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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 contacts and tethering of CD4 T cell targets.To analyze the consequence of VF contact, DCs were infected with HIV-iGFP and subsequently co-cultured with autologous CD4 T cells as outlined in Fig. 1. (A) HIV contacts proceed via Arc movements of single VF between cells. In A. Upper panel 6 frames have been taken from live imaging time lapse experiment with single particle tracking in red highlighting the movement of VF between two CD4 T cells (labeled 1 and 2). In A. lower panel VF velocities (blue) and distance from the point of origin (PO) (black) summarize the movement in the former panel, with CD4 T cell contacts indicated by red arrows. (B) Frequency of VF to CD4 Targets over time for 10 representative infected HIV iGFP DC. Each CD4 T cell in the co-culture is listed as “Target” and is represented over time by an open colored triangle (eg. Repeating black triangles, indicated VF contacting the same CD4 T cell “Target A”). CD4 T cell contact data is representative of in excess of 8 independent donors. (C)–(F) Multiple VF co-ordinate to tether CD4 T cells. C. Multiple filopodia (highlighted by arrows) are in contact with a neighboring CD4 T cells. Contact is a representative still image, where VF are revealed by F-actin staining using phalloidin (red) and HIV iGFP (white). D. CD4 tethering in real-time. Still images have been taken from Video S8 and time is present in seconds in the upper right corners. Viral filopodia have been outlined with white dotted lines as in Fig. 1. Within still images, CD4 T cell denoted “T1” is actively tethered and moved closer to the DC membrane. Whilst CD4 T cell “T2” remains tethered for the duration of the Video. E. Multiple VF contacts result in CD4 T cell tethering. In the left panel accumulative VF trajectories for a 3 minute infected DC-CD4 T cell co-culture. White arrows and asterix indicate CD4 T cells where either limited VF contact have occurred over this time (less than or equal to 1) or multiple contacts (>5) have occurred respectively. Image derived from Video S9. In the right panel, trajectories of CD4 T cell over the 3 minute co-culture are presented. Note CD4 T cell repositioning associated with high filopodial activity. Image derived from Video S10. F. Distance from the point of origin (upper panel) and velocities (lower panel) for CD4 T cells that are engaged in multiple VF contacts (“tethered”-asterix in E.) versus CD4 T cells with undetectable VF contact (“untethered”-arrows in E.). All data in representative of in excess of 8 independent donors DC-CD4 T cell co-cultures, where greater a minimum of 10 videos were acquired per donor.
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ppat-1002762-g003: VF contacts and tethering of CD4 T cell targets.To analyze the consequence of VF contact, DCs were infected with HIV-iGFP and subsequently co-cultured with autologous CD4 T cells as outlined in Fig. 1. (A) HIV contacts proceed via Arc movements of single VF between cells. In A. Upper panel 6 frames have been taken from live imaging time lapse experiment with single particle tracking in red highlighting the movement of VF between two CD4 T cells (labeled 1 and 2). In A. lower panel VF velocities (blue) and distance from the point of origin (PO) (black) summarize the movement in the former panel, with CD4 T cell contacts indicated by red arrows. (B) Frequency of VF to CD4 Targets over time for 10 representative infected HIV iGFP DC. Each CD4 T cell in the co-culture is listed as “Target” and is represented over time by an open colored triangle (eg. Repeating black triangles, indicated VF contacting the same CD4 T cell “Target A”). CD4 T cell contact data is representative of in excess of 8 independent donors. (C)–(F) Multiple VF co-ordinate to tether CD4 T cells. C. Multiple filopodia (highlighted by arrows) are in contact with a neighboring CD4 T cells. Contact is a representative still image, where VF are revealed by F-actin staining using phalloidin (red) and HIV iGFP (white). D. CD4 tethering in real-time. Still images have been taken from Video S8 and time is present in seconds in the upper right corners. Viral filopodia have been outlined with white dotted lines as in Fig. 1. Within still images, CD4 T cell denoted “T1” is actively tethered and moved closer to the DC membrane. Whilst CD4 T cell “T2” remains tethered for the duration of the Video. E. Multiple VF contacts result in CD4 T cell tethering. In the left panel accumulative VF trajectories for a 3 minute infected DC-CD4 T cell co-culture. White arrows and asterix indicate CD4 T cells where either limited VF contact have occurred over this time (less than or equal to 1) or multiple contacts (>5) have occurred respectively. Image derived from Video S9. In the right panel, trajectories of CD4 T cell over the 3 minute co-culture are presented. Note CD4 T cell repositioning associated with high filopodial activity. Image derived from Video S10. F. Distance from the point of origin (upper panel) and velocities (lower panel) for CD4 T cells that are engaged in multiple VF contacts (“tethered”-asterix in E.) versus CD4 T cells with undetectable VF contact (“untethered”-arrows in E.). All data in representative of in excess of 8 independent donors DC-CD4 T cell co-cultures, where greater a minimum of 10 videos were acquired per donor.

Mentions: VF contacts were either isolated to one to two filopodia engaging in Arc movement between CD4 T cells (Fig. 3A; Video S7), or several VF interacting simultaneously towards CD4 T cell targets (Fig. 3B & C). Overall estimated contacts between infected DCs and CD4 T cells ranged from 260 to 800 per hour; Average contacts = 460±167.hour−1; n = 45 (D = 3)). Given the speeds of VF movement and the limitations of fluorescence imaging, we must note this imaging represents a contact estimate. Furthermore the restriction of imaging to a limited Z-stack we predict to result in a somewhat conservative contact estimate. Continual and multiple VF activity preceded tethering and subsequent physical movement of CD4 T cells closer to the DC membrane (Fig. 3D–F. Video S8, S9, S10).


