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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

Complementary live imaging approaches reveal abundant HIV tipped filopodia (VF) on infected DC.(A) Detection strategy of HIV-T. The HIV Gag polyprotein is presented in the context of the HIV open reading frames. The biarsenical fluorescent dye FlAsH is shown and binds to a 12 amino acid motif (in bold) at the C-Terminus of Matrix. The protease cleavage site between HIV Matrix and Capsid is highlighted by black scissors. (B) Detection strategy using HIV-iGFP. HIV-iGFP constructs encode eGFP at the C-terminus of HIV matrix and are flanked by 5′ and 3′ HIV protease cleavage sites (highlighted by black scissors). For generation of cell-free virus with comparable infectivity to WT HIV Gag and Gag-Pol are expressed in trans to the HIV iGFP genome (see bottom of panel; HIV Gag only shown) to increase viral infectivity in one round. (C) & (D) Rescuing infectivity of HIV-iGFP. (C) HIV iGFP was prepared by co-transfecting WT HIV or WT Gag and Gag -POL (psPAX2) with HIV iGFP at an equimolar ratio into the 293T cell line. Three days post transfection, supernatants were harvested, diluted 1/1000 and 200 ml was added to 1×103 TZM-bl cells (HeLa HIV indicator cell line), seeded 24 hours prior in a 96 well plate. % infectivity is relative to wild type HIV and calculated as the (co-transfections)/(HIV-WT alone)×100. Statistical differences are presented as p values. Standard deviations are derived from assays in triplicate. (D) Further titration of psPAX2∶HIV-iGFP. As in C. HIV-iGFP was co-transfected with psPAX2, but here at as a titration. Supernatants were subsequently titered using the TZM-bl cell line as in C. Standard deviations and p values also as per C. % infectivity relative to wild type HIV is calculated as in C. (E) DC were infected with an MOI of 0.1 with either WT HIV (left panel), WT HIV rescued HIV-iGFP virus (middle panel) or psPAX2 rescued HIV-iGFP (right panel) as outlined in material and methods. To determine total infection, infected DC were stained with anti-HIV-p24 antibody KC57-RD1. Gates in panels reflect eGFP signal from infected p24 high cells, with percentages from gates presented in the lower right corners. Note WT rescued HIV iGFP virus generates infected DC with diluted eGFP signal. (F) Infected DCs expressing HIV-iGFP 4 days post infection and co-cultured with autologous resting CD4 T cells at a ratio of 1 DC to 3 CD4 T cells (images are also representative for HIV-T). Filopodia are highlighted by dotted lines. Neighboring CD4 T cells that are in contact with filopodia are marked as (T) (G) HIV iGFP infected cells (HIV in white) have been fixed and stained for F-actin using phalloidin dye (red). Note all filopodia stained red, bear HIV at their terminal tips (All scale bars are 5 µm). (H) Average lengths of filopodia & VF across multiple DC donors. Infected or uninfected DCs (untreated U/T) were co-cultured with CD4 T cells as in F. Length of filopodia from the base at the plasma membrane to the tip was calculated in live infected and uninfected DC donors. VF and Filopodia lengths in infected and uninfected co-cultures from D1 & D2 are presented as a comparison. Filopodial lengths are representative of greater than n = 20 donors. VF and filopodia from infected and uninfected U937 cell line are also presented as a comparison.
