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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 are capped and continous with immature HIV buds and do not associate with Arp2/3 antigens.(A) HIV envelope staining at the filopodial tip. An infected DC is presented bearing two long VF (HIV iGFP in white and Phalloidin staining in red) in excess of 20 µm (center panel boxed sections labeled 1 & 2). Left and right panels are magnified and HIV envelope stain is presented as blue. Scale bars in the center panel are 5 µm. Scale bars in left and right panels are 1 µm. (B)–(D) HIV at the tip of VF consists of cytosolic uncleave HIV Gag. (B) HIV-T FlAsH staining (white) at the tip of VF with F-actin staining using phalloidin (red) (scale bar is at 5 µm). (C) Further confirmation that HIV particles consist primarily of uncleaved HIV Gag. Image presented is HIV iGFP DC (all HIV particles will be detected) and counter-stained with the anti-p24/Capsid mAb 183 that specifically detects cleaved HIV p24/Capsid. Note the lack of mAb staining for HIV iGFP particles. Scale bar is at 5 µm. (C) Image is representative VF after imaging HIV infected DC via transmission electron microscopy (scale bar at 100 nm). Images in A-C are representative of n = 6 infected DC donors with HIV-T, HIV iGFP (fluorescence) or HIV (electron microscopy). (E) & (F) Virions unable to undergo fission from the plasma membrane (HIV GAG PTAP mutants) form VF. (E) A representative image of VF is presented in the fluorescent image, with HIV iGFP in white and phalloidin staining of actin in red. Note the mutation of the PTAP motif does not prevent VF formation. Data is representative of three independent infections. (F) DCs were infected with HIV iGFP or HIV iGFPPTAP-ve. VF lengths and average trajectory velocities are calculated as outlined in Fig. 1 & 2. Statistical significance is presented as p values. Data is representative of n = 3 independent experiments. (G) & (H) VF are enriched for filopodial antigens, but their antigens do not routinely co-localize with immature HIV particles. Immature DCs were infected as outlined in Fig. 1 and then fixed and counter stained for filopodial antigens (blue), (G) Arp2 and (H) phosphotyrosine (pTyr) staining are presented as overlays with F-actin (red) and HIV iGFP (white). HIV particles in close association with the antigenic stain are highlighted by asterix in volume projected images, whilst non-associated particles are marked by arrows. Staining for Wasp and Cortactin are presented for comparison in the supplementary Fig. S2. All scale bars are at 5 µm. Images are representative of n = 7 independent donors.
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ppat-1002762-g004: VF are capped and continous with immature HIV buds and do not associate with Arp2/3 antigens.(A) HIV envelope staining at the filopodial tip. An infected DC is presented bearing two long VF (HIV iGFP in white and Phalloidin staining in red) in excess of 20 µm (center panel boxed sections labeled 1 & 2). Left and right panels are magnified and HIV envelope stain is presented as blue. Scale bars in the center panel are 5 µm. Scale bars in left and right panels are 1 µm. (B)–(D) HIV at the tip of VF consists of cytosolic uncleave HIV Gag. (B) HIV-T FlAsH staining (white) at the tip of VF with F-actin staining using phalloidin (red) (scale bar is at 5 µm). (C) Further confirmation that HIV particles consist primarily of uncleaved HIV Gag. Image presented is HIV iGFP DC (all HIV particles will be detected) and counter-stained with the anti-p24/Capsid mAb 183 that specifically detects cleaved HIV p24/Capsid. Note the lack of mAb staining for HIV iGFP particles. Scale bar is at 5 µm. (C) Image is representative VF after imaging HIV infected DC via transmission electron microscopy (scale bar at 100 nm). Images in A-C are representative of n = 6 infected DC donors with HIV-T, HIV iGFP (fluorescence) or HIV (electron microscopy). (E) & (F) Virions unable to undergo fission from the plasma membrane (HIV GAG PTAP mutants) form VF. (E) A representative image of VF is presented in the fluorescent image, with HIV iGFP in white and phalloidin staining of actin in red. Note the mutation of the PTAP motif does not prevent VF formation. Data is representative of three independent infections. (F) DCs were infected with HIV iGFP or HIV iGFPPTAP-ve. VF lengths and average trajectory velocities are calculated as outlined in Fig. 1 & 2. Statistical significance is presented as p values. Data is representative of n = 3 independent experiments. (G) & (H) VF are enriched for filopodial antigens, but their antigens do not routinely co-localize with immature HIV particles. Immature DCs were infected as outlined in Fig. 1 and then fixed and counter stained for filopodial antigens (blue), (G) Arp2 and (H) phosphotyrosine (pTyr) staining are presented as overlays with F-actin (red) and HIV iGFP (white). HIV particles in close association with the antigenic stain are highlighted by asterix in volume projected images, whilst non-associated particles are marked by arrows. Staining for Wasp and Cortactin are presented for comparison in the supplementary Fig. S2. All scale bars are at 5 µm. Images are representative of n = 7 independent donors.

