Limits...
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

Kinesin-1 regulates nuclear entry of HIV-1 DNA(a–g) Kinesin-1 depletion affects early HIV-1 infection regardless of the route of viral entry. NHDF (a–b), CHME3 (c–d,g) or Jurkat (e–f) cells were transfected with control (ctrl), Kif5A or Kif5B siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-luc (a, c, e) or HIV-1-Ampho-luc (g) followed by measurements of luciferase activity in NHDF (a), CHME3 (c, g) or Jurkat (e) cells. (b, d and f). WB analysis demonstrated kinesin-1 depletion in samples from a, c and e using antibodies against kinesin-1 (Kif5A/B), Kif5B or β-actin (loading control). (h) Kinesin-1 knockdown does not inhibit VSV infection. NHDF cells treated with control, Kif5A or Kif5B siRNAs were infected 48h post-transfection with VSV at m.o.i. 10. 8h after infection cells were lysed and analyzed by WB using anti-VSV-G or anti-VSV-N antibodies. eIF4E was used as loading control. (i) Effects of kinesin-1 depletion on fusion of HIV-1 cores into the cytosol. NHDFs were treated with control, Kif5A or Kif5B siRNAs and then either mock infected (upper panels) or infected with HIV-1-VSV-luc containing BlaM-Vpr (lower panels). FACS analysis of cells showed ~14–18% shift from green (uncleaved CCF2) to blue (cleaved CCF2) cells in control and kinesin-1-depleted cultures. (j–l) Kinesin-1 regulates nuclear entry of HIV-1 DNA. (j) CHME3 cells treated with control (Ctrl), Kif5A or Kif5B siRNAs were infected with HIV-1-VSV-puro 48h post-transfection. Low molecular Hirt DNA was isolated 24h post-infection and levels of viral MSS-DNA, total viral DNA and 2-LTRs in samples were measured by qPCR using specific primers to MSS, puromycin or 2-LTRs, respectively. Copy numbers were calculated and normalized to input DNA in each sample. Data are presented as mean +/- SEM. (k–l) CHME3 cells were transfected with plasmids expressing either GFP or GFP-tagged dominant negative Kif5B (Kif5B-GFP-DN). 48h post-transfection cells were infected (k) and levels of MSS DNA, total DN and 2-LTRs were measured as described in j. or (l) cells were lysed in Laemmli buffer and analyzed by WB using anti-GFP antibody to detect GFP or dominant-negative GFP-Kif5 (Kif5B-GFP-DN). Molecular weight markers (in kDa) are shown to the right of WBs.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4380233&req=5

Figure 4: Kinesin-1 regulates nuclear entry of HIV-1 DNA(a–g) Kinesin-1 depletion affects early HIV-1 infection regardless of the route of viral entry. NHDF (a–b), CHME3 (c–d,g) or Jurkat (e–f) cells were transfected with control (ctrl), Kif5A or Kif5B siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-luc (a, c, e) or HIV-1-Ampho-luc (g) followed by measurements of luciferase activity in NHDF (a), CHME3 (c, g) or Jurkat (e) cells. (b, d and f). WB analysis demonstrated kinesin-1 depletion in samples from a, c and e using antibodies against kinesin-1 (Kif5A/B), Kif5B or β-actin (loading control). (h) Kinesin-1 knockdown does not inhibit VSV infection. NHDF cells treated with control, Kif5A or Kif5B siRNAs were infected 48h post-transfection with VSV at m.o.i. 10. 8h after infection cells were lysed and analyzed by WB using anti-VSV-G or anti-VSV-N antibodies. eIF4E was used as loading control. (i) Effects of kinesin-1 depletion on fusion of HIV-1 cores into the cytosol. NHDFs were treated with control, Kif5A or Kif5B siRNAs and then either mock infected (upper panels) or infected with HIV-1-VSV-luc containing BlaM-Vpr (lower panels). FACS analysis of cells showed ~14–18% shift from green (uncleaved CCF2) to blue (cleaved CCF2) cells in control and kinesin-1-depleted cultures. (j–l) Kinesin-1 regulates nuclear entry of HIV-1 DNA. (j) CHME3 cells treated with control (Ctrl), Kif5A or Kif5B siRNAs were infected with HIV-1-VSV-puro 48h post-transfection. Low molecular Hirt DNA was isolated 24h post-infection and levels of viral MSS-DNA, total viral DNA and 2-LTRs in samples were measured by qPCR using specific primers to MSS, puromycin or 2-LTRs, respectively. Copy numbers were calculated and normalized to input DNA in each sample. Data are presented as mean +/- SEM. (k–l) CHME3 cells were transfected with plasmids expressing either GFP or GFP-tagged dominant negative Kif5B (Kif5B-GFP-DN). 48h post-transfection cells were infected (k) and levels of MSS DNA, total DN and 2-LTRs were measured as described in j. or (l) cells were lysed in Laemmli buffer and analyzed by WB using anti-GFP antibody to detect GFP or dominant-negative GFP-Kif5 (Kif5B-GFP-DN). Molecular weight markers (in kDa) are shown to the right of WBs.

