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Viremic long-term nonprogressive HIV-1 infection is not associated with abnormalities in known Nef functions.

Heigele A, Camerini D, van't Wout AB, Kirchhoff F - Retrovirology (2014)

Bottom Line: Finally, we found that VNP-Nefs increased virion infectivity and enhanced HIV-1 replication and cytopathicity in primary human cells and in ex vivo infected lymphoid tissues.Our results show that nef alleles from VNPs and progressors of HIV-1 infection show only modest differences in established functions.Thus, the lack of chronic immune activation and disease progression in HIV-1-infected VNPs is apparently not associated with unusual functional properties of the accessory viral Nef protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany. frank.kirchhoff@uni-ulm.de.

ABSTRACT

Background: A small minority of HIV-1-infected individuals show low levels of immune activation and do not develop immunodeficiency despite high viral loads. Since the accessory viral Nef protein modulates T cell activation and plays a key role in the pathogenesis of AIDS, we investigated whether specific properties of Nef may be associated with this highly unusual clinical outcome of HIV-1 infection.

Findings: Comprehensive functional analyses of sequential HIV-1 strains from three viremic long-term non-progressors (VNP) showed that they encode full-length Nef proteins that are capable of modulating CD4, CD28, CD8ß, MHC-I and CD74 cell surface expression. Similar to Nef proteins from HIV-1-infected individuals with progressive infection (P-Nefs) and unlike Nefs from simian immunodeficiency viruses (SIVs) that do not cause chronic immune activation and disease in their natural simian hosts, VNP-Nefs were generally unable to down-modulate TCR-CD3 cell surface expression to block T cell activation and apoptosis. On average, VNP-Nefs suppressed NF-AT activation less effectively than P-Nefs and were slightly less active in enhancing NF-κB activity. Finally, we found that VNP-Nefs increased virion infectivity and enhanced HIV-1 replication and cytopathicity in primary human cells and in ex vivo infected lymphoid tissues.

Conclusions: Our results show that nef alleles from VNPs and progressors of HIV-1 infection show only modest differences in established functions. Thus, the lack of chronic immune activation and disease progression in HIV-1-infected VNPs is apparently not associated with unusual functional properties of the accessory viral Nef protein.

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VNP-Nefs cannot prevent activation-induced apoptosis of infected cells. (A, B) Expression of (A) CD69 and (B) CD25 at one or two days, respectively, after the second PHA stimulation in PBMCs transduced with HIV-1 IRES/eGFP constructs expressing the indicated groups of Nefs. (C, D) Correlations between expression of CD25 and (C) CD69 or (D) Nef-mediated CD28 down-modulation. (E) Quantitative assessment of levels of apoptosis two days post-stimulation in HIV-1-infected PBMCs. (F-H) Correlation between apoptosis and expression of (F) CD69 and (G) CD25 or (H) CD28 down-modulation by the respective Nefs. (I) PHA-induced NF-AT-dependent luciferase activity in HIV-1 infected Jurkat cells stably transfected with an NF-AT-dependent reporter gene[9]. (J) Correlation between CD69 expression and NF-AT-dependent luciferase expression. (K) Nef-mediated activation of NF-κB in 293Ts co-transfected with a NF-κB-dependent firefly luciferase construct, a pTAL promoter gaussia luciferase construct and an expression vector expressing the various Nef proteins in the presence of TNFα. The assay was performed as described elsewhere[21]. Panels A, B, E, I and K give average values (±SD) relative to those obtained with the nef-defective control HIV-1 construct and were derived from three independent experiments. Refer to the legend to Figure 2 for abbreviations, symbols, color coding and nef alleles analyzed.
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Figure 3: VNP-Nefs cannot prevent activation-induced apoptosis of infected cells. (A, B) Expression of (A) CD69 and (B) CD25 at one or two days, respectively, after the second PHA stimulation in PBMCs transduced with HIV-1 IRES/eGFP constructs expressing the indicated groups of Nefs. (C, D) Correlations between expression of CD25 and (C) CD69 or (D) Nef-mediated CD28 down-modulation. (E) Quantitative assessment of levels of apoptosis two days post-stimulation in HIV-1-infected PBMCs. (F-H) Correlation between apoptosis and expression of (F) CD69 and (G) CD25 or (H) CD28 down-modulation by the respective Nefs. (I) PHA-induced NF-AT-dependent luciferase activity in HIV-1 infected Jurkat cells stably transfected with an NF-AT-dependent reporter gene[9]. (J) Correlation between CD69 expression and NF-AT-dependent luciferase expression. (K) Nef-mediated activation of NF-κB in 293Ts co-transfected with a NF-κB-dependent firefly luciferase construct, a pTAL promoter gaussia luciferase construct and an expression vector expressing the various Nef proteins in the presence of TNFα. The assay was performed as described elsewhere[21]. Panels A, B, E, I and K give average values (±SD) relative to those obtained with the nef-defective control HIV-1 construct and were derived from three independent experiments. Refer to the legend to Figure 2 for abbreviations, symbols, color coding and nef alleles analyzed.

