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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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Related in: MedlinePlus

VF trajectories engage in fast overlapping Arc trajectories prior to engaging and scanning CD4 T cells.DC were infected with HIV-iGFP and subsequent live imaging proceeded with infected DCs co-culture with CD4 T cells at a ratio of 1 DC to 3 CD4 T cells as outlined in Fig. 1. (A) Untethered VF engage in sweeping Arc trajectories. To illustrate the overall movement of VF, 8 frames of the Supplementary Video S5 were superimposed. To highlight filopodia, a dashed line shadows the filopodial connection on VF as in Fig. 1. (B) 10 representative velocities of VF tips over time (lower graph) and corresponding movements from their point of origin (PO) (upper graph) (C) 10 representative velocities of filopodia from uninfected DC from the same donor. (D) Average velocities across entire VF trajectories across multiple DC donors and filopodial trajectories from untreated (U/T) controls (each point is the average VF Velocity over an entire 20 second trajectory). VF and filopodia from infected and untreated (U/T) U937 monocyte cell line is presented herein as a comparison. (E) Change in VF trajectories from Arc to Scan movements, when contacting CD4 T cells. The distance of the VF tip to the target T cells was calculated over time for 10 representative VF. Vertical red lines highlight the average scanning time of filopodia on the CD4 T cell membrane. Although scanning by normal filopdoia occurs, we could not resolve definitive trajectories as we did not have a tip marker equivalent to HIV on VF. (F) To illustrate the appearance of Scan trajectories in close contact with CD4 T cells, 3 frames have been taken from a live imaging time lapse experiment in Video S5, and single particle tracking over time highlighted in each frame. All scale bars are at 5 µm. All data is representative of in excess of 12 independent donors.
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ppat-1002762-g002: VF trajectories engage in fast overlapping Arc trajectories prior to engaging and scanning CD4 T cells.DC were infected with HIV-iGFP and subsequent live imaging proceeded with infected DCs co-culture with CD4 T cells at a ratio of 1 DC to 3 CD4 T cells as outlined in Fig. 1. (A) Untethered VF engage in sweeping Arc trajectories. To illustrate the overall movement of VF, 8 frames of the Supplementary Video S5 were superimposed. To highlight filopodia, a dashed line shadows the filopodial connection on VF as in Fig. 1. (B) 10 representative velocities of VF tips over time (lower graph) and corresponding movements from their point of origin (PO) (upper graph) (C) 10 representative velocities of filopodia from uninfected DC from the same donor. (D) Average velocities across entire VF trajectories across multiple DC donors and filopodial trajectories from untreated (U/T) controls (each point is the average VF Velocity over an entire 20 second trajectory). VF and filopodia from infected and untreated (U/T) U937 monocyte cell line is presented herein as a comparison. (E) Change in VF trajectories from Arc to Scan movements, when contacting CD4 T cells. The distance of the VF tip to the target T cells was calculated over time for 10 representative VF. Vertical red lines highlight the average scanning time of filopodia on the CD4 T cell membrane. Although scanning by normal filopdoia occurs, we could not resolve definitive trajectories as we did not have a tip marker equivalent to HIV on VF. (F) To illustrate the appearance of Scan trajectories in close contact with CD4 T cells, 3 frames have been taken from a live imaging time lapse experiment in Video S5, and single particle tracking over time highlighted in each frame. All scale bars are at 5 µm. All data is representative of in excess of 12 independent donors.