Mentions: To further explore how a kinesin-1 adaptor protein might regulate net inward movement of HIV-1 we examined whether kinesin-1 itself contributed to early infection. To do this, siRNAs were used to deplete different human cell types of either of two different isoforms of kinesin-1 heavy chain, Kif5A or Kif5B, followed by infection of these cells with HIV-1-VSV-luc reporter. Knockdown of either Kif5A or Kif5B inhibited infection in both primary fibroblasts (NHDFs) (Fig. 4a) and human microglia (CHME3) (Fig. 4c), and Kif5B was also found to be required for infection of T cell lines, Jurkat (Fig. 4e) and 174xCEM (Supplementary Fig. 4a). WB analysis confirmed Kif5 knockdown levels (Fig. 4b, 4d, 4f and Supplementary Fig. 4b). Importantly, kinesin-1-depleted cells exhibited no morphological changes or indications of apoptosis compared with control siRNA-treated cells (Supplementary Fig. 2a and 2b). Kif5A or Kif5B depletion in CHME3 cells also blocked infection by HIV-1-luc pseudotyped with MuLV amphotropic envelope glycoproteins (Fig. 4g). Furthermore, while infection by HIV-1-VSV-G was suppressed in Kif5A- or Kif5B-depleted cells, infection by VSV itself was not inhibited (Fig. 4h), further suggesting an envelope-independent block specific to HIV-1 that occurred at an early post-entry stage of infection. To confirm this, cells were treated with control, Kif5A or Kif5B siRNAs followed by infection with HIV-1-VSV-G carrying a β-lactamase-Vpr fusion protein, which allows monitoring of viral penetration into the cytosol23. Compared to control siRNA-treated samples, depletion of either Kif5A or Kif5B did not significantly affect fusion of HIV-1 into the cytoplasm of NHDFs (Fig. 4i), and did not correlate with the potent inhibition of early HIV-1 infection in cells depleted of either isoform (Fig. 4a–f). To further determine the point in the viral life cycle affected by kinesin-1, CHME3 cells were treated with control, Kif5A or Kif5B siRNAs. Cultures were then infected and levels of minus-strand strong stop (MSS) DNA, total viral DNA and 2-LTR circles were determined9. Quantitative real-time PCR (qPCR) analysis of viral DNA showed that while Kif5A or Kif5B depletion did not affect MSS or total viral DNA, 2-LTR circles were potently reduced (Fig. 4j), suggesting a block after reverse transcription but before nuclear entry of viral DNA. This block to 2LTR circles was also observed in CHME3 cells expressing a GFP-tagged dominant-negative form of Kif5B24, compared with GFP control (Fig. 4k and 4l). These findings demonstrated that kinesin-1 did not affect fusion of HIV-1 into the cytosol but was required for early post-entry stages of 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)

Kinesin-1 regulates nuclear entry of HIV-1 DNA(a–g) Kinesin-1 depletion affects early HIV-1 infection regardless of the route of viral entry. NHDF (a–b), CHME3 (c–d,g) or Jurkat (e–f) cells were transfected with control (ctrl), Kif5A or Kif5B siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-luc (a, c, e) or HIV-1-Ampho-luc (g) followed by measurements of luciferase activity in NHDF (a), CHME3 (c, g) or Jurkat (e) cells. (b, d and f). WB analysis demonstrated kinesin-1 depletion in samples from a, c and e using antibodies against kinesin-1 (Kif5A/B), Kif5B or β-actin (loading control). (h) Kinesin-1 knockdown does not inhibit VSV infection. NHDF cells treated with control, Kif5A or Kif5B siRNAs were infected 48h post-transfection with VSV at m.o.i. 10. 