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Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+ thymocytes.

Zou W, Denton PW, Watkins RL, Krisko JF, Nochi T, Foster JL, Garcia JV - Retrovirology (2012)

Bottom Line: Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared <1% for uninfected mice.We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing.This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infectious Diseases, University of North Carolina, Chapel Hill, NC 27599-7042, USA.

ABSTRACT

Background: The outcome of untreated HIV-1 infection is progression to AIDS and death in nearly all cases. Some important exceptions are the small number of patients infected with HIV-1 deleted for the accessory gene, nef. With these infections, disease progression is entirely suppressed or greatly delayed. Whether Nef is critical for high levels of replication or is directly cytotoxic remains controversial. The major problem in determining the role of Nef in HIV/AIDS has been the lack of tractable in vivo models where Nef's complex pathogenic phenotype can be recapitulated.

Results: Intravenous inoculation (3000 to 600,000 TCIU) of BLT humanized mice with HIV-1LAI reproducibly establishes a systemic infection. HIV-1LAI (LAI) replicates to high levels (peak viral load in blood 8,200,000 ± 1,800,000 copies of viral RNA/ml, range 3,600,000 to 20,400,000; n = 9) and exhaustively depletes CD4+ T cells in blood and tissues. CD4+CD8+ thymocytes were also efficiently depleted but CD4+CD8- thymocytes were partially resistant to cell killing by LAI. Infection with a nef-deleted LAI (LAINefdd) gave lower peak viral loads (1,220,000 ± 330,000, range 27,000 to 4,240,000; n = 17). For fourteen of seventeen LAINefdd-infected mice, there was little to no loss of either CD4+ T cells or thymocytes. Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared <1% for uninfected mice. Three exceptional LAINefdd-infected mice that lost CD4+ T cells received 600,000 TCIU. All three exhibited peak viral loads over 3,000,000 copies of LAINefdd RNA/ml. Over an extended time course, substantial systemic CD4+ T cell loss was observed for the three mice, but there was no loss of CD4+CD8+ or CD4+CD8- thymocytes.

Conclusion: We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing. Further, Nef was not necessary for the activation of peripheral blood CD8+ T cells following infection. However, CD4+CD8+ thymocyte killing was dependent on Nef even in cases of elevated LAINefdd replication and T cell loss. This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.

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Analysis of BLT humanized mice inoculated with a low dose ofnef(−) or wild-type LAI. (A) Each line depicts longitudinal plasma viral load data from individual BLT humanized mice infected with 3000 TCIU of LAINefdd (closed symbols) or LAI (open symbols). These data demonstrate delayed replication of LAINefdd relative to LAI following low dose inoculation. (B) Each line depicts the percentage of CD4+ T cells in peripheral blood over time where each animal’s symbol is matched to the mice in (A). Mice infected with a low dose of LAINefdd showed minimal changes in CD4+ T cell percentages when compared to BLT humanized mice inoculated with an equal dose of LAI. (C) Naïve BLT humanized mice (n = 5 in PB, spleen and HTO or n = 4 in LN, BM, lung and liver) and BLT humanized mice inoculated with 3000 TCIU of LAINefdd (n = 4) exhibited similar levels of CD4+ cells while mice inoculated with the same dose of LAI (n = 3) exhibited a drastic reduction in these cells. Shown are the percentages of human CD4+ T cells present in peripheral blood, lymph nodes, spleen, bone marrow, lung and liver, as well as the percentages of CD4+CD8- and CD4+CD8+ thymocytes in the human thymic organoid. The percent of CD4+ T cells in peripheral blood or tissues was relative to total CD3+ T cells while the percent of CD4+CD8- and CD4+CD8+ thymocytes was relative to total thymocytes. One-way ANOVA with three Bonferroni multiple comparisons tests was performed to compare the results within each tissue. If no difference was detected, the comparison is unmarked (alpha = 0.05). Comparisons yielding significant differences are represented by a line connecting the arrows above the respective bars (**p < 0.01).
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Figure 3: Analysis of BLT humanized mice inoculated with a low dose ofnef(−) or wild-type LAI. (A) Each line depicts longitudinal plasma viral load data from individual BLT humanized mice infected with 3000 TCIU of LAINefdd (closed symbols) or LAI (open symbols). These data demonstrate delayed replication of LAINefdd relative to LAI following low dose inoculation. (B) Each line depicts the percentage of CD4+ T cells in peripheral blood over time where each animal’s symbol is matched to the mice in (A). Mice infected with a low dose of LAINefdd showed minimal changes in CD4+ T cell percentages when compared to BLT humanized mice inoculated with an equal dose of LAI. (C) Naïve BLT humanized mice (n = 5 in PB, spleen and HTO or n = 4 in LN, BM, lung and liver) and BLT humanized mice inoculated with 3000 TCIU of LAINefdd (n = 4) exhibited similar levels of CD4+ cells while mice inoculated with the same dose of LAI (n = 3) exhibited a drastic reduction in these cells. Shown are the percentages of human CD4+ T cells present in peripheral blood, lymph nodes, spleen, bone marrow, lung and liver, as well as the percentages of CD4+CD8- and CD4+CD8+ thymocytes in the human thymic organoid. The percent of CD4+ T cells in peripheral blood or tissues was relative to total CD3+ T cells while the percent of CD4+CD8- and CD4+CD8+ thymocytes was relative to total thymocytes. One-way ANOVA with three Bonferroni multiple comparisons tests was performed to compare the results within each tissue. If no difference was detected, the comparison is unmarked (alpha = 0.05). Comparisons yielding significant differences are represented by a line connecting the arrows above the respective bars (**p < 0.01).

