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CD169-mediated trafficking of HIV to plasma membrane invaginations in dendritic cells attenuates efficacy of anti-gp120 broadly neutralizing antibodies.

Akiyama H, Ramirez NG, Gudheti MV, Gummuluru S - PLoS Pathog. (2015)

Bottom Line: We find HIV-1-induced formation of VCCs is restricted to myeloid cells, and that the cytoplasmic tail of CD169 is dispensable for HIV-1 trafficking and retention within VCCs and subsequent trans-infection to CD4⁺ T cells.Interestingly, introduction of a di-aromatic endocytic motif in the cytoplasmic tail of CD169 that results in endocytosis of HIV-1 particles, suppressed CD169-mediated HIV-1 trans-infection.Intriguingly, HIV-1 particles in deep VCCs were inefficiently accessed by anti-gp120 broadly neutralizing antibodies, VRC01 and NIH45-46 G54W, and thus were less susceptible to neutralization.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Myeloid dendritic cells (DCs) can capture HIV-1 via the receptor CD169/Siglec-1 that binds to the ganglioside, GM3, in the virus particle membrane. In turn, HIV-1 particles captured by CD169, an I-type lectin, whose expression on DCs is enhanced upon maturation with LPS, are protected from degradation in CD169+ virus-containing compartments (VCCs) and disseminated to CD4⁺ T cells, a mechanism of DC-mediated HIV-1 trans-infection. In this study, we describe the mechanism of VCC formation and its role in immune evasion mechanisms of HIV-1. We find HIV-1-induced formation of VCCs is restricted to myeloid cells, and that the cytoplasmic tail of CD169 is dispensable for HIV-1 trafficking and retention within VCCs and subsequent trans-infection to CD4⁺ T cells. Interestingly, introduction of a di-aromatic endocytic motif in the cytoplasmic tail of CD169 that results in endocytosis of HIV-1 particles, suppressed CD169-mediated HIV-1 trans-infection. Furthermore, super-resolution microscopy revealed close association of CD169 and HIV-1 particles in surface-accessible but deep plasma membrane invaginations. Intriguingly, HIV-1 particles in deep VCCs were inefficiently accessed by anti-gp120 broadly neutralizing antibodies, VRC01 and NIH45-46 G54W, and thus were less susceptible to neutralization. Our study suggests that HIV-1 capture by CD169 can provide virus evasion from both innate (phagocytosis) and adaptive immune responses.

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The cytoplasmic tail (CT) of CD169 is dispensable for mediating HIV-1 trans-infection.(A) Sequences of wild type and mutant CD169 CTs. The asterisks represent stop codons introduced into the ORFs of the two CT mutants. (B) Western blot analysis of THP-1 cell lysates expressing either wild type or mutant CD169. (C) Cell surface expression of CD169 on THP-1 cells was measured by flow cytometry. (D) Relative cell surface expression of CD169 CT mutants was quantified and normalized to that observed with THP-1/CD169 cells. (E) Cells were challenged with HIV-1, washed and cell-associated p24gag was measured. The data shown is the virus capture by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) normalized to that observed with THP-1/CD169 cells. (F) THP-1/CD169- or THP-1/CD169 CT mutant-mediated trans-infection was determined by measuring luciferase activity in THP—CD4+ T cell co-cultures 2 days post initiation of co-culture. The data shown is the relative virus transmission by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) to that observed with THP-1/CD169 cells. (G) Efficacy of trans-infection was calculated as trans-infection (luciferase activity) per amount of virus captured (cell-associated p24gag) and normalized to that observed with THP-1/CD169 cells (set as 100). The data shown are the means ± SEM of three (D to F) or four (G) independent experiments. (H) THP-1/CD169 or THP-1/CD169ΔCT4R cells were incubated with Gag-mCherry VLPs (red), washed, fixed and stained for CD169 (green) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. WT: THP-1/CD169, ΔCT: THP-1/CD169ΔCT, ΔCT4R: THP-1/CD169ΔCT4R and Vec: empty vector transduced THP-1.
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ppat.1004751.g002: The cytoplasmic tail (CT) of CD169 is dispensable for mediating HIV-1 trans-infection.(A) Sequences of wild type and mutant CD169 CTs. The asterisks represent stop codons introduced into the ORFs of the two CT mutants. (B) Western blot analysis of THP-1 cell lysates expressing either wild type or mutant CD169. (C) Cell surface expression of CD169 on THP-1 cells was measured by flow cytometry. (D) Relative cell surface expression of CD169 CT mutants was quantified and normalized to that observed with THP-1/CD169 cells. (E) Cells were challenged with HIV-1, washed and cell-associated p24gag was measured. The data shown is the virus capture by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) normalized to that observed with THP-1/CD169 cells. (F) THP-1/CD169- or THP-1/CD169 CT mutant-mediated trans-infection was determined by measuring luciferase activity in THP—CD4+ T cell co-cultures 2 days post initiation of co-culture. The data shown is the relative virus transmission by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) to that observed with THP-1/CD169 cells. (G) Efficacy of trans-infection was calculated as trans-infection (luciferase activity) per amount of virus captured (cell-associated p24gag) and normalized to that observed with THP-1/CD169 cells (set as 100). The data shown are the means ± SEM of three (D to F) or four (G) independent experiments. (H) THP-1/CD169 or THP-1/CD169ΔCT4R cells were incubated with Gag-mCherry VLPs (red), washed, fixed and stained for CD169 (green) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. WT: THP-1/CD169, ΔCT: THP-1/CD169ΔCT, ΔCT4R: THP-1/CD169ΔCT4R and Vec: empty vector transduced THP-1.