8h after infection cells were lysed and analyzed by WB using anti-VSV-G or anti-VSV-N antibodies. eIF4E was used as loading control. (i) Effects of kinesin-1 depletion on fusion of HIV-1 cores into the cytosol. NHDFs were treated with control, Kif5A or Kif5B siRNAs and then either mock infected (upper panels) or infected with HIV-1-VSV-luc containing BlaM-Vpr (lower panels). FACS analysis of cells showed ~14–18% shift from green (uncleaved CCF2) to blue (cleaved CCF2) cells in control and kinesin-1-depleted cultures. (j–l) Kinesin-1 regulates nuclear entry of HIV-1 DNA. (j) CHME3 cells treated with control (Ctrl), Kif5A or Kif5B siRNAs were infected with HIV-1-VSV-puro 48h post-transfection. Low molecular Hirt DNA was isolated 24h post-infection and levels of viral MSS-DNA, total viral DNA and 2-LTRs in samples were measured by qPCR using specific primers to MSS, puromycin or 2-LTRs, respectively. Copy numbers were calculated and normalized to input DNA in each sample. Data are presented as mean +/- SEM. (k–l) CHME3 cells were transfected with plasmids expressing either GFP or GFP-tagged dominant negative Kif5B (Kif5B-GFP-DN). 48h post-transfection cells were infected (k) and levels of MSS DNA, total DN and 2-LTRs were measured as described in j. or (l) cells were lysed in Laemmli buffer and analyzed by WB using anti-GFP antibody to detect GFP or dominant-negative GFP-Kif5 (Kif5B-GFP-DN). Molecular weight markers (in kDa) are shown to the right of WBs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4380233&req=5

Figure 4: Kinesin-1 regulates nuclear entry of HIV-1 DNA(a–g) Kinesin-1 depletion affects early HIV-1 infection regardless of the route of viral entry. NHDF (a–b), CHME3 (c–d,g) or Jurkat (e–f) cells were transfected with control (ctrl), Kif5A or Kif5B siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-luc (a, c, e) or HIV-1-Ampho-luc (g) followed by measurements of luciferase activity in NHDF (a), CHME3 (c, g) or Jurkat (e) cells. (b, d and f). WB analysis demonstrated kinesin-1 depletion in samples from a, c and e using antibodies against kinesin-1 (Kif5A/B), Kif5B or β-actin (loading control). (h) Kinesin-1 knockdown does not inhibit VSV infection. NHDF cells treated with control, Kif5A or Kif5B siRNAs were infected 48h post-transfection with VSV at m.o.i. 10. 8h after infection cells were lysed and analyzed by WB using anti-VSV-G or anti-VSV-N antibodies. eIF4E was used as loading control. (i) Effects of kinesin-1 depletion on fusion of HIV-1 cores into the cytosol. NHDFs were treated with control, Kif5A or Kif5B siRNAs and then either mock infected (upper panels) or infected with HIV-1-VSV-luc containing BlaM-Vpr (lower panels). FACS analysis of cells showed ~14–18% shift from green (uncleaved CCF2) to blue (cleaved CCF2) cells in control and kinesin-1-depleted cultures. (j–l) Kinesin-1 regulates nuclear entry of HIV-1 DNA. (j) CHME3 cells treated with control (Ctrl), Kif5A or Kif5B siRNAs were infected with HIV-1-VSV-puro 48h post-transfection. Low molecular Hirt DNA was isolated 24h post-infection and levels of viral MSS-DNA, total viral DNA and 2-LTRs in samples were measured by qPCR using specific primers to MSS, puromycin or 2-LTRs, respectively. Copy numbers were calculated and normalized to input DNA in each sample. Data are presented as mean +/- SEM. (k–l) CHME3 cells were transfected with plasmids expressing either GFP or GFP-tagged dominant negative Kif5B (Kif5B-GFP-DN). 48h post-transfection cells were infected (k) and levels of MSS DNA, total DN and 2-LTRs were measured as described in j. or (l) cells were lysed in Laemmli buffer and analyzed by WB using anti-GFP antibody to detect GFP or dominant-negative GFP-Kif5 (Kif5B-GFP-DN). Molecular weight markers (in kDa) are shown to the right of WBs.
