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Mobilization of HIV spread by diaphanous 2 dependent filopodia in infected dendritic cells.

Aggarwal A, Iemma TL, Shih I, Newsome TP, McAllery S, Cunningham AL, Turville SG - PLoS Pathog. (2012)

Bottom Line: Long viral filopodial formation was dependent on the formin diaphanous 2 (Diaph2), and not a dominant Arp2/3 filopodial pathway often associated with pathogenic actin polymerization.Manipulation of HIV Nef reduced HIV transfer 25-fold by reducing viral filopodia frequency, supporting the potency of DC HIV transfer was dependent on viral filopodia abundance.Thus our observations show HIV corrupts DC to CD4 T cell interactions by physically embedding at the leading edge contacts of long DC filopodial networks.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of HIV Biology, Immunovirology and Pathogenesis Program, The Kirby Institute, University of New South Wales, Sydney, New South Wales, Australia.

ABSTRACT
Paramount to the success of persistent viral infection is the ability of viruses to navigate hostile environments en route to future targets. In response to such obstacles, many viruses have developed the ability of establishing actin rich-membrane bridges to aid in future infections. Herein through dynamic imaging of HIV infected dendritic cells, we have observed how viral high-jacking of the actin/membrane network facilitates one of the most efficient forms of HIV spread. Within infected DC, viral egress is coupled to viral filopodia formation, with more than 90% of filopodia bearing immature HIV on their tips at extensions of 10 to 20 µm. Live imaging showed HIV filopodia routinely pivoting at their base, and projecting HIV virions at µm.sec⁻¹ along repetitive arc trajectories. HIV filopodial dynamics lead to up to 800 DC to CD4 T cell contacts per hour, with selection of T cells culminating in multiple filopodia tethering and converging to envelope the CD4 T-cell membrane with budding HIV particles. Long viral filopodial formation was dependent on the formin diaphanous 2 (Diaph2), and not a dominant Arp2/3 filopodial pathway often associated with pathogenic actin polymerization. Manipulation of HIV Nef reduced HIV transfer 25-fold by reducing viral filopodia frequency, supporting the potency of DC HIV transfer was dependent on viral filopodia abundance. Thus our observations show HIV corrupts DC to CD4 T cell interactions by physically embedding at the leading edge contacts of long DC filopodial networks.