Mentions: Four mice inoculated with 3000 TCIU of LAINefdd (0.56 ng p24gag) became systemically infected. The appearance of virus in peripheral blood was greatly delayed from the 7–14 days seen for wild-type LAI-infected mice (Figure 3A, LAI-1,-2 and −3) to 30–55 days for the four LAINefdd infected mice (p = 0.014, log rank test). These results demonstrate a generally reduced level of replication by the nef-deleted virus. However, in two of four mice (LAINefdd-2 and LAINefdd-4) viral loads did reach 106 copies of viral RNA/ml of plasma demonstrating the in vivo fitness of nef-defective virus (Figure 3A). The other two mice (LAINefdd-1 and LAINefdd-3) had depressed viral replication with viral loads clearly under 106 after sixty days. In contrast to the mice infected with wild-type LAI, the LAINefdd infected mice did not show significant depletion of their circulating CD4+ T cells (Figure 3B). Even the two mice whose viral loads reached 106 copies/ml maintained high levels of CD4+ T cells in peripheral blood (Figure 3B). Therefore, infection with a low dose of nef-deleted virus results in delayed replication and most strikingly a minimal capacity to induce peripheral CD4+ T cell depletion.


Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+ thymocytes.

Zou W, Denton PW, Watkins RL, Krisko JF, Nochi T, Foster JL, Garcia JV - Retrovirology (2012)

