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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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Viral filopodial contacts precedes the formation of an enveloping DC-CD4 T cell contact.(A)–(D) Long-term contact by an infected DC leads to enveloping of the neighboring CD4 T cells. (A) Long-term enveloping in real-time. To follow the consequence of initial VF contact and tethering, HIV iGFP infected DC initially engaged in VF contacts with CD4 T cells were imaged over a period of 2 hours. Stills were taken from Video S15, with time elapsed presented in the upper left corner as minutes. In the final frame, two CD4 T cells are labeled 1 & 2 that have been enveloped by the infected DC. Scale bar at 5 µm. (B) Enveloping of multiple CD4 T cells. DCs were infected with HIV-T and 5 days post infection co-cultured with CD4 T cells at a ratio of 1 DC to 3 CD4 T cells for 2 hours. HIV particles were then imaged using FlAsH staining (green), samples fixed/permeabilized and nuclei countered stained with DAPI (Blue). Note CD4 T cells numbered 1 through to 4 have distal and proximal FlasH positive viral particles across their membrane. (C) Viral synapses between infected DC and CD4 T cells mature into extensive enveloping contacts. DC-T cell co-cultures are generated as per conditions in B, with the exception of the use of HIV iGFP (green). After 2 hours of co-culture, cells were stained with the DC membrane marker CD209 (red) and nuclei counterstained with DAPI (blue). Upper panel is representative of an infected DC engaging multiple CD4 T-cells as in B. In the lower panel, the infected DC is limited to two CD4 T cells, with the T cell conjugated on the right with a representative infected DC-T cell viral synapse. Scale bars are at 5 µm. Images are representative from 4 independent donors. (D) Long-term enveloping precedes viral fission across the synaptic cleft and seeding of the CD4 T cell with mature HIV. DCs were infected and co-cultured with autologous CD4 T cells as in (C). Total HIV (iGFP - green), mature HIV (Anti-p24/Capsid clone 183 in red) staining was carried out as per materials and methods. Nuclei are stained with DAPI as in (B). Arrow denotes accumulation of mature HIV particles at the viral synapse and also at the distal CD4 T cell membrane. Note HIV iGFP staining at the viral synapse exceeds mature HIV particles. Scale bar at 5 µm. (E) Electron microscopy confirms the presence of both immature/actively budding (black arrows) and mature HIV virions (white arrows) between the DC-T cell mature contact zone. The appearance of a rare immature HIV particle is marked by *. Scale bar, 500 nm. Data from A–D is representative of n = 5 donors. (F–H) Schematic of the sequential contacts by VF (F), leading to selection and tethering (G) and enveloping of the T cells target coinciding with viral release (H). Whilst upper panels are schematics, lower panels have included primary representative data to support schematics.
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ppat-1002762-g006: Viral filopodial contacts precedes the formation of an enveloping DC-CD4 T cell contact.(A)–(D) Long-term contact by an infected DC leads to enveloping of the neighboring CD4 T cells. (A) Long-term enveloping in real-time. To follow the consequence of initial VF contact and tethering, HIV iGFP infected DC initially engaged in VF contacts with CD4 T cells were imaged over a period of 2 hours. Stills were taken from Video S15, with time elapsed presented in the upper left corner as minutes. In the final frame, two CD4 T cells are labeled 1 & 2 that have been enveloped by the infected DC. Scale bar at 5 µm. (B) Enveloping of multiple CD4 T cells. DCs were infected with HIV-T and 5 days post infection co-cultured with CD4 T cells at a ratio of 1 DC to 3 CD4 T cells for 2 hours. HIV particles were then imaged using FlAsH staining (green), samples fixed/permeabilized and nuclei countered stained with DAPI (Blue). Note CD4 T cells numbered 1 through to 4 have distal and proximal FlasH positive viral particles across their membrane. (C) Viral synapses between infected DC and CD4 T cells mature into extensive enveloping contacts. DC-T cell co-cultures are generated as per conditions in B, with the exception of the use of HIV iGFP (green). After 2 hours of co-culture, cells were stained with the DC membrane marker CD209 (red) and nuclei counterstained with DAPI (blue). Upper panel is representative of an infected DC engaging multiple CD4 T-cells as in B. In the lower panel, the infected DC is limited to two CD4 T cells, with the T cell conjugated on the right with a representative infected DC-T cell viral synapse. Scale bars are at 5 µm. Images are representative from 4 independent donors. (D) Long-term enveloping precedes viral fission across the synaptic cleft and seeding of the CD4 T cell with mature HIV. DCs were infected and co-cultured with autologous CD4 T cells as in (C). Total HIV (iGFP - green), mature HIV (Anti-p24/Capsid clone 183 in red) staining was carried out as per materials and methods. Nuclei are stained with DAPI as in (B). Arrow denotes accumulation of mature HIV particles at the viral synapse and also at the distal CD4 T cell membrane. Note HIV iGFP staining at the viral synapse exceeds mature HIV particles. Scale bar at 5 µm. (E) Electron microscopy confirms the presence of both immature/actively budding (black arrows) and mature HIV virions (white arrows) between the DC-T cell mature contact zone. The appearance of a rare immature HIV particle is marked by *. Scale bar, 500 nm. Data from A–D is representative of n = 5 donors. (F–H) Schematic of the sequential contacts by VF (F), leading to selection and tethering (G) and enveloping of the T cells target coinciding with viral release (H). Whilst upper panels are schematics, lower panels have included primary representative data to support schematics.