Mentions: For initial phenotyping of viral particles at the tips of filopodia, we first determined whether HIV envelope was present at the filopodial tip. Unfortunately staining of HIV envelope using the 2G12 antibody observed significant signal on the plasma membrane of HIV iGFP infected DCs and U937 cells. Unlike the HIV Gag signal, where there theoretically would be approximately 5000 copies [29], HIV envelope copies have recently been estimated as 14+/−7 trimers per virion [30]. Whilst amplification of the signal using a combination of increased exposure time and CCD/EMCCD camera electronic amplification can permit detection, this was counter balanced by the significant signal of the infected cell membrane. To overcome this, we focused on filopodia that were in excess of 20 µm in length, thereby focusing on a HIV particle that was sufficiently distanced from fluorescence of the cell membrane. Using this approach we could readily detect the presence of HIV Env on the HIV particle capping the filopodial extension (Fig. 4A).


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 are capped and continous with immature HIV buds and do not associate with Arp2/3 antigens.(A) HIV envelope staining at the filopodial tip. An infected DC is presented bearing two long VF (HIV iGFP in white and Phalloidin staining in red) in excess of 20 µm (center panel boxed sections labeled 1 & 2). Left and right panels are magnified and HIV envelope stain is presented as blue. Scale bars in the center panel are 5 µm. Scale bars in left and right panels are 1 µm. (B)–(D) HIV at the tip of VF consists of cytosolic uncleave HIV Gag. (B) HIV-T FlAsH staining (white) at the tip of VF with F-actin staining using phalloidin (red) (scale bar is at 5 µm). (C) Further confirmation that HIV particles consist primarily of uncleaved HIV Gag. Image presented is HIV iGFP DC (all HIV particles will be detected) and counter-stained with the anti-p24/Capsid mAb 183 that specifically detects cleaved HIV p24/Capsid. Note the lack of mAb staining for HIV iGFP particles. Scale bar is at 5 µm. (C) Image is representative VF after imaging HIV infected DC via transmission electron microscopy (scale bar at 100 nm). Images in A-C are representative of n = 6 infected DC donors with HIV-T, HIV iGFP (fluorescence) or HIV (electron microscopy). (E) & (F) Virions unable to undergo fission from the plasma membrane (HIV GAG PTAP mutants) form VF. (E) A representative image of VF is presented in the fluorescent image, with HIV iGFP in white and phalloidin staining of actin in red. Note the mutation of the PTAP motif does not prevent VF formation. Data is representative of three independent infections. (F) DCs were infected with HIV iGFP or HIV iGFPPTAP-ve. VF lengths and average trajectory velocities are calculated as outlined in Fig. 1 & 2. Statistical significance is presented as p values. Data is representative of n = 3 independent experiments. (G) & (H) VF are enriched for filopodial antigens, but their antigens do not routinely co-localize with immature HIV particles. Immature DCs were infected as outlined in Fig. 1 and then fixed and counter stained for filopodial antigens (blue), (G) Arp2 and (H) phosphotyrosine (pTyr) staining are presented as overlays with F-actin (red) and HIV iGFP (white). HIV particles in close association with the antigenic stain are highlighted by asterix in volume projected images, whilst non-associated particles are marked by arrows. Staining for Wasp and Cortactin are presented for comparison in the supplementary Fig. S2. All scale bars are at 5 µm. Images are representative of n = 7 independent donors.