Mentions: From hereon we use HIV iGFP as the primary tool in live imaging, as they are significantly brighter and photo bleach tolerant, which are key criteria for lower exposure times for fast acquisition of events during live imaging and the recording of longer trajectories for single particle analysis. Also at this point it is important to clarify the images in live cell acquisition versus the higher resolution images in fixed samples. In live cell acquisition there is readily detectable diffuse eGFP staining throughout the cell body, with low-level expression of eGFP at the filopodial tips. In contrast, fixed cell imaging there is resolution of HIV particles across the plasma membrane and at filopodial tips. This discrepancy is reasoned two-fold. Firstly, higher resolution fixed imaging is through the acquisition of entire infected cell volumes and subsequent 3- dimensional deconvolution [26]. In contrast in live cell imaging there was only acquisition of one Z-plane over time, thus lower resolution images due to the lack of entire Z-stack acquisition and subsequent deconvolution. The acquisition of only one Z-plane was a factor of VF dynamics, as acquisition of trajectories at velocities in excess of 1 µm.sec−1 limited the time needed for acquisition of a significant Z-stack. Secondly, the majority of fluorescence is within the cell body and was obviously limited at filopodial tips due to the relative small size of the virion. Thus exposure times that allow detection of virions on VF results in image acquisition that appear to overexpose the fluorescence in the cell body. Whilst the abovementioned fixed imaging conditions can remove the majority of out of focus light at the cell body, the restricted conditions of live imaging cannot. That said even with lower resolution images in live cell imaging, HIV particle detection at filopodial tips can be readily achieved, as the signal is of sufficient distance from the cell body to allow resolution. As we could readily resolve HIV particles on filopodia, we next characterized VF velocities and trajectories and the capacity of VF to be involved in HIV spread. We infected DCs and four days post infection we co-cultured them with autologous CD4 T cells. VF, when DC were not in immediate contact with CD4 T cells, displayed trajectories in arc-like movements, termed arc velocities, which included movements towards or away from CD4 T cells. Live acquisition of uninfected DCs observed similar arc trajectories, but only when DCs were co-cultured with CD4 T cells (Video S3). The trajectory of Arc velocities followed a single sweep, where the Arc velocity would slow or stop at the end of the trajectory and then often reversed and took the same path (Fig. 2A; Video S5). Analysis of velocity of both VF and uninfected DC filopodia observed trajectories with both acceleration to speeds >6 µm.sec−1 and deceleration with brief interludes of stationary pauses (Fig. 2B & C) with an average of 1.11±0.75 µm.sec−1 for VF (Fig. 2D; n = 124 trajectories (D = 4)) and 1.156±0.75 µm.sec−1 for uninfected DC filopodia (Fig. 2D; n = 113 trajectories (D = 4); p = 0.766 versus VF). VF Arc velocities terminated once in contact with the CD4 T cell membrane (Fig. 2E–F; Video S6 & S7). At that time, we have observed a movement significantly slower used by VF to scan CD4 membrane surface that we have termed Scan velocity (Fig. 2D–E; p<0.0001 for Arc versus Scan velocities; n = 52 (D = 4); Video S7). VF Scan velocity was 0.3422±0.196 µm.sec−1 for an average duration of 13.717±10.577 seconds (n = 52) (D = 4)). Unfortunately the tips of filopodia expressed on uninfected DCs could not be resolved when in the vicinity of CD4 T cells (given they lacked an equivalent tip marker to HIV on VF) and thus the equivalent dynamics of CD4 T cell contact could not be observed. To determine if VF Arc and Scan trajectories were simply a function of random Brownian motion, we calculated their respective Hurst Exponent as previously described [27], [28]. The Hurst Exponent (H) mathematically classifies trajectories as random (H = 0.5), directional (H>0.5), or confined movements (H<0.5). Whereas Arc velocities had limited variation in H and were significantly directional (H = 0.740±0.134; p<0.0001; n = 52), scan velocities ranged from confined to directional movements (H = 0.428±0.234; n = 52).


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 trajectories engage in fast overlapping Arc trajectories prior to engaging and scanning CD4 T cells.DC were infected with HIV-iGFP and subsequent live imaging proceeded with infected DCs co-culture with CD4 T cells at a ratio of 1 DC to 3 CD4 T cells as outlined in Fig. 1. (A) Untethered VF engage in sweeping Arc trajectories. To illustrate the overall movement of VF, 8 frames of the Supplementary Video S5 were superimposed. To highlight filopodia, a dashed line shadows the filopodial connection on VF as in Fig. 1. (B) 10 representative velocities of VF tips over time (lower graph) and corresponding movements from their point of origin (PO) (upper graph) (C) 10 representative velocities of filopodia from uninfected DC from the same donor. (D) Average velocities across entire VF trajectories across multiple DC donors and filopodial trajectories from untreated (U/T) controls (each point is the average VF Velocity over an entire 20 second trajectory). VF and filopodia from infected and untreated (U/T) U937 monocyte cell line is presented herein as a comparison. (E) Change in VF trajectories from Arc to Scan movements, when contacting CD4 T cells. The distance of the VF tip to the target T cells was calculated over time for 10 representative VF. Vertical red lines highlight the average scanning time of filopodia on the CD4 T cell membrane. Although scanning by normal filopdoia occurs, we could not resolve definitive trajectories as we did not have a tip marker equivalent to HIV on VF. (F) To illustrate the appearance of Scan trajectories in close contact with CD4 T cells, 3 frames have been taken from a live imaging time lapse experiment in Video S5, and single particle tracking over time highlighted in each frame. All scale bars are at 5 µm. All data is representative of in excess of 12 independent donors.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369929&req=5