Mentions: To further explore how a kinesin-1 adaptor protein might regulate net inward movement of HIV-1 we examined whether kinesin-1 itself contributed to early infection. To do this, siRNAs were used to deplete different human cell types of either of two different isoforms of kinesin-1 heavy chain, Kif5A or Kif5B, followed by infection of these cells with HIV-1-VSV-luc reporter. Knockdown of either Kif5A or Kif5B inhibited infection in both primary fibroblasts (NHDFs) (Fig. 4a) and human microglia (CHME3) (Fig. 4c), and Kif5B was also found to be required for infection of T cell lines, Jurkat (Fig. 4e) and 174xCEM (Supplementary Fig. 4a). WB analysis confirmed Kif5 knockdown levels (Fig. 4b, 4d, 4f and Supplementary Fig. 4b). Importantly, kinesin-1-depleted cells exhibited no morphological changes or indications of apoptosis compared with control siRNA-treated cells (Supplementary Fig. 2a and 2b). Kif5A or Kif5B depletion in CHME3 cells also blocked infection by HIV-1-luc pseudotyped with MuLV amphotropic envelope glycoproteins (Fig. 4g). Furthermore, while infection by HIV-1-VSV-G was suppressed in Kif5A- or Kif5B-depleted cells, infection by VSV itself was not inhibited (Fig. 4h), further suggesting an envelope-independent block specific to HIV-1 that occurred at an early post-entry stage of infection. To confirm this, cells were treated with control, Kif5A or Kif5B siRNAs followed by infection with HIV-1-VSV-G carrying a β-lactamase-Vpr fusion protein, which allows monitoring of viral penetration into the cytosol23. Compared to control siRNA-treated samples, depletion of either Kif5A or Kif5B did not significantly affect fusion of HIV-1 into the cytoplasm of NHDFs (Fig. 4i), and did not correlate with the potent inhibition of early HIV-1 infection in cells depleted of either isoform (Fig. 4a–f). To further determine the point in the viral life cycle affected by kinesin-1, CHME3 cells were treated with control, Kif5A or Kif5B siRNAs. Cultures were then infected and levels of minus-strand strong stop (MSS) DNA, total viral DNA and 2-LTR circles were determined9. Quantitative real-time PCR (qPCR) analysis of viral DNA showed that while Kif5A or Kif5B depletion did not affect MSS or total viral DNA, 2-LTR circles were potently reduced (Fig. 4j), suggesting a block after reverse transcription but before nuclear entry of viral DNA. This block to 2LTR circles was also observed in CHME3 cells expressing a GFP-tagged dominant-negative form of Kif5B24, compared with GFP control (Fig. 4k and 4l). These findings demonstrated that kinesin-1 did not affect fusion of HIV-1 into the cytosol but was required for early post-entry stages of 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