Mentions: CD169 has been reported as a phagocytic receptor on porcine macrophages that can mediate endocytosis of PRSSV [26]. However, to date, no previously defined endocytosis signaling motifs have been described in the CT of human CD169. Since CD169 was trafficked to and colocalized with HIV-1 in surface-accessible compartments in myeloid cells (Fig. 1), we postulated that there was an unidentified trafficking motif in the CT that contributed to colocalization of CD169 and HIV-1 in VCCs. Two CD169 CT truncation mutants were constructed (Fig. 2A), one of which has a stop codon right after the transmembrane domain of CD169 (CD169ΔCT) [15]. Since previous studies have demonstrated severe reduction in cell surface expression of plasma membrane targeted proteins upon deletion of cytoplasmic tails [27,28], we constructed a second CD169 CT mutant that expressed the first four amino acids of CT (CD169ΔCT4R). These CD169 CT mutants were transduced into THP-1 cells and the ability of these stably transduced cell lines expressing CD169 mutants to capture HIV and form VCCs was compared to that observed with THP-1 cells expressing wild type CD169 (THP-1/CD169) (Fig. 1B). Deletion of the cytoplasmic tail (CD169ΔCT) resulted in decreased expression of CD169 in in THP-1 cells (Fig. 2B and S1 Fig B). Furthermore, cell surface expression of CD169ΔCT was further reduced (Fig. 2C and D) and resulted in severe attenuation of HIV-1 capture (Fig. 2E). Interestingly, inclusion of the membrane proximal 4 arginine residues in the cytoplasmic tail resulted in higher expression of CD169 in cells and partial rescue of cell surface expression of CD169 (Fig. 2C, 2D and S1 Fig B), and importantly, capture of HIV-1 particles (Fig. 2E). The efficiency of virus capture by THP-1/CD169ΔCT4R cells was much lower than that exhibited by wt THP-1/CD169 cells (Fig. 2E), in correlation with CD169 expression level on the cell surface (Fig. 2C and D). We next co-cultured CD4+ T cells with THP-1 cells expressing CD169 CT mutants to investigate the role of CD169 CT in mediating HIV-1 trans-infection. Interestingly, THP-1/CD169ΔCT4R but not THP-1/CD169ΔCT cells could transmit HIV-1 to CD4+ T cells (Fig. 2F). Furthermore, there was no significant difference in the efficiency of trans-infection (T cell infection per amount of virus captured by THP-1 cells) mediated by THP-1/CD169 and THP-1/CD169ΔCT4R cells (Fig. 2G). Finally, CD169+ VCCs were also observed in THP-1/CD169ΔCT4R cells (Fig. 2H), suggesting that the CD169 CT sequences downstream of the four arginine residues were dispensable for the formation of VCCs and CD169-mediated HIV-1 trans-infection.