ppat-1002762-g002: VF trajectories engage in fast overlapping Arc trajectories prior to engaging and scanning CD4 T cells.DC were infected with HIV-iGFP and subsequent live imaging proceeded with infected DCs co-culture with CD4 T cells at a ratio of 1 DC to 3 CD4 T cells as outlined in Fig. 1. (A) Untethered VF engage in sweeping Arc trajectories. To illustrate the overall movement of VF, 8 frames of the Supplementary Video S5 were superimposed. To highlight filopodia, a dashed line shadows the filopodial connection on VF as in Fig. 1. (B) 10 representative velocities of VF tips over time (lower graph) and corresponding movements from their point of origin (PO) (upper graph) (C) 10 representative velocities of filopodia from uninfected DC from the same donor. (D) Average velocities across entire VF trajectories across multiple DC donors and filopodial trajectories from untreated (U/T) controls (each point is the average VF Velocity over an entire 20 second trajectory). VF and filopodia from infected and untreated (U/T) U937 monocyte cell line is presented herein as a comparison. (E) Change in VF trajectories from Arc to Scan movements, when contacting CD4 T cells. The distance of the VF tip to the target T cells was calculated over time for 10 representative VF. Vertical red lines highlight the average scanning time of filopodia on the CD4 T cell membrane. Although scanning by normal filopdoia occurs, we could not resolve definitive trajectories as we did not have a tip marker equivalent to HIV on VF. (F) To illustrate the appearance of Scan trajectories in close contact with CD4 T cells, 3 frames have been taken from a live imaging time lapse experiment in Video S5, and single particle tracking over time highlighted in each frame. All scale bars are at 5 µm. All data is representative of in excess of 12 independent donors.
Mentions: From hereon we use HIV iGFP as the primary tool in live imaging, as they are significantly brighter and photo bleach tolerant, which are key criteria for lower exposure times for fast acquisition of events during live imaging and the recording of longer trajectories for single particle analysis. Also at this point it is important to clarify the images in live cell acquisition versus the higher resolution images in fixed samples. In live cell acquisition there is readily detectable diffuse eGFP staining throughout the cell body, with low-level expression of eGFP at the filopodial tips. In contrast, fixed cell imaging there is resolution of HIV particles across the plasma membrane and at filopodial tips. This discrepancy is reasoned two-fold. Firstly, higher resolution fixed imaging is through the acquisition of entire infected cell volumes and subsequent 3- dimensional deconvolution [26]. In contrast in live cell imaging there was only acquisition of one Z-plane over time, thus lower resolution images due to the lack of entire Z-stack acquisition and subsequent deconvolution. The acquisition of only one Z-plane was a factor of VF dynamics, as acquisition of trajectories at velocities in excess of 1 µm.sec−1 limited the time needed for acquisition of a significant Z-stack. Secondly, the majority of fluorescence is within the cell body and was obviously limited at filopodial tips due to the relative small size of the virion. Thus exposure times that allow detection of virions on VF results in image acquisition that appear to overexpose the fluorescence in the cell body. Whilst the abovementioned fixed imaging conditions can remove the majority of out of focus light at the cell body, the restricted conditions of live imaging cannot. That said even with lower resolution images in live cell imaging, HIV particle detection at filopodial tips can be readily achieved, as the signal is of sufficient distance from the cell body to allow resolution. As we could readily resolve HIV particles on filopodia, we next characterized VF velocities and trajectories and the capacity of VF to be involved in HIV spread. We infected DCs and four days post infection we co-cultured them with autologous CD4 T cells. VF, when DC were not in immediate contact with CD4 T cells, displayed trajectories in arc-like movements, termed arc velocities, which included movements towards or away from CD4 T cells. Live acquisition of uninfected DCs observed similar arc trajectories, but only when DCs were co-cultured with CD4 T cells (Video S3). The trajectory of Arc velocities followed a single sweep, where the Arc velocity would slow or stop at the end of the trajectory and then often reversed and took the same path (Fig. 2A; Video S5). Analysis of velocity of both VF and uninfected DC filopodia observed trajectories with both acceleration to speeds >6 µm.sec−1 and deceleration with brief interludes of stationary pauses (Fig. 2B & C) with an average of 1.11±0.75 µm.sec−1 for VF (Fig. 2D; n = 124 trajectories (D = 4)) and 1.156±0.75 µm.sec−1 for uninfected DC filopodia (Fig. 2D; n = 113 trajectories (D = 4); p = 0.766 versus VF). VF Arc velocities terminated once in contact with the CD4 T cell membrane (Fig. 2E–F; Video S6 & S7). At that time, we have observed a movement significantly slower used by VF to scan CD4 membrane surface that we have termed Scan velocity (Fig. 2D–E; p<0.0001 for Arc versus Scan velocities; n = 52 (D = 4); Video S7). VF Scan velocity was 0.3422±0.196 µm.sec−1 for an average duration of 13.717±10.577 seconds (n = 52) (D = 4)). Unfortunately the tips of filopodia expressed on uninfected DCs could not be resolved when in the vicinity of CD4 T cells (given they lacked an equivalent tip marker to HIV on VF) and thus the equivalent dynamics of CD4 T cell contact could not be observed. To determine if VF Arc and Scan trajectories were simply a function of random Brownian motion, we calculated their respective Hurst Exponent as previously described [27], [28]. The Hurst Exponent (H) mathematically classifies trajectories as random (H = 0.5), directional (H>0.5), or confined movements (H<0.5). Whereas Arc velocities had limited variation in H and were significantly directional (H = 0.740±0.134; p<0.0001; n = 52), scan velocities ranged from confined to directional movements (H = 0.428±0.234; n = 52).

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