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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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High VF frequencies correlate with the efficiency of immature DC viral transfer.(A) Schematic of the DC-CD4 T cell transfer assay. DC are infected with either HIVWT (high VF frequency) or HIV-NEF-ve (low VF frequency) pseudotyped with the VSVg glycoprotein, to ensure equal infection frequencies. After 4 days, DC infections are normalized to 5% with uninfected DC. Normalized populations are serially diluted below 1 infected DC per co-culture. 4 days post co-culture, CD4 T cell infections are resolved by staining cells for of HIV capsid and resolution by flow cytometry. (B) Flow cytometry detection of HIV p24 within CD4 T cell recipients when input infected DC are limiting (upper panel) versus (C) when input infected CD4 T cells are limiting (lower panel). Approximate infected cell number input into co-cultures is indicated at “Approx. Input*” on the X-axis. CD4 T cell infection frequencies are detected by the accumulation of a high HIV p24 population as indicated by the square gate in each dot-plot. Statistical difference is presented in upper HIV WT panels, and is calculated from data acquired from the same assay performed in triplicate. CD4 T cell infection frequency versus infected donor input is further summarized in right panels for two representative donors. Standard deviations represent co-cultures in triplicate. Data is representative of n = 8 independent donors.
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ppat-1002762-g007: High VF frequencies correlate with the efficiency of immature DC viral transfer.(A) Schematic of the DC-CD4 T cell transfer assay. DC are infected with either HIVWT (high VF frequency) or HIV-NEF-ve (low VF frequency) pseudotyped with the VSVg glycoprotein, to ensure equal infection frequencies. After 4 days, DC infections are normalized to 5% with uninfected DC. Normalized populations are serially diluted below 1 infected DC per co-culture. 4 days post co-culture, CD4 T cell infections are resolved by staining cells for of HIV capsid and resolution by flow cytometry. (B) Flow cytometry detection of HIV p24 within CD4 T cell recipients when input infected DC are limiting (upper panel) versus (C) when input infected CD4 T cells are limiting (lower panel). Approximate infected cell number input into co-cultures is indicated at “Approx. Input*” on the X-axis. CD4 T cell infection frequencies are detected by the accumulation of a high HIV p24 population as indicated by the square gate in each dot-plot. Statistical difference is presented in upper HIV WT panels, and is calculated from data acquired from the same assay performed in triplicate. CD4 T cell infection frequency versus infected donor input is further summarized in right panels for two representative donors. Standard deviations represent co-cultures in triplicate. Data is representative of n = 8 independent donors.

Mentions: The significantly lower VF number in HIVNEF-ve-iGFP infected DCs gave the unique opportunity to determine the potential role of VF in absolute viral transfer efficiency in primary DC, whilst not significantly influencing their viability and/or phenotype. Thus we tested the ability of immature DC infected with HIVNEF-ve(low VF frequency) versus HIVWT (high VF frequency) to transfer virus to a permissive (activated) autologous CD4 T cell population. Given CD4 T cells are VF low/absent expressing cell types, we also tested CD4 T cell to CD4 T cell transfer as a control. We stringently normalized both HIVNEF-ve and HIVWT infected populations (Fig. 7A schematic) prior to dilution and addition to target CD4 T cell populations. After 5 days co-culture, we utilized flow cytometry to not only resolved infected T cell population, but also determine the proportion infected. To enumerate the latter we take advantage of HIV capsid staining using the directly conjugated mAb clone KC57. In infected populations (both DCs and CD4 T cells), HIV capsid accumulates to significantly high levels to enable resolution of uninfected and infected populations as previously described [16]. Resolution of productive infection (versus only carriage of the virus) is further supported by the down-regulation of CD4 only observed in the capsid high population [11]. To unequivocally rule out this population is a result of CD4 T cell Gag acquisition independent of infection, we incubated DC-T cell co-cultures with 10 µM of AZT and confirmed the appearance of the p24/Capsid high population to be a result of productive infection (Fig. S2H).