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ppat-1002762-g001: Complementary live imaging approaches reveal abundant HIV tipped filopodia (VF) on infected DC.(A) Detection strategy of HIV-T. The HIV Gag polyprotein is presented in the context of the HIV open reading frames. The biarsenical fluorescent dye FlAsH is shown and binds to a 12 amino acid motif (in bold) at the C-Terminus of Matrix. The protease cleavage site between HIV Matrix and Capsid is highlighted by black scissors. (B) Detection strategy using HIV-iGFP. HIV-iGFP constructs encode eGFP at the C-terminus of HIV matrix and are flanked by 5′ and 3′ HIV protease cleavage sites (highlighted by black scissors). For generation of cell-free virus with comparable infectivity to WT HIV Gag and Gag-Pol are expressed in trans to the HIV iGFP genome (see bottom of panel; HIV Gag only shown) to increase viral infectivity in one round. (C) & (D) Rescuing infectivity of HIV-iGFP. (C) HIV iGFP was prepared by co-transfecting WT HIV or WT Gag and Gag -POL (psPAX2) with HIV iGFP at an equimolar ratio into the 293T cell line. Three days post transfection, supernatants were harvested, diluted 1/1000 and 200 ml was added to 1×103 TZM-bl cells (HeLa HIV indicator cell line), seeded 24 hours prior in a 96 well plate. % infectivity is relative to wild type HIV and calculated as the (co-transfections)/(HIV-WT alone)×100. Statistical differences are presented as p values. Standard deviations are derived from assays in triplicate. (D) Further titration of psPAX2∶HIV-iGFP. As in C. HIV-iGFP was co-transfected with psPAX2, but here at as a titration. Supernatants were subsequently titered using the TZM-bl cell line as in C. Standard deviations and p values also as per C. % infectivity relative to wild type HIV is calculated as in C. (E) DC were infected with an MOI of 0.1 with either WT HIV (left panel), WT HIV rescued HIV-iGFP virus (middle panel) or psPAX2 rescued HIV-iGFP (right panel) as outlined in material and methods. To determine total infection, infected DC were stained with anti-HIV-p24 antibody KC57-RD1. Gates in panels reflect eGFP signal from infected p24 high cells, with percentages from gates presented in the lower right corners. Note WT rescued HIV iGFP virus generates infected DC with diluted eGFP signal. (F) Infected DCs expressing HIV-iGFP 4 days post infection and co-cultured with autologous resting CD4 T cells at a ratio of 1 DC to 3 CD4 T cells (images are also representative for HIV-T). Filopodia are highlighted by dotted lines. Neighboring CD4 T cells that are in contact with filopodia are marked as (T) (G) HIV iGFP infected cells (HIV in white) have been fixed and stained for F-actin using phalloidin dye (red). Note all filopodia stained red, bear HIV at their terminal tips (All scale bars are 5 µm). (H) Average lengths of filopodia & VF across multiple DC donors. Infected or uninfected DCs (untreated U/T) were co-cultured with CD4 T cells as in F. Length of filopodia from the base at the plasma membrane to the tip was calculated in live infected and uninfected DC donors. VF and Filopodia lengths in infected and uninfected co-cultures from D1 & D2 are presented as a comparison. Filopodial lengths are representative of greater than n = 20 donors. VF and filopodia from infected and uninfected U937 cell line are also presented as a comparison.

Mentions: To investigate the dynamics of HIV transfer from infected DC to CD4 T cells, we attempted to map this process in real-time. To do so we needed to undertake imaging of the de novo viral pool present in infected primary DC. To date imaging of the de novo pool has remained limited as markers amenable to imaging need to be integrated within the size sensitive HIV genome. The generation of HIV constructs by others and ourselves have addressed this short-coming by using small genetic tags that work in conjunction with cell permeable fluorescent dyes (termed hereon as HIV-T, Fig. 1A) [21], [22], or by overcoming the complications that large fluorescent proteins create when inserted into HIV structural proteins (termed HIV-iGFP, Fig. 1B) [23]. To persist with the use of traditional fluorescent proteins, we needed to rescue viral infectivity at two levels. Firstly we flanked the large fluorescent proteins with HIV protease cleavage sites (Fig. 1B) as previously described [23], thus giving the virus the ability to untether from the fluorescent protein when maturing. Secondly, we rescued this virus further by supplying wild type (WT) Gag and Gag-Pol in trans (Fig. 1C & D). Initially attempts of supplying WT Gag and Gag-Pol by co-transfecting WT HIV with HIV-iGFP did increase infectivity to levels comparable to WT HIV (Fig. 1C). However subsequent analysis of infected DC populations, highlight the dominance of the WT genome within the de novo pool (Fig. 1E HIV+HIV-iGFP). Thus to avoid a secondary WT competing genome in future de novo virus, we supplied WT HIV Gag and Gag-Pol in trans only at the protein level using the 2nd generation lentiviral vector psPAX2 (Fig. 1D & E). When using this latter approach, we could not only rescue HIV-iGFP (Fig. 1C & D), but also readily infect primary DC (Fig. 1E; HIV+psPAX2 & Fig. S1A). More importantly, as there was no competing WT HIV genome, we could detect every cell that was infected by high levels of Gag-iGFP expression.