CD169-mediated trafficking of HIV to plasma membrane invaginations in dendritic cells attenuates efficacy of anti-gp120 broadly neutralizing antibodies.

Akiyama H, Ramirez NG, Gudheti MV, Gummuluru S - PLoS Pathog. (2015)

The cytoplasmic tail (CT) of CD169 is dispensable for mediating HIV-1 trans-infection.(A) Sequences of wild type and mutant CD169 CTs. The asterisks represent stop codons introduced into the ORFs of the two CT mutants. (B) Western blot analysis of THP-1 cell lysates expressing either wild type or mutant CD169. (C) Cell surface expression of CD169 on THP-1 cells was measured by flow cytometry. (D) Relative cell surface expression of CD169 CT mutants was quantified and normalized to that observed with THP-1/CD169 cells. (E) Cells were challenged with HIV-1, washed and cell-associated p24gag was measured. The data shown is the virus capture by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) normalized to that observed with THP-1/CD169 cells. (F) THP-1/CD169- or THP-1/CD169 CT mutant-mediated trans-infection was determined by measuring luciferase activity in THP—CD4+ T cell co-cultures 2 days post initiation of co-culture. The data shown is the relative virus transmission by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) to that observed with THP-1/CD169 cells. (G) Efficacy of trans-infection was calculated as trans-infection (luciferase activity) per amount of virus captured (cell-associated p24gag) and normalized to that observed with THP-1/CD169 cells (set as 100). The data shown are the means ± SEM of three (D to F) or four (G) independent experiments. (H) THP-1/CD169 or THP-1/CD169ΔCT4R cells were incubated with Gag-mCherry VLPs (red), washed, fixed and stained for CD169 (green) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. WT: THP-1/CD169, ΔCT: THP-1/CD169ΔCT, ΔCT4R: THP-1/CD169ΔCT4R and Vec: empty vector transduced THP-1.
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Related In: Results  -  Collection

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ppat.1004751.g002: The cytoplasmic tail (CT) of CD169 is dispensable for mediating HIV-1 trans-infection.(A) Sequences of wild type and mutant CD169 CTs. The asterisks represent stop codons introduced into the ORFs of the two CT mutants. (B) Western blot analysis of THP-1 cell lysates expressing either wild type or mutant CD169. (C) Cell surface expression of CD169 on THP-1 cells was measured by flow cytometry. (D) Relative cell surface expression of CD169 CT mutants was quantified and normalized to that observed with THP-1/CD169 cells. (E) Cells were challenged with HIV-1, washed and cell-associated p24gag was measured. The data shown is the virus capture by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) normalized to that observed with THP-1/CD169 cells. (F) THP-1/CD169- or THP-1/CD169 CT mutant-mediated trans-infection was determined by measuring luciferase activity in THP—CD4+ T cell co-cultures 2 days post initiation of co-culture. The data shown is the relative virus transmission by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) to that observed with THP-1/CD169 cells. (G) Efficacy of trans-infection was calculated as trans-infection (luciferase activity) per amount of virus captured (cell-associated p24gag) and normalized to that observed with THP-1/CD169 cells (set as 100). The data shown are the means ± SEM of three (D to F) or four (G) independent experiments. (H) THP-1/CD169 or THP-1/CD169ΔCT4R cells were incubated with Gag-mCherry VLPs (red), washed, fixed and stained for CD169 (green) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. WT: THP-1/CD169, ΔCT: THP-1/CD169ΔCT, ΔCT4R: THP-1/CD169ΔCT4R and Vec: empty vector transduced THP-1.
Mentions: CD169 has been reported as a phagocytic receptor on porcine macrophages that can mediate endocytosis of PRSSV [26]. However, to date, no previously defined endocytosis signaling motifs have been described in the CT of human CD169. Since CD169 was trafficked to and colocalized with HIV-1 in surface-accessible compartments in myeloid cells (Fig. 1), we postulated that there was an unidentified trafficking motif in the CT that contributed to colocalization of CD169 and HIV-1 in VCCs. Two CD169 CT truncation mutants were constructed (Fig. 2A), one of which has a stop codon right after the transmembrane domain of CD169 (CD169ΔCT) [15]. Since previous studies have demonstrated severe reduction in cell surface expression of plasma membrane targeted proteins upon deletion of cytoplasmic tails [27,28], we constructed a second CD169 CT mutant that expressed the first four amino acids of CT (CD169ΔCT4R). These CD169 CT mutants were transduced into THP-1 cells and the ability of these stably transduced cell lines expressing CD169 mutants to capture HIV and form VCCs was compared to that observed with THP-1 cells expressing wild type CD169 (THP-1/CD169) (Fig. 1B). Deletion of the cytoplasmic tail (CD169ΔCT) resulted in decreased expression of CD169 in in THP-1 cells (Fig. 2B and S1 Fig B). Furthermore, cell surface expression of CD169ΔCT was further reduced (Fig. 2C and D) and resulted in severe attenuation of HIV-1 capture (Fig. 2E). Interestingly, inclusion of the membrane proximal 4 arginine residues in the cytoplasmic tail resulted in higher expression of CD169 in cells and partial rescue of cell surface expression of CD169 (Fig. 2C, 2D and S1 Fig B), and importantly, capture of HIV-1 particles (Fig. 2E). The efficiency of virus capture by THP-1/CD169ΔCT4R cells was much lower than that exhibited by wt THP-1/CD169 cells (Fig. 2E), in correlation with CD169 expression level on the cell surface (Fig. 2C and D). We next co-cultured CD4+ T cells with THP-1 cells expressing CD169 CT mutants to investigate the role of CD169 CT in mediating HIV-1 trans-infection. Interestingly, THP-1/CD169ΔCT4R but not THP-1/CD169ΔCT cells could transmit HIV-1 to CD4+ T cells (Fig. 2F). Furthermore, there was no significant difference in the efficiency of trans-infection (T cell infection per amount of virus captured by THP-1 cells) mediated by THP-1/CD169 and THP-1/CD169ΔCT4R cells (Fig. 2G). Finally, CD169+ VCCs were also observed in THP-1/CD169ΔCT4R cells (Fig. 2H), suggesting that the CD169 CT sequences downstream of the four arginine residues were dispensable for the formation of VCCs and CD169-mediated HIV-1 trans-infection.