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VF form by a Diaph2 dependent pathway with frequency regulated by HIV Nef and not HIV Env.(A) To further delineate how VF pathway are formed, Wasp and Diaph2 was knockdown in the U937 cell line using shRNA. After 2 weeks of puromycin selection, resistant U937 cell lines were infected with HIV iGFP and VF lengths and trajectory velocities enumerated as in Fig. 4. P values are included to highlight significant differences in each variable. Protein knock-down for Wasp and Diaph2 are presented in Fig. S2E. The house-keeping protein Gapdh is present below to normalize lysate loading. Data is representative of 4 independent infections using HIV iGFP. (B) To rule out manipulation of the Arp2/3 filopodial pathway, the U937 cell line was infected with HIV iGFP and two days post infection, infected cells were treated with the Abl/Src kinase inhibitor Dasatinib at 10 µM for 4 hours. Note, under these conditions Vaccinia actin tails do not form (data not shown). VF were then enumerated for lengths and trajectory velocities as outlined Fig. 3. (C) Accumulative single particle tracking for 1 minute of VF trajectories in scrambled controls (upper panel) versus Diaph2 knockdowns (lower panel). Note the confined trajectories in the absence of Diaph2. Diaph2 knockdown particle tracking is derived from Video S12. Scale bars are 5 µm. Data from knockdown experiments is representative of 4 independent HIV iGFP infections. (D) Fixed cell images of control shRNA (upper panel) and Diaph2 (lower panel) transduced cells infected with HIV iGFP. Note the significantly shorter VF lengths in Diaph2 knockdown U937 cells. Scale bars are 5 µm. Images are representative of 4 independent infections with HIV iGFP. (E) Attenuation of cell-cell transfer in Diaph2 knockdown U937. U937 were infected with HIV and 2 days post infection were stained for HIV p24 and enumerated by flow cytometry. After infections were verified to be equivalent, infected U937 cells were co-cultured at a ratio of 1∶5 with the T cell HIV indicator cell line JLTR-R5. Four days post infection, fluorescent images were acquired for the entire well and enumerated using Image J. Standard deviations represent co-cultures in triplicate. Data is representative of 3 independent infections. (F) VF form in the absence of HIV envelope. DCs were infected with either VSVg pseudotyped HIV iGFP or HIV iGFP-ENV-ve as outlined in Fig. 1. VF were then enumerated for lengths and trajectory velocities as outlined B. Data is representative of four independent infections. P values are presented for significant differences. (G) Deletion of HIV Nef leads to significantly lower VF frequency on DC. Enumeration of VF numbers over time in uninfected DCs (U/T), or HIV infected DCS with HIV−iGFP or HIV-NEF-iGFP. Each point represents live imaging of a VF bearing DC over a period of 2 minutes under imaging conditions outlined in materials and methods. Accumulative data presented is equally drawn from 5 independent donors. Statistical significance is indicated by p values.
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ppat-1002762-g005: VF form by a Diaph2 dependent pathway with frequency regulated by HIV Nef and not HIV Env.(A) To further delineate how VF pathway are formed, Wasp and Diaph2 was knockdown in the U937 cell line using shRNA. After 2 weeks of puromycin selection, resistant U937 cell lines were infected with HIV iGFP and VF lengths and trajectory velocities enumerated as in Fig. 4. P values are included to highlight significant differences in each variable. Protein knock-down for Wasp and Diaph2 are presented in Fig. S2E. The house-keeping protein Gapdh is present below to normalize lysate loading. Data is representative of 4 independent infections using HIV iGFP. (B) To rule out manipulation of the Arp2/3 filopodial pathway, the U937 cell line was infected with HIV iGFP and two days post infection, infected cells were treated with the Abl/Src kinase inhibitor Dasatinib at 10 µM for 4 hours. Note, under these conditions Vaccinia actin tails do not form (data not shown). VF were then enumerated for lengths and trajectory velocities as outlined Fig. 3. (C) Accumulative single particle tracking for 1 minute of VF trajectories in scrambled controls (upper panel) versus Diaph2 knockdowns (lower panel). Note the confined trajectories in the absence of Diaph2. Diaph2 knockdown particle tracking is derived from Video S12. Scale bars are 5 µm. Data from knockdown experiments is representative of 4 independent HIV iGFP infections. (D) Fixed cell images of control shRNA (upper panel) and Diaph2 (lower panel) transduced cells infected with HIV iGFP. Note the significantly shorter VF lengths in Diaph2 knockdown U937 cells. Scale bars are 5 µm. Images are representative of 4 independent infections with HIV iGFP. (E) Attenuation of cell-cell transfer in Diaph2 knockdown U937. U937 were infected with HIV and 2 days post infection were stained for HIV p24 and enumerated by flow cytometry. After infections were verified to be equivalent, infected U937 cells were co-cultured at a ratio of 1∶5 with the T cell HIV indicator cell line JLTR-R5. Four days post infection, fluorescent images were acquired for the entire well and enumerated using Image J. Standard deviations represent co-cultures in triplicate. Data is representative of 3 independent infections. (F) VF form in the absence of HIV envelope. DCs were infected with either VSVg pseudotyped HIV iGFP or HIV iGFP-ENV-ve as outlined in Fig. 1. VF were then enumerated for lengths and trajectory velocities as outlined B. Data is representative of four independent infections. P values are presented for significant differences. (G) Deletion of HIV Nef leads to significantly lower VF frequency on DC. Enumeration of VF numbers over time in uninfected DCs (U/T), or HIV infected DCS with HIV−iGFP or HIV-NEF-iGFP. Each point represents live imaging of a VF bearing DC over a period of 2 minutes under imaging conditions outlined in materials and methods. Accumulative data presented is equally drawn from 5 independent donors. Statistical significance is indicated by p values.