Mentions: The above mentioned results demonstrated that the low levels of immune activation in VNPs were not associated with an increased activity of Nef to modulate receptors involved in the activation of T cells by APCs. It is known, however, that the HIV-1 Nef may also affect T cell activation by modulating downstream signaling pathways[19,20]. To directly examine the effects of Nef on the responsiveness of primary human cells to activation, we infected PBMCs with various HIV-1 IRES/eGFP constructs and stimulated them by PHA treatment as described previously[10]. The results showed that virally infected cells expressing VNP- and P-Nefs expressed higher surface levels of CD69 and CD25 than cells producing HIV-2 or SIV Nefs (Figure 3A, B). Expression of the early and late T cell activation markers correlated with one another (Figure 3C) and inversely with the efficiency of Nef-mediated modulation of CD28 (Figure 3D). Similarly, the levels of apoptosis were higher in HIV-1-infected PBMCs expressing VNP- and P-Nefs, compared to those expressing SIV or HIV-2 Nefs (Figure 3E) and correlated directly with the expression of T cell activation markers and inversely (albeit imperfectly) with CD28 cell surface expression (Figure 3F-H). These results are in agreement with previous studies showing that Nef-mediated down-modulation of TCR-CD3 and (to a lesser extent) CD28 suppresses the responsiveness of virally infected T cells to stimulation and activation-induced cell death[10,12,13].


Viremic long-term nonprogressive HIV-1 infection is not associated with abnormalities in known Nef functions.

Heigele A, Camerini D, van't Wout AB, Kirchhoff F - Retrovirology (2014)

