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Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection.

Stantchev TS, Paciga M, Lankford CR, Schwartzkopff F, Broder CC, Clouse KA - Retrovirology (2012)

Bottom Line: Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM.However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells.They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cell Biology, Division of Monoclonal Antibodies, U.S. Food and Drug Administration, Bethesda, MD 20892, USA.

ABSTRACT

Background: The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process.

Results: We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells.

Conclusions: Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies. More importantly, from an in vivo perspective, the preferential utilization of PDI may be relevant to the HIV-1 entry and establishment of virus reservoirs in resting CD4+ cells, while the elevated levels of Trx reported in the chronic stages of HIV-1 infection may facilitate the virus entry in macrophages and help to sustain high viremia during the decline of T lymphocytes.

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Cell surface expression of PDI and/or Thioredoxin. PM-1 T cells (panel A), primary human MDM (panel B) and PBLs (panel C) were isolated and cultured as described in Methods. Subsequently, the cells were fixed with 1.6% paraformaldehyde and treated with NH4Cl to quench the reactive aldehyde groups before labeling. The PDI and Trx levels on the cell surface were detected by a combination of directly labeled anti-PDI-FITC and anti-Trx-APC mAbs from the same clones used in the HIV-1 infection experiments. In addition, the unstimulated and PHA-P stimulated PBLs were labeled with anti-CD4-Pacific blue conjugated mAb, and the PDI and Trx expression were analyzed on the CD4+ lymphocyte (Ly) population. The results were analyzed and the final graphs were created using FlowJoe software.
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Figure 6: Cell surface expression of PDI and/or Thioredoxin. PM-1 T cells (panel A), primary human MDM (panel B) and PBLs (panel C) were isolated and cultured as described in Methods. Subsequently, the cells were fixed with 1.6% paraformaldehyde and treated with NH4Cl to quench the reactive aldehyde groups before labeling. The PDI and Trx levels on the cell surface were detected by a combination of directly labeled anti-PDI-FITC and anti-Trx-APC mAbs from the same clones used in the HIV-1 infection experiments. In addition, the unstimulated and PHA-P stimulated PBLs were labeled with anti-CD4-Pacific blue conjugated mAb, and the PDI and Trx expression were analyzed on the CD4+ lymphocyte (Ly) population. The results were analyzed and the final graphs were created using FlowJoe software.

Mentions: In an attempt to delineate the basis for the observed cell-type specific requirements of the thiol/disulfide exchange during virus entry, cell surface expression of PDI and Trx was assessed in the various cell types used for infection. The same mAb clones used to inhibit HIV-1 infection were directly conjugated to different fluorescent dyes (anti-PDI- DyLight 488 and anti-Trx-APC), and used for Flow Cytometry analysis. Although PDI and Trx are predominantly found as intracellular proteins, it is well-documented that these molecules can also be secreted and associate with the plasma membrane[34-36]. Given that cell surface PDI and Trx molecules are peripheral plasma membrane proteins, the cells prepared for Flow Cytometry analysis of cell surface PDI and Trx were washed with DPBS, fixed with 1.6% paraformaldehyde prior to labeling to prevent dissociation from the cell surface and then treated with NH4Cl to quench the reactive aldehyde groups before incubation with the specific mAbs as described in Methods. In addition to the anti-PDI and anti-Trx mAbs, PBLs were also incubated with a specific anti-CD4 mAb, directly labeled with the fluorescent marker, Pacific blue, and only those cells that stained positive for CD4 were further analyzed for their PDI and Trx cell surface levels. We found that PM-1 cells and primary MDM expressed both PDI and Trx on their surface (Figure6A,B). Intriguingly, in contrast to PDI which was present to comparable levels on both unstimulated and PHA-activated CD4+ T cells, Trx expression was very low on unstimulated CD4+ lymphocytes, but increased significantly after PHA treatment (Figure6C). Taken together our findings strongly suggest that the cell type specific effects of anti-PDI and anti-Trx mAbs observed in PM-1 cells, primary MDM, and PHA activated PBLs cannot be attributed to the absence of any enzyme on the plasma membrane of the various cells that have been tested.


Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection.