Mentions: The capacity of VF to mediate tethering and co-ordination of neighboring CD4 T cell contacts supports their role as viral synapse (VS) precursors, with VS formation triggered by HIV envelope and eventually mediating CD4 T cell infection. To image closer synapse formation, we identified infected DC with VF in close proximity to CD4 T cells and imaged the potential for synapse formation. Using both and HIV-iGFP (Fig. 6A) and HIV-T (Fig. 6B), we observed viral transfer across to the CD4 T cell membrane. We observed dynamic movement of newly produced HIV virions (HIV-T and HIV iGFP positive) over the entire CD4 T cell membrane (Fig. 6A & 6B; Video S15). Viral movement was over and around the CD4 T cell membrane (Video S15), with continual seeding and repositioning of the virus for up to 3 hours (the duration of the video acquisition). In addition, this phenomena could proceed from one infected DC over multiple CD4 T cells simultaneously (Fig. 6B & C). Of note co-culture of CD4 T cells with DCs infected with VSVg HIVENV-ve (HIV envelope negative) did not result in seeding of virus over the CD4 T cell surface. Whilst HIV iGFP would detect all virions, the detection of HIV-T on the CD4 T cell membrane supports either covering of the CD4 T cell membrane by DC in a form of cytophagocytosis or assembly, release and dissemination of HIV from the DC cytosol (as HIV-T only stains reduced cytosolic HIV Gag). To determine the extent of DC membrane that cover CD4 T cells at the viral synapse, we fixed and stained HIV infected DC-CD4 T cell co-cultures with the abundant DC membrane antigen CD209. Whilst the DC membrane covered substantial surface areas of the CD4 T cells engaged in viral synapses, the distal areas of the CD4 T cell membrane were not CD209 positive and thus we conclude although CD4 T cells were engulfed, they were not cytophagocytosed (Fig. 6C).


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)

Viral filopodial contacts precedes the formation of an enveloping DC-CD4 T cell contact.(A)–(D) Long-term contact by an infected DC leads to enveloping of the neighboring CD4 T cells. (A) Long-term enveloping in real-time. To follow the consequence of initial VF contact and tethering, HIV iGFP infected DC initially engaged in VF contacts with CD4 T cells were imaged over a period of 2 hours. Stills were taken from Video S15, with time elapsed presented in the upper left corner as minutes. In the final frame, two CD4 T cells are labeled 1 & 2 that have been enveloped by the infected DC. Scale bar at 5 µm. (B) Enveloping of multiple CD4 T cells. DCs were infected with HIV-T and 5 days post infection co-cultured with CD4 T cells at a ratio of 1 DC to 3 CD4 T cells for 2 hours. HIV particles were then imaged using FlAsH staining (green), samples fixed/permeabilized and nuclei countered stained with DAPI (Blue). Note CD4 T cells numbered 1 through to 4 have distal and proximal FlasH positive viral particles across their membrane. (C) Viral synapses between infected DC and CD4 T cells mature into extensive enveloping contacts. DC-T cell co-cultures are generated as per conditions in B, with the exception of the use of HIV iGFP (green). After 2 hours of co-culture, cells were stained with the DC membrane marker CD209 (red) and nuclei counterstained with DAPI (blue). Upper panel is representative of an infected DC engaging multiple CD4 T-cells as in B. In the lower panel, the infected DC is limited to two CD4 T cells, with the T cell conjugated on the right with a representative infected DC-T cell viral synapse. Scale bars are at 5 µm. Images are representative from 4 independent donors. (D) Long-term enveloping precedes viral fission across the synaptic cleft and seeding of the CD4 T cell with mature HIV. DCs were infected and co-cultured with autologous CD4 T cells as in (C). Total HIV (iGFP - green), mature HIV (Anti-p24/Capsid clone 183 in red) staining was carried out as per materials and methods. Nuclei are stained with DAPI as in (B). Arrow denotes accumulation of mature HIV particles at the viral synapse and also at the distal CD4 T cell membrane. Note HIV iGFP staining at the viral synapse exceeds mature HIV particles. Scale bar at 5 µm. (E) Electron microscopy confirms the presence of both immature/actively budding (black arrows) and mature HIV virions (white arrows) between the DC-T cell mature contact zone. The appearance of a rare immature HIV particle is marked by *. Scale bar, 500 nm. Data from A–D is representative of n = 5 donors. (F–H) Schematic of the sequential contacts by VF (F), leading to selection and tethering (G) and enveloping of the T cells target coinciding with viral release (H). Whilst upper panels are schematics, lower panels have included primary representative data to support schematics.
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Related In: Results  -  Collection