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)

High VF frequencies correlate with the efficiency of immature DC viral transfer.(A) Schematic of the DC-CD4 T cell transfer assay. DC are infected with either HIVWT (high VF frequency) or HIV-NEF-ve (low VF frequency) pseudotyped with the VSVg glycoprotein, to ensure equal infection frequencies. After 4 days, DC infections are normalized to 5% with uninfected DC. Normalized populations are serially diluted below 1 infected DC per co-culture. 4 days post co-culture, CD4 T cell infections are resolved by staining cells for of HIV capsid and resolution by flow cytometry. (B) Flow cytometry detection of HIV p24 within CD4 T cell recipients when input infected DC are limiting (upper panel) versus (C) when input infected CD4 T cells are limiting (lower panel). Approximate infected cell number input into co-cultures is indicated at “Approx. Input*” on the X-axis. CD4 T cell infection frequencies are detected by the accumulation of a high HIV p24 population as indicated by the square gate in each dot-plot. Statistical difference is presented in upper HIV WT panels, and is calculated from data acquired from the same assay performed in triplicate. CD4 T cell infection frequency versus infected donor input is further summarized in right panels for two representative donors. Standard deviations represent co-cultures in triplicate. Data is representative of n = 8 independent donors.
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Related In: Results  -  Collection

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ppat-1002762-g007: High VF frequencies correlate with the efficiency of immature DC viral transfer.(A) Schematic of the DC-CD4 T cell transfer assay. DC are infected with either HIVWT (high VF frequency) or HIV-NEF-ve (low VF frequency) pseudotyped with the VSVg glycoprotein, to ensure equal infection frequencies. After 4 days, DC infections are normalized to 5% with uninfected DC. Normalized populations are serially diluted below 1 infected DC per co-culture. 4 days post co-culture, CD4 T cell infections are resolved by staining cells for of HIV capsid and resolution by flow cytometry. (B) Flow cytometry detection of HIV p24 within CD4 T cell recipients when input infected DC are limiting (upper panel) versus (C) when input infected CD4 T cells are limiting (lower panel). Approximate infected cell number input into co-cultures is indicated at “Approx. Input*” on the X-axis. CD4 T cell infection frequencies are detected by the accumulation of a high HIV p24 population as indicated by the square gate in each dot-plot. Statistical difference is presented in upper HIV WT panels, and is calculated from data acquired from the same assay performed in triplicate. CD4 T cell infection frequency versus infected donor input is further summarized in right panels for two representative donors. Standard deviations represent co-cultures in triplicate. Data is representative of n = 8 independent donors.
Mentions: The significantly lower VF number in HIVNEF-ve-iGFP infected DCs gave the unique opportunity to determine the potential role of VF in absolute viral transfer efficiency in primary DC, whilst not significantly influencing their viability and/or phenotype. Thus we tested the ability of immature DC infected with HIVNEF-ve(low VF frequency) versus HIVWT (high VF frequency) to transfer virus to a permissive (activated) autologous CD4 T cell population. Given CD4 T cells are VF low/absent expressing cell types, we also tested CD4 T cell to CD4 T cell transfer as a control. We stringently normalized both HIVNEF-ve and HIVWT infected populations (Fig. 7A schematic) prior to dilution and addition to target CD4 T cell populations. After 5 days co-culture, we utilized flow cytometry to not only resolved infected T cell population, but also determine the proportion infected. To enumerate the latter we take advantage of HIV capsid staining using the directly conjugated mAb clone KC57. In infected populations (both DCs and CD4 T cells), HIV capsid accumulates to significantly high levels to enable resolution of uninfected and infected populations as previously described [16]. Resolution of productive infection (versus only carriage of the virus) is further supported by the down-regulation of CD4 only observed in the capsid high population [11]. To unequivocally rule out this population is a result of CD4 T cell Gag acquisition independent of infection, we incubated DC-T cell co-cultures with 10 µM of AZT and confirmed the appearance of the p24/Capsid high population to be a result of productive infection (Fig. S2H).

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