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)

Complementary live imaging approaches reveal abundant HIV tipped filopodia (VF) on infected DC.(A) Detection strategy of HIV-T. The HIV Gag polyprotein is presented in the context of the HIV open reading frames. The biarsenical fluorescent dye FlAsH is shown and binds to a 12 amino acid motif (in bold) at the C-Terminus of Matrix. The protease cleavage site between HIV Matrix and Capsid is highlighted by black scissors. (B) Detection strategy using HIV-iGFP. HIV-iGFP constructs encode eGFP at the C-terminus of HIV matrix and are flanked by 5′ and 3′ HIV protease cleavage sites (highlighted by black scissors). For generation of cell-free virus with comparable infectivity to WT HIV Gag and Gag-Pol are expressed in trans to the HIV iGFP genome (see bottom of panel; HIV Gag only shown) to increase viral infectivity in one round. (C) & (D) Rescuing infectivity of HIV-iGFP. (C) HIV iGFP was prepared by co-transfecting WT HIV or WT Gag and Gag -POL (psPAX2) with HIV iGFP at an equimolar ratio into the 293T cell line. Three days post transfection, supernatants were harvested, diluted 1/1000 and 200 ml was added to 1×103 TZM-bl cells (HeLa HIV indicator cell line), seeded 24 hours prior in a 96 well plate. % infectivity is relative to wild type HIV and calculated as the (co-transfections)/(HIV-WT alone)×100. Statistical differences are presented as p values. Standard deviations are derived from assays in triplicate. (D) Further titration of psPAX2∶HIV-iGFP. As in C. HIV-iGFP was co-transfected with psPAX2, but here at as a titration. Supernatants were subsequently titered using the TZM-bl cell line as in C. Standard deviations and p values also as per C. % infectivity relative to wild type HIV is calculated as in C. (E) DC were infected with an MOI of 0.1 with either WT HIV (left panel), WT HIV rescued HIV-iGFP virus (middle panel) or psPAX2 rescued HIV-iGFP (right panel) as outlined in material and methods. To determine total infection, infected DC were stained with anti-HIV-p24 antibody KC57-RD1. Gates in panels reflect eGFP signal from infected p24 high cells, with percentages from gates presented in the lower right corners. Note WT rescued HIV iGFP virus generates infected DC with diluted eGFP signal. (F) Infected DCs expressing HIV-iGFP 4 days post infection and co-cultured with autologous resting CD4 T cells at a ratio of 1 DC to 3 CD4 T cells (images are also representative for HIV-T). Filopodia are highlighted by dotted lines. Neighboring CD4 T cells that are in contact with filopodia are marked as (T) (G) HIV iGFP infected cells (HIV in white) have been fixed and stained for F-actin using phalloidin dye (red). Note all filopodia stained red, bear HIV at their terminal tips (All scale bars are 5 µm). (H) Average lengths of filopodia & VF across multiple DC donors. Infected or uninfected DCs (untreated U/T) were co-cultured with CD4 T cells as in F. Length of filopodia from the base at the plasma membrane to the tip was calculated in live infected and uninfected DC donors. VF and Filopodia lengths in infected and uninfected co-cultures from D1 & D2 are presented as a comparison. Filopodial lengths are representative of greater than n = 20 donors. VF and filopodia from infected and uninfected U937 cell line are also presented as a comparison.
