used by HIV-110,11. Measurements of luciferase activity revealed that compared to control siRNA-treated cultures, FEZ1 depletion using either of two independent siRNAs potently reduced HIV-1 infection in all three cell types (Fig. 1b, 1d and 1f, respectively). Furthermore, WB analysis showed that effects on infection correlated with the levels of FEZ1 depletion achieved with each siRNA (Fig. 1c, 1e and 1g). While FEZ1 siRNAs did not affect cell morphology or induce apoptosis (Supplementary Fig. 2a and 2b, respectively), to further rule out potential off-target effects the ability of exogenously-expressed siRNA-resistant flag-tagged FEZ1 to rescue infection was tested. In control Flag-expressing NHDFs FEZ1 siRNAs reduced FEZ1 levels and suppressed HIV-1 infection (Fig. 1h–i). By contrast, FEZ1 levels were maintained in FEZ1-Flag-expressing NHDFs in the presence of FEZ1 siRNAs and infectivity was restored (Fig. 1h–i). Again, the levels of viral infection correlated directly with the levels of FEZ1 expression.


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)

FEZ1 is a positive regulator of HIV-1 infection in non-neuronal human cells(a) Endogenous levels of FEZ1 in neuroblastoma cells (Sh-SY5Y), microglia (CHME3) or primary normal human dermal fibroblasts (NHDF). Equal cell numbers were lysed and analyzed by WB using anti-FEZ1 antibody. Equal loading was confirmed using anti-GAPDH antibody. (b–i) FEZ1 is required for early HIV-1 infection regardless of the route of viral entry. NHDF (b–c), CHME3 (d–e) or Thp-1 differentiated into macrophages (f–g) were transfected with control siRNA (control) or independent FEZ1 siRNAs (FEZ1-B or FEZ1-C). 48h post-transfection cells were infected with HIV-1-VSV-luc followed by measurements of luciferase activity to determine levels of infection in NHDF (b), CHME3 (d) or differentiated Thp-1 (f) cells. (c, e and g) WB analysis showing the extent of FEZ1 depletion in samples from b, d and f, respectively. β-actin or eIF4E were used as loading controls. (h and i) Rescue of RNAi-mediated FEZ1 depletion restores HIV-1 infection. (h) Pools of NHDF cells expressing either Flag control or FEZ1-Flag were treated with either control or FEZ1-C siRNAs followed by HIV-1-VSV-luc infection and measurements of infectivity as in b–d. (i) WB analysis showing the levels of FEZ1 in samples from h. eIF4E was used as a loading control. (j and k) NHDF or CHME3 cells were transfected with control or FEZ1-C siRNAs followed by infection with HIV-1-Ampho-luc 48h post-transfection. Luciferase assays were used to determine levels of infection in NHDF (j) or CHME3 (k). Results are representative of 3 or more independent experiments, and error bars represent standard deviation. Molecular weight markers (in kDa) are shown to the right of WBs.
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Figure 1: FEZ1 is a positive regulator of HIV-1 infection in non-neuronal human cells(a) Endogenous levels of FEZ1 in neuroblastoma cells (Sh-SY5Y), microglia (CHME3) or primary normal human dermal fibroblasts (NHDF). Equal cell numbers were lysed and analyzed by WB using anti-FEZ1 antibody. Equal loading was confirmed using anti-GAPDH antibody. (b–i) FEZ1 is required for early HIV-1 infection regardless of the route of viral entry. NHDF (b–c), CHME3 (d–e) or Thp-1 differentiated into macrophages (f–g) were transfected with control siRNA (control) or independent FEZ1 siRNAs (FEZ1-B or FEZ1-C). 48h post-transfection cells were infected with HIV-1-VSV-luc followed by measurements of luciferase activity to determine levels of infection in NHDF (b), CHME3 (d) or differentiated Thp-1 (f) cells. (c, e and g) WB analysis showing the extent of FEZ1 depletion in samples from b, d and f, respectively. β-actin or eIF4E were used as loading controls. (h and i) Rescue of RNAi-mediated FEZ1 depletion restores HIV-1 infection. (h) Pools of NHDF cells expressing either Flag control or FEZ1-Flag were treated with either control or FEZ1-C siRNAs followed by HIV-1-VSV-luc infection and measurements of infectivity as in b–d. (i) WB analysis showing the levels of FEZ1 in samples from h. eIF4E was used as a loading control. (j and k) NHDF or CHME3 cells were transfected with control or FEZ1-C siRNAs followed by infection with HIV-1-Ampho-luc 48h post-transfection. Luciferase assays were used to determine levels of infection in NHDF (j) or CHME3 (k). Results are representative of 3 or more independent experiments, and error bars represent standard deviation. Molecular weight markers (in kDa) are shown to the right of WBs.
Mentions: While naturally high FEZ1 levels contribute to neuronal resistance to HIV-1 infection8,9, the potential contribution of FEZ1 to infection in other cell types remains unknown. To begin exploring this, endogenous FEZ1 levels were measured in different human cell types by western blotting (WB). In line with our previous work, FEZ1 was naturally expressed at high levels in the neuroblastoma line, SH-SY5Y compared to either the human microglia line, CHME3 or primary normal human dermal fibroblasts (NHDFs) (Fig. 1a). FEZ1 levels in a variety of T cell and monocyte lines were also found to be either comparable with or lower than those in NHDFs (Supplementary Fig. 1), demonstrating that FEZ1 expression in various natural target cell types (microglia, T cells and monocytes) and NHDFs is notably lower than in neuronal cells. To test if endogenous FEZ1 levels in non-neuronal cells could influence their susceptibility to infection, FEZ1 was depleted in NHDFs, microglia (CHME3) or macrophages (differentiated Thp-1) using RNAi. siRNA-treated cells were then infected with HIV-1 carrying a luciferase reporter gene and pseudotyped with vesicular stomatitis virus G (VSV-G) envelope glycoprotein, widely used to improve HIV-1 infectivity and which mediates endocytic fusion, one of the entry pathways used by HIV-110,11. Measurements of luciferase activity revealed that compared to control siRNA-treated cultures, FEZ1 depletion using either of two independent siRNAs potently reduced HIV-1 infection in all three cell types (Fig. 1b, 1d and 1f, respectively). Furthermore, WB analysis showed that effects on infection correlated with the levels of FEZ1 depletion achieved with each siRNA (Fig. 1c, 1e and 1g). While FEZ1 siRNAs did not affect cell morphology or induce apoptosis (Supplementary Fig. 2a and 2b, respectively), to further rule out potential off-target effects the ability of exogenously-expressed siRNA-resistant flag-tagged FEZ1 to rescue infection was tested. In control Flag-expressing NHDFs FEZ1 siRNAs reduced FEZ1 levels and suppressed HIV-1 infection (Fig. 1h–i). By contrast, FEZ1 levels were maintained in FEZ1-Flag-expressing NHDFs in the presence of FEZ1 siRNAs and infectivity was restored (Fig. 1h–i). Again, the levels of viral infection correlated directly with the levels of FEZ1 expression.

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