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ppat-1002762-g006: Viral filopodial contacts precedes the formation of an enveloping DC-CD4 T cell contact.(A)–(D) Long-term contact by an infected DC leads to enveloping of the neighboring CD4 T cells. (A) Long-term enveloping in real-time. To follow the consequence of initial VF contact and tethering, HIV iGFP infected DC initially engaged in VF contacts with CD4 T cells were imaged over a period of 2 hours. Stills were taken from Video S15, with time elapsed presented in the upper left corner as minutes. In the final frame, two CD4 T cells are labeled 1 & 2 that have been enveloped by the infected DC. Scale bar at 5 µm. (B) Enveloping of multiple CD4 T cells. DCs were infected with HIV-T and 5 days post infection co-cultured with CD4 T cells at a ratio of 1 DC to 3 CD4 T cells for 2 hours. HIV particles were then imaged using FlAsH staining (green), samples fixed/permeabilized and nuclei countered stained with DAPI (Blue). Note CD4 T cells numbered 1 through to 4 have distal and proximal FlasH positive viral particles across their membrane. (C) Viral synapses between infected DC and CD4 T cells mature into extensive enveloping contacts. DC-T cell co-cultures are generated as per conditions in B, with the exception of the use of HIV iGFP (green). After 2 hours of co-culture, cells were stained with the DC membrane marker CD209 (red) and nuclei counterstained with DAPI (blue). Upper panel is representative of an infected DC engaging multiple CD4 T-cells as in B. In the lower panel, the infected DC is limited to two CD4 T cells, with the T cell conjugated on the right with a representative infected DC-T cell viral synapse. Scale bars are at 5 µm. Images are representative from 4 independent donors. (D) Long-term enveloping precedes viral fission across the synaptic cleft and seeding of the CD4 T cell with mature HIV. DCs were infected and co-cultured with autologous CD4 T cells as in (C). Total HIV (iGFP - green), mature HIV (Anti-p24/Capsid clone 183 in red) staining was carried out as per materials and methods. Nuclei are stained with DAPI as in (B). Arrow denotes accumulation of mature HIV particles at the viral synapse and also at the distal CD4 T cell membrane. Note HIV iGFP staining at the viral synapse exceeds mature HIV particles. Scale bar at 5 µm. (E) Electron microscopy confirms the presence of both immature/actively budding (black arrows) and mature HIV virions (white arrows) between the DC-T cell mature contact zone. The appearance of a rare immature HIV particle is marked by *. Scale bar, 500 nm. Data from A–D is representative of n = 5 donors. (F–H) Schematic of the sequential contacts by VF (F), leading to selection and tethering (G) and enveloping of the T cells target coinciding with viral release (H). Whilst upper panels are schematics, lower panels have included primary representative data to support schematics.
Mentions: The capacity of VF to mediate tethering and co-ordination of neighboring CD4 T cell contacts supports their role as viral synapse (VS) precursors, with VS formation triggered by HIV envelope and eventually mediating CD4 T cell infection. To image closer synapse formation, we identified infected DC with VF in close proximity to CD4 T cells and imaged the potential for synapse formation. Using both and HIV-iGFP (Fig. 6A) and HIV-T (Fig. 6B), we observed viral transfer across to the CD4 T cell membrane. We observed dynamic movement of newly produced HIV virions (HIV-T and HIV iGFP positive) over the entire CD4 T cell membrane (Fig. 6A & 6B; Video S15). Viral movement was over and around the CD4 T cell membrane (Video S15), with continual seeding and repositioning of the virus for up to 3 hours (the duration of the video acquisition). In addition, this phenomena could proceed from one infected DC over multiple CD4 T cells simultaneously (Fig. 6B & C). Of note co-culture of CD4 T cells with DCs infected with VSVg HIVENV-ve (HIV envelope negative) did not result in seeding of virus over the CD4 T cell surface. Whilst HIV iGFP would detect all virions, the detection of HIV-T on the CD4 T cell membrane supports either covering of the CD4 T cell membrane by DC in a form of cytophagocytosis or assembly, release and dissemination of HIV from the DC cytosol (as HIV-T only stains reduced cytosolic HIV Gag). To determine the extent of DC membrane that cover CD4 T cells at the viral synapse, we fixed and stained HIV infected DC-CD4 T cell co-cultures with the abundant DC membrane antigen CD209. Whilst the DC membrane covered substantial surface areas of the CD4 T cells engaged in viral synapses, the distal areas of the CD4 T cell membrane were not CD209 positive and thus we conclude although CD4 T cells were engulfed, they were not cytophagocytosed (Fig. 6C).

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