Mentions: To explore how VF were formed, we investigated two major pathways of filopodial nucleation/elongation; the F-actin regulators Arp2/Wasp-dependent pathway, and the formin Diaph2 pathway. Whilst initial immunophenotyping observed both Arp2 and Wasp along filopodia, it was difficult to determine in this setting whether they were in fact the dominant regulators of VF. We also attempted immunostaining Diaph2 in a similar manner to Arp2 and Wasp, yet with currently commercially available antibodies, we were unable to specifically detect Diaph2 in fixed samples. Given the limitations of immunophenotyping of filopodia, we generated stable U937 clones expressing shRNA targeting Wasp and Diaph2, and we characterized VF after successful knockdown at the protein level (Fig. S2). When Wasp was knocked-down, VF lengths and velocities remained unchanged compared to the scrambled shRNA controls (Fig. 5A). We obtained similar results when we treated the infected cells with the Bcr/Abl and Src family tyrosine kinase inhibitor Dasatinib under concentrations and conditions that readily prevent Arp2/3 complex activation by Vaccinia (Fig. 5B[34], confirming that Arp2/Wasp was not involved in VF formation/elongation. In contrast, when shRNA knockdown of the formin Diaph2 were used, significantly reduced VF lengths and velocities were observed (Length: 1.95 µm+/−1.683; n = 181, versus control p<0.0001. Velocity: 0.198 µm.sec−1; n = 164, versus control p<0.0001) (Fig. 5A, C & D; Video S12). Using the same shRNA lentiviral pool, we could not significantly knockdown Diaph2 in primary DCs (Fig. S2F), even after priming DCs with SIV Vpx as previously described [35]. Thus we utilized the TRIPZ lentiviral vector encoding shRNA towards Diaph2, as transduced cells could be identified with the aid of red fluorescent protein. Whilst transduction rates in primary DCs using lentiviral vectors were low, we could identify TRIPZ transduced and HIV infected DC populations. Using this approach, we also observed primary DCs to express significantly shorter VF in comparison to non-transduced and scramble shRNA transduced cultures (2.86 µm+/−1.83 & 9.37+/−3.98 for Diaph2 versus Scrambled shRNA respectively n = 79; p<0.0001; Video S13). Although the low primary DC transduction rates using the TRIPZ lentiviral vector limited further study of Diaph2 depleted cells. As the U937 cell line expressed equivalent VF and was homogenously depleted of Diaph2, we utilized this cell line model to dissect the role of VF in cell-cell transfer. Diaph2-dependent VF formation was important for cell-cell transfer as subsequent co-culture of Diaph2 shRNA U937 cells with permissive CD4 T cells line showed significant attenuation in cell-cell transfer compared to control shRNA conditions (control U937 versus diaph2; p = 0.00278, n = 3Fig. 5E), supporting a direct functional role of long VF in HIV spread. Of note, both stable knockdown of Wasp and Diaph2 in the context of the U937 cell line had no effect on their overall viability, as viability assays using either Alamar Blue or trypan blue exclusion were not significantly different to scrambled shRNA controls. To further control for defects in HIV assembly and release we infected Diaph2 and Sr transduced cells, normalized their infection 3 days post infection to 10% and then harvested viral supernatant 3 days post normalization. Using both detection of reverse transcription (to detect particles in the supernatant) versus infectivity by titering the supernatant on the TZMbl indicator cell line, we did not observe any significant difference in HIV release or infectivity (Supplementary Fig. S2G).


Mobilization of HIV spread by diaphanous 2 dependent filopodia in infected dendritic cells.

Aggarwal A, Iemma TL, Shih I, Newsome TP, McAllery S, Cunningham AL, Turville SG - PLoS Pathog. (2012)