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Related In: Results  -  Collection

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ppat-1002762-g001: Complementary live imaging approaches reveal abundant HIV tipped filopodia (VF) on infected DC.(A) Detection strategy of HIV-T. The HIV Gag polyprotein is presented in the context of the HIV open reading frames. The biarsenical fluorescent dye FlAsH is shown and binds to a 12 amino acid motif (in bold) at the C-Terminus of Matrix. The protease cleavage site between HIV Matrix and Capsid is highlighted by black scissors. (B) Detection strategy using HIV-iGFP. HIV-iGFP constructs encode eGFP at the C-terminus of HIV matrix and are flanked by 5′ and 3′ HIV protease cleavage sites (highlighted by black scissors). For generation of cell-free virus with comparable infectivity to WT HIV Gag and Gag-Pol are expressed in trans to the HIV iGFP genome (see bottom of panel; HIV Gag only shown) to increase viral infectivity in one round. (C) & (D) Rescuing infectivity of HIV-iGFP. (C) HIV iGFP was prepared by co-transfecting WT HIV or WT Gag and Gag -POL (psPAX2) with HIV iGFP at an equimolar ratio into the 293T cell line. Three days post transfection, supernatants were harvested, diluted 1/1000 and 200 ml was added to 1×103 TZM-bl cells (HeLa HIV indicator cell line), seeded 24 hours prior in a 96 well plate. % infectivity is relative to wild type HIV and calculated as the (co-transfections)/(HIV-WT alone)×100. Statistical differences are presented as p values. Standard deviations are derived from assays in triplicate. (D) Further titration of psPAX2∶HIV-iGFP. As in C. HIV-iGFP was co-transfected with psPAX2, but here at as a titration. Supernatants were subsequently titered using the TZM-bl cell line as in C. Standard deviations and p values also as per C. % infectivity relative to wild type HIV is calculated as in C. (E) DC were infected with an MOI of 0.1 with either WT HIV (left panel), WT HIV rescued HIV-iGFP virus (middle panel) or psPAX2 rescued HIV-iGFP (right panel) as outlined in material and methods. To determine total infection, infected DC were stained with anti-HIV-p24 antibody KC57-RD1. Gates in panels reflect eGFP signal from infected p24 high cells, with percentages from gates presented in the lower right corners. Note WT rescued HIV iGFP virus generates infected DC with diluted eGFP signal. (F) Infected DCs expressing HIV-iGFP 4 days post infection and co-cultured with autologous resting CD4 T cells at a ratio of 1 DC to 3 CD4 T cells (images are also representative for HIV-T). Filopodia are highlighted by dotted lines. Neighboring CD4 T cells that are in contact with filopodia are marked as (T) (G) HIV iGFP infected cells (HIV in white) have been fixed and stained for F-actin using phalloidin dye (red). Note all filopodia stained red, bear HIV at their terminal tips (All scale bars are 5 µm). (H) Average lengths of filopodia & VF across multiple DC donors. Infected or uninfected DCs (untreated U/T) were co-cultured with CD4 T cells as in F. Length of filopodia from the base at the plasma membrane to the tip was calculated in live infected and uninfected DC donors. VF and Filopodia lengths in infected and uninfected co-cultures from D1 & D2 are presented as a comparison. Filopodial lengths are representative of greater than n = 20 donors. VF and filopodia from infected and uninfected U937 cell line are also presented as a comparison.
Mentions: To investigate the dynamics of HIV transfer from infected DC to CD4 T cells, we attempted to map this process in real-time. To do so we needed to undertake imaging of the de novo viral pool present in infected primary DC. To date imaging of the de novo pool has remained limited as markers amenable to imaging need to be integrated within the size sensitive HIV genome. The generation of HIV constructs by others and ourselves have addressed this short-coming by using small genetic tags that work in conjunction with cell permeable fluorescent dyes (termed hereon as HIV-T, Fig. 1A) [21], [22], or by overcoming the complications that large fluorescent proteins create when inserted into HIV structural proteins (termed HIV-iGFP, Fig. 1B) [23]. To persist with the use of traditional fluorescent proteins, we needed to rescue viral infectivity at two levels. Firstly we flanked the large fluorescent proteins with HIV protease cleavage sites (Fig. 1B) as previously described [23], thus giving the virus the ability to untether from the fluorescent protein when maturing. Secondly, we rescued this virus further by supplying wild type (WT) Gag and Gag-Pol in trans (Fig. 1C & D). Initially attempts of supplying WT Gag and Gag-Pol by co-transfecting WT HIV with HIV-iGFP did increase infectivity to levels comparable to WT HIV (Fig. 1C). However subsequent analysis of infected DC populations, highlight the dominance of the WT genome within the de novo pool (Fig. 1E HIV+HIV-iGFP). Thus to avoid a secondary WT competing genome in future de novo virus, we supplied WT HIV Gag and Gag-Pol in trans only at the protein level using the 2nd generation lentiviral vector psPAX2 (Fig. 1D & E). When using this latter approach, we could not only rescue HIV-iGFP (Fig. 1C & D), but also readily infect primary DC (Fig. 1E; HIV+psPAX2 & Fig. S1A). More importantly, as there was no competing WT HIV genome, we could detect every cell that was infected by high levels of Gag-iGFP expression.

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