VNP-Nefs cannot prevent activation-induced apoptosis of infected cells. (A, B) Expression of (A) CD69 and (B) CD25 at one or two days, respectively, after the second PHA stimulation in PBMCs transduced with HIV-1 IRES/eGFP constructs expressing the indicated groups of Nefs. (C, D) Correlations between expression of CD25 and (C) CD69 or (D) Nef-mediated CD28 down-modulation. (E) Quantitative assessment of levels of apoptosis two days post-stimulation in HIV-1-infected PBMCs. (F-H) Correlation between apoptosis and expression of (F) CD69 and (G) CD25 or (H) CD28 down-modulation by the respective Nefs. (I) PHA-induced NF-AT-dependent luciferase activity in HIV-1 infected Jurkat cells stably transfected with an NF-AT-dependent reporter gene[9]. (J) Correlation between CD69 expression and NF-AT-dependent luciferase expression. (K) Nef-mediated activation of NF-κB in 293Ts co-transfected with a NF-κB-dependent firefly luciferase construct, a pTAL promoter gaussia luciferase construct and an expression vector expressing the various Nef proteins in the presence of TNFα. The assay was performed as described elsewhere[21]. Panels A, B, E, I and K give average values (±SD) relative to those obtained with the nef-defective control HIV-1 construct and were derived from three independent experiments. Refer to the legend to Figure 2 for abbreviations, symbols, color coding and nef alleles analyzed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: VNP-Nefs cannot prevent activation-induced apoptosis of infected cells. (A, B) Expression of (A) CD69 and (B) CD25 at one or two days, respectively, after the second PHA stimulation in PBMCs transduced with HIV-1 IRES/eGFP constructs expressing the indicated groups of Nefs. (C, D) Correlations between expression of CD25 and (C) CD69 or (D) Nef-mediated CD28 down-modulation. (E) Quantitative assessment of levels of apoptosis two days post-stimulation in HIV-1-infected PBMCs. (F-H) Correlation between apoptosis and expression of (F) CD69 and (G) CD25 or (H) CD28 down-modulation by the respective Nefs. (I) PHA-induced NF-AT-dependent luciferase activity in HIV-1 infected Jurkat cells stably transfected with an NF-AT-dependent reporter gene[9]. (J) Correlation between CD69 expression and NF-AT-dependent luciferase expression. (K) Nef-mediated activation of NF-κB in 293Ts co-transfected with a NF-κB-dependent firefly luciferase construct, a pTAL promoter gaussia luciferase construct and an expression vector expressing the various Nef proteins in the presence of TNFα. The assay was performed as described elsewhere[21]. Panels A, B, E, I and K give average values (±SD) relative to those obtained with the nef-defective control HIV-1 construct and were derived from three independent experiments. Refer to the legend to Figure 2 for abbreviations, symbols, color coding and nef alleles analyzed.
Mentions: The above mentioned results demonstrated that the low levels of immune activation in VNPs were not associated with an increased activity of Nef to modulate receptors involved in the activation of T cells by APCs. It is known, however, that the HIV-1 Nef may also affect T cell activation by modulating downstream signaling pathways[19,20]. To directly examine the effects of Nef on the responsiveness of primary human cells to activation, we infected PBMCs with various HIV-1 IRES/eGFP constructs and stimulated them by PHA treatment as described previously[10]. The results showed that virally infected cells expressing VNP- and P-Nefs expressed higher surface levels of CD69 and CD25 than cells producing HIV-2 or SIV Nefs (Figure 3A, B). Expression of the early and late T cell activation markers correlated with one another (Figure 3C) and inversely with the efficiency of Nef-mediated modulation of CD28 (Figure 3D). Similarly, the levels of apoptosis were higher in HIV-1-infected PBMCs expressing VNP- and P-Nefs, compared to those expressing SIV or HIV-2 Nefs (Figure 3E) and correlated directly with the expression of T cell activation markers and inversely (albeit imperfectly) with CD28 cell surface expression (Figure 3F-H). These results are in agreement with previous studies showing that Nef-mediated down-modulation of TCR-CD3 and (to a lesser extent) CD28 suppresses the responsiveness of virally infected T cells to stimulation and activation-induced cell death[10,12,13].

Bottom Line: Finally, we found that VNP-Nefs increased virion infectivity and enhanced HIV-1 replication and cytopathicity in primary human cells and in ex vivo infected lymphoid tissues.Our results show that nef alleles from VNPs and progressors of HIV-1 infection show only modest differences in established functions.Thus, the lack of chronic immune activation and disease progression in HIV-1-infected VNPs is apparently not associated with unusual functional properties of the accessory viral Nef protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany. frank.kirchhoff@uni-ulm.de.

ABSTRACT

Background: A small minority of HIV-1-infected individuals show low levels of immune activation and do not develop immunodeficiency despite high viral loads. Since the accessory viral Nef protein modulates T cell activation and plays a key role in the pathogenesis of AIDS, we investigated whether specific properties of Nef may be associated with this highly unusual clinical outcome of HIV-1 infection.

Findings: Comprehensive functional analyses of sequential HIV-1 strains from three viremic long-term non-progressors (VNP) showed that they encode full-length Nef proteins that are capable of modulating CD4, CD28, CD8ß, MHC-I and CD74 cell surface expression. Similar to Nef proteins from HIV-1-infected individuals with progressive infection (P-Nefs) and unlike Nefs from simian immunodeficiency viruses (SIVs) that do not cause chronic immune activation and disease in their natural simian hosts, VNP-Nefs were generally unable to down-modulate TCR-CD3 cell surface expression to block T cell activation and apoptosis. On average, VNP-Nefs suppressed NF-AT activation less effectively than P-Nefs and were slightly less active in enhancing NF-κB activity. Finally, we found that VNP-Nefs increased virion infectivity and enhanced HIV-1 replication and cytopathicity in primary human cells and in ex vivo infected lymphoid tissues.

Conclusions: Our results show that nef alleles from VNPs and progressors of HIV-1 infection show only modest differences in established functions. Thus, the lack of chronic immune activation and disease progression in HIV-1-infected VNPs is apparently not associated with unusual functional properties of the accessory viral Nef protein.

Show MeSH
Related in: MedlinePlus