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 contacts and tethering of CD4 T cell targets.To analyze the consequence of VF contact, DCs were infected with HIV-iGFP and subsequently co-cultured with autologous CD4 T cells as outlined in Fig. 1. (A) HIV contacts proceed via Arc movements of single VF between cells. In A. Upper panel 6 frames have been taken from live imaging time lapse experiment with single particle tracking in red highlighting the movement of VF between two CD4 T cells (labeled 1 and 2). In A. lower panel VF velocities (blue) and distance from the point of origin (PO) (black) summarize the movement in the former panel, with CD4 T cell contacts indicated by red arrows. (B) Frequency of VF to CD4 Targets over time for 10 representative infected HIV iGFP DC. Each CD4 T cell in the co-culture is listed as “Target” and is represented over time by an open colored triangle (eg. Repeating black triangles, indicated VF contacting the same CD4 T cell “Target A”). CD4 T cell contact data is representative of in excess of 8 independent donors. (C)–(F) Multiple VF co-ordinate to tether CD4 T cells. C. Multiple filopodia (highlighted by arrows) are in contact with a neighboring CD4 T cells. Contact is a representative still image, where VF are revealed by F-actin staining using phalloidin (red) and HIV iGFP (white). D. CD4 tethering in real-time. Still images have been taken from Video S8 and time is present in seconds in the upper right corners. Viral filopodia have been outlined with white dotted lines as in Fig. 1. Within still images, CD4 T cell denoted “T1” is actively tethered and moved closer to the DC membrane. Whilst CD4 T cell “T2” remains tethered for the duration of the Video. E. Multiple VF contacts result in CD4 T cell tethering. In the left panel accumulative VF trajectories for a 3 minute infected DC-CD4 T cell co-culture. White arrows and asterix indicate CD4 T cells where either limited VF contact have occurred over this time (less than or equal to 1) or multiple contacts (>5) have occurred respectively. Image derived from Video S9. In the right panel, trajectories of CD4 T cell over the 3 minute co-culture are presented. Note CD4 T cell repositioning associated with high filopodial activity. Image derived from Video S10. F. Distance from the point of origin (upper panel) and velocities (lower panel) for CD4 T cells that are engaged in multiple VF contacts (“tethered”-asterix in E.) versus CD4 T cells with undetectable VF contact (“untethered”-arrows in E.). All data in representative of in excess of 8 independent donors DC-CD4 T cell co-cultures, where greater a minimum of 10 videos were acquired per donor.
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Related In: Results  -  Collection

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ppat-1002762-g003: VF contacts and tethering of CD4 T cell targets.To analyze the consequence of VF contact, DCs were infected with HIV-iGFP and subsequently co-cultured with autologous CD4 T cells as outlined in Fig. 1. (A) HIV contacts proceed via Arc movements of single VF between cells. In A. Upper panel 6 frames have been taken from live imaging time lapse experiment with single particle tracking in red highlighting the movement of VF between two CD4 T cells (labeled 1 and 2). In A. lower panel VF velocities (blue) and distance from the point of origin (PO) (black) summarize the movement in the former panel, with CD4 T cell contacts indicated by red arrows. (B) Frequency of VF to CD4 Targets over time for 10 representative infected HIV iGFP DC. Each CD4 T cell in the co-culture is listed as “Target” and is represented over time by an open colored triangle (eg. Repeating black triangles, indicated VF contacting the same CD4 T cell “Target A”). CD4 T cell contact data is representative of in excess of 8 independent donors. (C)–(F) Multiple VF co-ordinate to tether CD4 T cells. C. Multiple filopodia (highlighted by arrows) are in contact with a neighboring CD4 T cells. Contact is a representative still image, where VF are revealed by F-actin staining using phalloidin (red) and HIV iGFP (white). D. CD4 tethering in real-time. Still images have been taken from Video S8 and time is present in seconds in the upper right corners. Viral filopodia have been outlined with white dotted lines as in Fig. 1. Within still images, CD4 T cell denoted “T1” is actively tethered and moved closer to the DC membrane. Whilst CD4 T cell “T2” remains tethered for the duration of the Video. E. Multiple VF contacts result in CD4 T cell tethering. In the left panel accumulative VF trajectories for a 3 minute infected DC-CD4 T cell co-culture. White arrows and asterix indicate CD4 T cells where either limited VF contact have occurred over this time (less than or equal to 1) or multiple contacts (>5) have occurred respectively. Image derived from Video S9. In the right panel, trajectories of CD4 T cell over the 3 minute co-culture are presented. Note CD4 T cell repositioning associated with high filopodial activity. Image derived from Video S10. F. Distance from the point of origin (upper panel) and velocities (lower panel) for CD4 T cells that are engaged in multiple VF contacts (“tethered”-asterix in E.) versus CD4 T cells with undetectable VF contact (“untethered”-arrows in E.). All data in representative of in excess of 8 independent donors DC-CD4 T cell co-cultures, where greater a minimum of 10 videos were acquired per donor.
Mentions: VF contacts were either isolated to one to two filopodia engaging in Arc movement between CD4 T cells (Fig. 3A; Video S7), or several VF interacting simultaneously towards CD4 T cell targets (Fig. 3B & C). Overall estimated contacts between infected DCs and CD4 T cells ranged from 260 to 800 per hour; Average contacts = 460±167.hour−1; n = 45 (D = 3)). Given the speeds of VF movement and the limitations of fluorescence imaging, we must note this imaging represents a contact estimate. Furthermore the restriction of imaging to a limited Z-stack we predict to result in a somewhat conservative contact estimate. Continual and multiple VF activity preceded tethering and subsequent physical movement of CD4 T cells closer to the DC membrane (Fig. 3D–F. Video S8, S9, S10).

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