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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 enhance virion infectivity and viral replication. (A) P4-CCR5 (left) and TZM-bl (right) cells were infected with recombinant HIV-1 IRES-EGFP constructs expressing the indicated groups of nef alleles. Infections were performed in triplicate with two independent virus stocks. All panels give average values ± SD. RLU, relative light units. (B) Average levels of p24 antigen and (C) cumulative p24 production by PBMCs over 11 days of culture. PBMCs were either stimulated with PHA (1 μg/ml) for 3 days prior to infection with the X4-tropic HIV-1 NL4-3 clone or an R5-tropic derivative thereof[28] or infected immediately after isolation and stimulated three days later. Supernatants were collected at 2- or 3-day intervals, and productive HIV-1 infection was assessed by measuring p24 antigen content. The values were derived from three independent experiments. (D) Cumulative virus production in human lymphoid tissue (HLT) infected ex vivo. (E) Depletion of CD4+ T cells in HLT infected ex vivo. Tissues from six donors were infected with X4 HIV-1 NL4-3 expressing the indicated nef alleles, and cumulative p24 production by the tissue blocks over 15 days or CD4+ T-cell depletion at the end of culture was determined as described previously[25,27]. Refer to the legend to Figure 2 for abbreviations, symbols, color coding and nef alleles analyzed.
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Figure 4: VNP Nefs enhance virion infectivity and viral replication. (A) P4-CCR5 (left) and TZM-bl (right) cells were infected with recombinant HIV-1 IRES-EGFP constructs expressing the indicated groups of nef alleles. Infections were performed in triplicate with two independent virus stocks. All panels give average values ± SD. RLU, relative light units. (B) Average levels of p24 antigen and (C) cumulative p24 production by PBMCs over 11 days of culture. PBMCs were either stimulated with PHA (1 μg/ml) for 3 days prior to infection with the X4-tropic HIV-1 NL4-3 clone or an R5-tropic derivative thereof[28] or infected immediately after isolation and stimulated three days later. Supernatants were collected at 2- or 3-day intervals, and productive HIV-1 infection was assessed by measuring p24 antigen content. The values were derived from three independent experiments. (D) Cumulative virus production in human lymphoid tissue (HLT) infected ex vivo. (E) Depletion of CD4+ T cells in HLT infected ex vivo. Tissues from six donors were infected with X4 HIV-1 NL4-3 expressing the indicated nef alleles, and cumulative p24 production by the tissue blocks over 15 days or CD4+ T-cell depletion at the end of culture was determined as described previously[25,27]. Refer to the legend to Figure 2 for abbreviations, symbols, color coding and nef alleles analyzed.

Mentions: Finally, we examined the ability of VNP-Nefs to enhance HIV-1 infection, replication and cytopathicity. Infection of P4-CCR5 and TZM-bl indicator cells[24] with virus stocks containing normalized quantities of p24 antigen derived from 293T cells transiently transfected with the different proviral constructs[24], showed that most VNP- and P-Nefs enhanced virion infectivity, albeit with variable efficacy (Figure 4A, Additional file1: Figure S3). In agreement with published data[25] the magnitude of the Nef effect on HIV-1 infection was much higher in P4-CCR5 cells than in TZM-bl cells that are highly susceptible to infection. To determine the effect of Nef on HIV-1 replication in primary human cells, PBMCs were infected with HIV-1 constructs and virus production in the supernatant was determined as described previously[26]. In agreement with the high viral loads, VNP-Nefs increased virus production (Figure 4B), irrespectively of the viral coreceptor tropism, and of whether or not the PBMCs were pre-stimulated or stimulated 3 days after infection (Figure 4C). Since PBMC cultures may not faithfully recapitulate the viral phenotype in vivo, we also determined the levels of viral replication and CD4+ T cell depletion in ex vivo human tonsillary tissues that allow determination of the cytopathicity and replication capacity of HIV-1 without exogenous stimulation[27]. Since the number of cultures that could be set up per tonsil was limited, we only compared the effect of the VNP-Nefs with the HIV-1 control Nefs. We found that HIV-1 constructs expressing VNP-Nefs replicated efficiently in human lymphoid tissue (HLT) and depleted CD4+ T cells as effectively as otherwise isogenic viral strains expressing the control NL4-3 and NA7 Nefs (Figure 4D, E). These results are in agreement with the previous finding that biologically cloned HIV-1 isolates from these three VNPs are cytopathic and replication competent in ex vivo infected human lymphoid tissue[3].