VF form by a Diaph2 dependent pathway with frequency regulated by HIV Nef and not HIV Env.(A) To further delineate how VF pathway are formed, Wasp and Diaph2 was knockdown in the U937 cell line using shRNA. After 2 weeks of puromycin selection, resistant U937 cell lines were infected with HIV iGFP and VF lengths and trajectory velocities enumerated as in Fig. 4. P values are included to highlight significant differences in each variable. Protein knock-down for Wasp and Diaph2 are presented in Fig. S2E. The house-keeping protein Gapdh is present below to normalize lysate loading. Data is representative of 4 independent infections using HIV iGFP. (B) To rule out manipulation of the Arp2/3 filopodial pathway, the U937 cell line was infected with HIV iGFP and two days post infection, infected cells were treated with the Abl/Src kinase inhibitor Dasatinib at 10 µM for 4 hours. Note, under these conditions Vaccinia actin tails do not form (data not shown). VF were then enumerated for lengths and trajectory velocities as outlined Fig. 3. (C) Accumulative single particle tracking for 1 minute of VF trajectories in scrambled controls (upper panel) versus Diaph2 knockdowns (lower panel). Note the confined trajectories in the absence of Diaph2. Diaph2 knockdown particle tracking is derived from Video S12. Scale bars are 5 µm. Data from knockdown experiments is representative of 4 independent HIV iGFP infections. (D) Fixed cell images of control shRNA (upper panel) and Diaph2 (lower panel) transduced cells infected with HIV iGFP. Note the significantly shorter VF lengths in Diaph2 knockdown U937 cells. Scale bars are 5 µm. Images are representative of 4 independent infections with HIV iGFP. (E) Attenuation of cell-cell transfer in Diaph2 knockdown U937. U937 were infected with HIV and 2 days post infection were stained for HIV p24 and enumerated by flow cytometry. After infections were verified to be equivalent, infected U937 cells were co-cultured at a ratio of 1∶5 with the T cell HIV indicator cell line JLTR-R5. Four days post infection, fluorescent images were acquired for the entire well and enumerated using Image J. Standard deviations represent co-cultures in triplicate. Data is representative of 3 independent infections. (F) VF form in the absence of HIV envelope. DCs were infected with either VSVg pseudotyped HIV iGFP or HIV iGFP-ENV-ve as outlined in Fig. 1. VF were then enumerated for lengths and trajectory velocities as outlined B. Data is representative of four independent infections. P values are presented for significant differences. (G) Deletion of HIV Nef leads to significantly lower VF frequency on DC. Enumeration of VF numbers over time in uninfected DCs (U/T), or HIV infected DCS with HIV−iGFP or HIV-NEF-iGFP. Each point represents live imaging of a VF bearing DC over a period of 2 minutes under imaging conditions outlined in materials and methods. Accumulative data presented is equally drawn from 5 independent donors. Statistical significance is indicated by p values.
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ppat-1002762-g005: VF form by a Diaph2 dependent pathway with frequency regulated by HIV Nef and not HIV Env.(A) To further delineate how VF pathway are formed, Wasp and Diaph2 was knockdown in the U937 cell line using shRNA. After 2 weeks of puromycin selection, resistant U937 cell lines were infected with HIV iGFP and VF lengths and trajectory velocities enumerated as in Fig. 4. P values are included to highlight significant differences in each variable. Protein knock-down for Wasp and Diaph2 are presented in Fig. S2E. The house-keeping protein Gapdh is present below to normalize lysate loading. Data is representative of 4 independent infections using HIV iGFP. (B) To rule out manipulation of the Arp2/3 filopodial pathway, the U937 cell line was infected with HIV iGFP and two days post infection, infected cells were treated with the Abl/Src kinase inhibitor Dasatinib at 10 µM for 4 hours. Note, under these conditions Vaccinia actin tails do not form (data not shown). VF were then enumerated for lengths and trajectory velocities as outlined Fig. 3. (C) Accumulative single particle tracking for 1 minute of VF trajectories in scrambled controls (upper panel) versus Diaph2 knockdowns (lower panel). Note the confined trajectories in the absence of Diaph2. Diaph2 knockdown particle tracking is derived from Video S12. Scale bars are 5 µm. Data from knockdown experiments is representative of 4 independent HIV iGFP infections. (D) Fixed cell images of control shRNA (upper panel) and Diaph2 (lower panel) transduced cells infected with HIV iGFP. Note the significantly shorter VF lengths in Diaph2 knockdown U937 cells. Scale bars are 5 µm. Images are representative of 4 independent infections with HIV iGFP. (E) Attenuation of cell-cell transfer in Diaph2 knockdown U937. U937 were infected with HIV and 2 days post infection were stained for HIV p24 and enumerated by flow cytometry. After infections were verified to be equivalent, infected U937 cells were co-cultured at a ratio of 1∶5 with the T cell HIV indicator cell line JLTR-R5. Four days post infection, fluorescent images were acquired for the entire well and enumerated using Image J. Standard deviations represent co-cultures in triplicate. Data is representative of 3 independent infections. (F) VF form in the absence of HIV envelope. DCs were infected with either VSVg pseudotyped HIV iGFP or HIV iGFP-ENV-ve as outlined in Fig. 1. VF were then enumerated for lengths and trajectory velocities as outlined B. Data is representative of four independent infections. P values are presented for significant differences. (G) Deletion of HIV Nef leads to significantly lower VF frequency on DC. Enumeration of VF numbers over time in uninfected DCs (U/T), or HIV infected DCS with HIV−iGFP or HIV-NEF-iGFP. Each point represents live imaging of a VF bearing DC over a period of 2 minutes under imaging conditions outlined in materials and methods. Accumulative data presented is equally drawn from 5 independent donors. Statistical significance is indicated by p values.
Mentions: To explore how VF were formed, we investigated two major pathways of filopodial nucleation/elongation; the F-actin regulators Arp2/Wasp-dependent pathway, and the formin Diaph2 pathway. Whilst initial immunophenotyping observed both Arp2 and Wasp along filopodia, it was difficult to determine in this setting whether they were in fact the dominant regulators of VF. We also attempted immunostaining Diaph2 in a similar manner to Arp2 and Wasp, yet with currently commercially available antibodies, we were unable to specifically detect Diaph2 in fixed samples. Given the limitations of immunophenotyping of filopodia, we generated stable U937 clones expressing shRNA targeting Wasp and Diaph2, and we characterized VF after successful knockdown at the protein level (Fig. S2). When Wasp was knocked-down, VF lengths and velocities remained unchanged compared to the scrambled shRNA controls (Fig. 5A). We obtained similar results when we treated the infected cells with the Bcr/Abl and Src family tyrosine kinase inhibitor Dasatinib under concentrations and conditions that readily prevent Arp2/3 complex activation by Vaccinia (Fig. 5B[34], confirming that Arp2/Wasp was not involved in VF formation/elongation. In contrast, when shRNA knockdown of the formin Diaph2 were used, significantly reduced VF lengths and velocities were observed (Length: 1.95 µm+/−1.683; n = 181, versus control p<0.0001. Velocity: 0.198 µm.sec−1; n = 164, versus control p<0.0001) (Fig. 5A, C & D; Video S12). Using the same shRNA lentiviral pool, we could not significantly knockdown Diaph2 in primary DCs (Fig. S2F), even after priming DCs with SIV Vpx as previously described [35]. Thus we utilized the TRIPZ lentiviral vector encoding shRNA towards Diaph2, as transduced cells could be identified with the aid of red fluorescent protein. Whilst transduction rates in primary DCs using lentiviral vectors were low, we could identify TRIPZ transduced and HIV infected DC populations. Using this approach, we also observed primary DCs to express significantly shorter VF in comparison to non-transduced and scramble shRNA transduced cultures (2.86 µm+/−1.83 & 9.37+/−3.98 for Diaph2 versus Scrambled shRNA respectively n = 79; p<0.0001; Video S13). Although the low primary DC transduction rates using the TRIPZ lentiviral vector limited further study of Diaph2 depleted cells. As the U937 cell line expressed equivalent VF and was homogenously depleted of Diaph2, we utilized this cell line model to dissect the role of VF in cell-cell transfer. Diaph2-dependent VF formation was important for cell-cell transfer as subsequent co-culture of Diaph2 shRNA U937 cells with permissive CD4 T cells line showed significant attenuation in cell-cell transfer compared to control shRNA conditions (control U937 versus diaph2; p = 0.00278, n = 3Fig. 5E), supporting a direct functional role of long VF in HIV spread. Of note, both stable knockdown of Wasp and Diaph2 in the context of the U937 cell line had no effect on their overall viability, as viability assays using either Alamar Blue or trypan blue exclusion were not significantly different to scrambled shRNA controls. To further control for defects in HIV assembly and release we infected Diaph2 and Sr transduced cells, normalized their infection 3 days post infection to 10% and then harvested viral supernatant 3 days post normalization. Using both detection of reverse transcription (to detect particles in the supernatant) versus infectivity by titering the supernatant on the TZMbl indicator cell line, we did not observe any significant difference in HIV release or infectivity (Supplementary Fig. S2G).