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Related In: Results  -  Collection

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ppat-1002762-g004: VF are capped and continous with immature HIV buds and do not associate with Arp2/3 antigens.(A) HIV envelope staining at the filopodial tip. An infected DC is presented bearing two long VF (HIV iGFP in white and Phalloidin staining in red) in excess of 20 µm (center panel boxed sections labeled 1 & 2). Left and right panels are magnified and HIV envelope stain is presented as blue. Scale bars in the center panel are 5 µm. Scale bars in left and right panels are 1 µm. (B)–(D) HIV at the tip of VF consists of cytosolic uncleave HIV Gag. (B) HIV-T FlAsH staining (white) at the tip of VF with F-actin staining using phalloidin (red) (scale bar is at 5 µm). (C) Further confirmation that HIV particles consist primarily of uncleaved HIV Gag. Image presented is HIV iGFP DC (all HIV particles will be detected) and counter-stained with the anti-p24/Capsid mAb 183 that specifically detects cleaved HIV p24/Capsid. Note the lack of mAb staining for HIV iGFP particles. Scale bar is at 5 µm. (C) Image is representative VF after imaging HIV infected DC via transmission electron microscopy (scale bar at 100 nm). Images in A-C are representative of n = 6 infected DC donors with HIV-T, HIV iGFP (fluorescence) or HIV (electron microscopy). (E) & (F) Virions unable to undergo fission from the plasma membrane (HIV GAG PTAP mutants) form VF. (E) A representative image of VF is presented in the fluorescent image, with HIV iGFP in white and phalloidin staining of actin in red. Note the mutation of the PTAP motif does not prevent VF formation. Data is representative of three independent infections. (F) DCs were infected with HIV iGFP or HIV iGFPPTAP-ve. VF lengths and average trajectory velocities are calculated as outlined in Fig. 1 & 2. Statistical significance is presented as p values. Data is representative of n = 3 independent experiments. (G) & (H) VF are enriched for filopodial antigens, but their antigens do not routinely co-localize with immature HIV particles. Immature DCs were infected as outlined in Fig. 1 and then fixed and counter stained for filopodial antigens (blue), (G) Arp2 and (H) phosphotyrosine (pTyr) staining are presented as overlays with F-actin (red) and HIV iGFP (white). HIV particles in close association with the antigenic stain are highlighted by asterix in volume projected images, whilst non-associated particles are marked by arrows. Staining for Wasp and Cortactin are presented for comparison in the supplementary Fig. S2. All scale bars are at 5 µm. Images are representative of n = 7 independent donors.
Mentions: For initial phenotyping of viral particles at the tips of filopodia, we first determined whether HIV envelope was present at the filopodial tip. Unfortunately staining of HIV envelope using the 2G12 antibody observed significant signal on the plasma membrane of HIV iGFP infected DCs and U937 cells. Unlike the HIV Gag signal, where there theoretically would be approximately 5000 copies [29], HIV envelope copies have recently been estimated as 14+/−7 trimers per virion [30]. Whilst amplification of the signal using a combination of increased exposure time and CCD/EMCCD camera electronic amplification can permit detection, this was counter balanced by the significant signal of the infected cell membrane. To overcome this, we focused on filopodia that were in excess of 20 µm in length, thereby focusing on a HIV particle that was sufficiently distanced from fluorescence of the cell membrane. Using this approach we could readily detect the presence of HIV Env on the HIV particle capping the filopodial extension (Fig. 4A).

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