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 enhance virion infectivity and viral replication. (A) P4-CCR5 (left) and TZM-bl (right) cells were infected with recombinant HIV-1 IRES-EGFP constructs expressing the indicated groups of nef alleles. Infections were performed in triplicate with two independent virus stocks. All panels give average values ± SD. RLU, relative light units. (B) Average levels of p24 antigen and (C) cumulative p24 production by PBMCs over 11 days of culture. PBMCs were either stimulated with PHA (1 μg/ml) for 3 days prior to infection with the X4-tropic HIV-1 NL4-3 clone or an R5-tropic derivative thereof[28] or infected immediately after isolation and stimulated three days later. Supernatants were collected at 2- or 3-day intervals, and productive HIV-1 infection was assessed by measuring p24 antigen content. The values were derived from three independent experiments. (D) Cumulative virus production in human lymphoid tissue (HLT) infected ex vivo. (E) Depletion of CD4+ T cells in HLT infected ex vivo. Tissues from six donors were infected with X4 HIV-1 NL4-3 expressing the indicated nef alleles, and cumulative p24 production by the tissue blocks over 15 days or CD4+ T-cell depletion at the end of culture was determined as described previously[25,27]. 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 4: VNP Nefs enhance virion infectivity and viral replication. (A) P4-CCR5 (left) and TZM-bl (right) cells were infected with recombinant HIV-1 IRES-EGFP constructs expressing the indicated groups of nef alleles. Infections were performed in triplicate with two independent virus stocks. All panels give average values ± SD. RLU, relative light units. (B) Average levels of p24 antigen and (C) cumulative p24 production by PBMCs over 11 days of culture. PBMCs were either stimulated with PHA (1 μg/ml) for 3 days prior to infection with the X4-tropic HIV-1 NL4-3 clone or an R5-tropic derivative thereof[28] or infected immediately after isolation and stimulated three days later. Supernatants were collected at 2- or 3-day intervals, and productive HIV-1 infection was assessed by measuring p24 antigen content. The values were derived from three independent experiments. (D) Cumulative virus production in human lymphoid tissue (HLT) infected ex vivo. (E) Depletion of CD4+ T cells in HLT infected ex vivo. Tissues from six donors were infected with X4 HIV-1 NL4-3 expressing the indicated nef alleles, and cumulative p24 production by the tissue blocks over 15 days or CD4+ T-cell depletion at the end of culture was determined as described previously[25,27]. Refer to the legend to Figure 2 for abbreviations, symbols, color coding and nef alleles analyzed.
Mentions: Finally, we examined the ability of VNP-Nefs to enhance HIV-1 infection, replication and cytopathicity. Infection of P4-CCR5 and TZM-bl indicator cells[24] with virus stocks containing normalized quantities of p24 antigen derived from 293T cells transiently transfected with the different proviral constructs[24], showed that most VNP- and P-Nefs enhanced virion infectivity, albeit with variable efficacy (Figure 4A, Additional file1: Figure S3). In agreement with published data[25] the magnitude of the Nef effect on HIV-1 infection was much higher in P4-CCR5 cells than in TZM-bl cells that are highly susceptible to infection. To determine the effect of Nef on HIV-1 replication in primary human cells, PBMCs were infected with HIV-1 constructs and virus production in the supernatant was determined as described previously[26]. In agreement with the high viral loads, VNP-Nefs increased virus production (Figure 4B), irrespectively of the viral coreceptor tropism, and of whether or not the PBMCs were pre-stimulated or stimulated 3 days after infection (Figure 4C). Since PBMC cultures may not faithfully recapitulate the viral phenotype in vivo, we also determined the levels of viral replication and CD4+ T cell depletion in ex vivo human tonsillary tissues that allow determination of the cytopathicity and replication capacity of HIV-1 without exogenous stimulation[27]. Since the number of cultures that could be set up per tonsil was limited, we only compared the effect of the VNP-Nefs with the HIV-1 control Nefs. We found that HIV-1 constructs expressing VNP-Nefs replicated efficiently in human lymphoid tissue (HLT) and depleted CD4+ T cells as effectively as otherwise isogenic viral strains expressing the control NL4-3 and NA7 Nefs (Figure 4D, E). These results are in agreement with the previous finding that biologically cloned HIV-1 isolates from these three VNPs are cytopathic and replication competent in ex vivo infected human lymphoid tissue[3].

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