Bottom Line: We find HIV-1-induced formation of VCCs is restricted to myeloid cells, and that the cytoplasmic tail of CD169 is dispensable for HIV-1 trafficking and retention within VCCs and subsequent trans-infection to CD4⁺ T cells.Interestingly, introduction of a di-aromatic endocytic motif in the cytoplasmic tail of CD169 that results in endocytosis of HIV-1 particles, suppressed CD169-mediated HIV-1 trans-infection.Intriguingly, HIV-1 particles in deep VCCs were inefficiently accessed by anti-gp120 broadly neutralizing antibodies, VRC01 and NIH45-46 G54W, and thus were less susceptible to neutralization.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Myeloid dendritic cells (DCs) can capture HIV-1 via the receptor CD169/Siglec-1 that binds to the ganglioside, GM3, in the virus particle membrane. In turn, HIV-1 particles captured by CD169, an I-type lectin, whose expression on DCs is enhanced upon maturation with LPS, are protected from degradation in CD169+ virus-containing compartments (VCCs) and disseminated to CD4⁺ T cells, a mechanism of DC-mediated HIV-1 trans-infection. In this study, we describe the mechanism of VCC formation and its role in immune evasion mechanisms of HIV-1. We find HIV-1-induced formation of VCCs is restricted to myeloid cells, and that the cytoplasmic tail of CD169 is dispensable for HIV-1 trafficking and retention within VCCs and subsequent trans-infection to CD4⁺ T cells. Interestingly, introduction of a di-aromatic endocytic motif in the cytoplasmic tail of CD169 that results in endocytosis of HIV-1 particles, suppressed CD169-mediated HIV-1 trans-infection. Furthermore, super-resolution microscopy revealed close association of CD169 and HIV-1 particles in surface-accessible but deep plasma membrane invaginations. Intriguingly, HIV-1 particles in deep VCCs were inefficiently accessed by anti-gp120 broadly neutralizing antibodies, VRC01 and NIH45-46 G54W, and thus were less susceptible to neutralization. Our study suggests that HIV-1 capture by CD169 can provide virus evasion from both innate (phagocytosis) and adaptive immune responses.

Show MeSH
Related in: MedlinePlus