Stantchev TS, Paciga M, Lankford CR, Schwartzkopff F, Broder CC, Clouse KA - Retrovirology (2012)

Cell surface expression of PDI and/or Thioredoxin. PM-1 T cells (panel A), primary human MDM (panel B) and PBLs (panel C) were isolated and cultured as described in Methods. Subsequently, the cells were fixed with 1.6% paraformaldehyde and treated with NH4Cl to quench the reactive aldehyde groups before labeling. The PDI and Trx levels on the cell surface were detected by a combination of directly labeled anti-PDI-FITC and anti-Trx-APC mAbs from the same clones used in the HIV-1 infection experiments. In addition, the unstimulated and PHA-P stimulated PBLs were labeled with anti-CD4-Pacific blue conjugated mAb, and the PDI and Trx expression were analyzed on the CD4+ lymphocyte (Ly) population. The results were analyzed and the final graphs were created using FlowJoe software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526565&req=5

Figure 6: Cell surface expression of PDI and/or Thioredoxin. PM-1 T cells (panel A), primary human MDM (panel B) and PBLs (panel C) were isolated and cultured as described in Methods. Subsequently, the cells were fixed with 1.6% paraformaldehyde and treated with NH4Cl to quench the reactive aldehyde groups before labeling. The PDI and Trx levels on the cell surface were detected by a combination of directly labeled anti-PDI-FITC and anti-Trx-APC mAbs from the same clones used in the HIV-1 infection experiments. In addition, the unstimulated and PHA-P stimulated PBLs were labeled with anti-CD4-Pacific blue conjugated mAb, and the PDI and Trx expression were analyzed on the CD4+ lymphocyte (Ly) population. The results were analyzed and the final graphs were created using FlowJoe software.
Mentions: In an attempt to delineate the basis for the observed cell-type specific requirements of the thiol/disulfide exchange during virus entry, cell surface expression of PDI and Trx was assessed in the various cell types used for infection. The same mAb clones used to inhibit HIV-1 infection were directly conjugated to different fluorescent dyes (anti-PDI- DyLight 488 and anti-Trx-APC), and used for Flow Cytometry analysis. Although PDI and Trx are predominantly found as intracellular proteins, it is well-documented that these molecules can also be secreted and associate with the plasma membrane[34-36]. Given that cell surface PDI and Trx molecules are peripheral plasma membrane proteins, the cells prepared for Flow Cytometry analysis of cell surface PDI and Trx were washed with DPBS, fixed with 1.6% paraformaldehyde prior to labeling to prevent dissociation from the cell surface and then treated with NH4Cl to quench the reactive aldehyde groups before incubation with the specific mAbs as described in Methods. In addition to the anti-PDI and anti-Trx mAbs, PBLs were also incubated with a specific anti-CD4 mAb, directly labeled with the fluorescent marker, Pacific blue, and only those cells that stained positive for CD4 were further analyzed for their PDI and Trx cell surface levels. We found that PM-1 cells and primary MDM expressed both PDI and Trx on their surface (Figure6A,B). Intriguingly, in contrast to PDI which was present to comparable levels on both unstimulated and PHA-activated CD4+ T cells, Trx expression was very low on unstimulated CD4+ lymphocytes, but increased significantly after PHA treatment (Figure6C). Taken together our findings strongly suggest that the cell type specific effects of anti-PDI and anti-Trx mAbs observed in PM-1 cells, primary MDM, and PHA activated PBLs cannot be attributed to the absence of any enzyme on the plasma membrane of the various cells that have been tested.

Bottom Line: Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM.However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells.They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cell Biology, Division of Monoclonal Antibodies, U.S. Food and Drug Administration, Bethesda, MD 20892, USA.

ABSTRACT

Background: The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process.

Results: We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells.

Conclusions: Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies. More importantly, from an in vivo perspective, the preferential utilization of PDI may be relevant to the HIV-1 entry and establishment of virus reservoirs in resting CD4+ cells, while the elevated levels of Trx reported in the chronic stages of HIV-1 infection may facilitate the virus entry in macrophages and help to sustain high viremia during the decline of T lymphocytes.

Show MeSH
Related in: MedlinePlus