Bottom Line: Long viral filopodial formation was dependent on the formin diaphanous 2 (Diaph2), and not a dominant Arp2/3 filopodial pathway often associated with pathogenic actin polymerization.Manipulation of HIV Nef reduced HIV transfer 25-fold by reducing viral filopodia frequency, supporting the potency of DC HIV transfer was dependent on viral filopodia abundance.Thus our observations show HIV corrupts DC to CD4 T cell interactions by physically embedding at the leading edge contacts of long DC filopodial networks.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of HIV Biology, Immunovirology and Pathogenesis Program, The Kirby Institute, University of New South Wales, Sydney, New South Wales, Australia.

ABSTRACT
Paramount to the success of persistent viral infection is the ability of viruses to navigate hostile environments en route to future targets. In response to such obstacles, many viruses have developed the ability of establishing actin rich-membrane bridges to aid in future infections. Herein through dynamic imaging of HIV infected dendritic cells, we have observed how viral high-jacking of the actin/membrane network facilitates one of the most efficient forms of HIV spread. Within infected DC, viral egress is coupled to viral filopodia formation, with more than 90% of filopodia bearing immature HIV on their tips at extensions of 10 to 20 µm. Live imaging showed HIV filopodia routinely pivoting at their base, and projecting HIV virions at µm.sec⁻¹ along repetitive arc trajectories. HIV filopodial dynamics lead to up to 800 DC to CD4 T cell contacts per hour, with selection of T cells culminating in multiple filopodia tethering and converging to envelope the CD4 T-cell membrane with budding HIV particles. Long viral filopodial formation was dependent on the formin diaphanous 2 (Diaph2), and not a dominant Arp2/3 filopodial pathway often associated with pathogenic actin polymerization. Manipulation of HIV Nef reduced HIV transfer 25-fold by reducing viral filopodia frequency, supporting the potency of DC HIV transfer was dependent on viral filopodia abundance. Thus our observations show HIV corrupts DC to CD4 T cell interactions by physically embedding at the leading edge contacts of long DC filopodial networks.

Show MeSH
Related in: MedlinePlus