Analysis of BLT humanized mice inoculated with a low dose ofnef(−) or wild-type LAI. (A) Each line depicts longitudinal plasma viral load data from individual BLT humanized mice infected with 3000 TCIU of LAINefdd (closed symbols) or LAI (open symbols). These data demonstrate delayed replication of LAINefdd relative to LAI following low dose inoculation. (B) Each line depicts the percentage of CD4+ T cells in peripheral blood over time where each animal’s symbol is matched to the mice in (A). Mice infected with a low dose of LAINefdd showed minimal changes in CD4+ T cell percentages when compared to BLT humanized mice inoculated with an equal dose of LAI. (C) Naïve BLT humanized mice (n = 5 in PB, spleen and HTO or n = 4 in LN, BM, lung and liver) and BLT humanized mice inoculated with 3000 TCIU of LAINefdd (n = 4) exhibited similar levels of CD4+ cells while mice inoculated with the same dose of LAI (n = 3) exhibited a drastic reduction in these cells. Shown are the percentages of human CD4+ T cells present in peripheral blood, lymph nodes, spleen, bone marrow, lung and liver, as well as the percentages of CD4+CD8- and CD4+CD8+ thymocytes in the human thymic organoid. The percent of CD4+ T cells in peripheral blood or tissues was relative to total CD3+ T cells while the percent of CD4+CD8- and CD4+CD8+ thymocytes was relative to total thymocytes. One-way ANOVA with three Bonferroni multiple comparisons tests was performed to compare the results within each tissue. If no difference was detected, the comparison is unmarked (alpha = 0.05). Comparisons yielding significant differences are represented by a line connecting the arrows above the respective bars (**p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Analysis of BLT humanized mice inoculated with a low dose ofnef(−) or wild-type LAI. (A) Each line depicts longitudinal plasma viral load data from individual BLT humanized mice infected with 3000 TCIU of LAINefdd (closed symbols) or LAI (open symbols). These data demonstrate delayed replication of LAINefdd relative to LAI following low dose inoculation. (B) Each line depicts the percentage of CD4+ T cells in peripheral blood over time where each animal’s symbol is matched to the mice in (A). Mice infected with a low dose of LAINefdd showed minimal changes in CD4+ T cell percentages when compared to BLT humanized mice inoculated with an equal dose of LAI. (C) Naïve BLT humanized mice (n = 5 in PB, spleen and HTO or n = 4 in LN, BM, lung and liver) and BLT humanized mice inoculated with 3000 TCIU of LAINefdd (n = 4) exhibited similar levels of CD4+ cells while mice inoculated with the same dose of LAI (n = 3) exhibited a drastic reduction in these cells. Shown are the percentages of human CD4+ T cells present in peripheral blood, lymph nodes, spleen, bone marrow, lung and liver, as well as the percentages of CD4+CD8- and CD4+CD8+ thymocytes in the human thymic organoid. The percent of CD4+ T cells in peripheral blood or tissues was relative to total CD3+ T cells while the percent of CD4+CD8- and CD4+CD8+ thymocytes was relative to total thymocytes. One-way ANOVA with three Bonferroni multiple comparisons tests was performed to compare the results within each tissue. If no difference was detected, the comparison is unmarked (alpha = 0.05). Comparisons yielding significant differences are represented by a line connecting the arrows above the respective bars (**p < 0.01).
Mentions: Four mice inoculated with 3000 TCIU of LAINefdd (0.56 ng p24gag) became systemically infected. The appearance of virus in peripheral blood was greatly delayed from the 7–14 days seen for wild-type LAI-infected mice (Figure 3A, LAI-1,-2 and −3) to 30–55 days for the four LAINefdd infected mice (p = 0.014, log rank test). These results demonstrate a generally reduced level of replication by the nef-deleted virus. However, in two of four mice (LAINefdd-2 and LAINefdd-4) viral loads did reach 106 copies of viral RNA/ml of plasma demonstrating the in vivo fitness of nef-defective virus (Figure 3A). The other two mice (LAINefdd-1 and LAINefdd-3) had depressed viral replication with viral loads clearly under 106 after sixty days. In contrast to the mice infected with wild-type LAI, the LAINefdd infected mice did not show significant depletion of their circulating CD4+ T cells (Figure 3B). Even the two mice whose viral loads reached 106 copies/ml maintained high levels of CD4+ T cells in peripheral blood (Figure 3B). Therefore, infection with a low dose of nef-deleted virus results in delayed replication and most strikingly a minimal capacity to induce peripheral CD4+ T cell depletion.

Bottom Line: Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared <1% for uninfected mice.We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing.This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infectious Diseases, University of North Carolina, Chapel Hill, NC 27599-7042, USA.

ABSTRACT

Background: The outcome of untreated HIV-1 infection is progression to AIDS and death in nearly all cases. Some important exceptions are the small number of patients infected with HIV-1 deleted for the accessory gene, nef. With these infections, disease progression is entirely suppressed or greatly delayed. Whether Nef is critical for high levels of replication or is directly cytotoxic remains controversial. The major problem in determining the role of Nef in HIV/AIDS has been the lack of tractable in vivo models where Nef's complex pathogenic phenotype can be recapitulated.

Results: Intravenous inoculation (3000 to 600,000 TCIU) of BLT humanized mice with HIV-1LAI reproducibly establishes a systemic infection. HIV-1LAI (LAI) replicates to high levels (peak viral load in blood 8,200,000 ± 1,800,000 copies of viral RNA/ml, range 3,600,000 to 20,400,000; n = 9) and exhaustively depletes CD4+ T cells in blood and tissues. CD4+CD8+ thymocytes were also efficiently depleted but CD4+CD8- thymocytes were partially resistant to cell killing by LAI. Infection with a nef-deleted LAI (LAINefdd) gave lower peak viral loads (1,220,000 ± 330,000, range 27,000 to 4,240,000; n = 17). For fourteen of seventeen LAINefdd-infected mice, there was little to no loss of either CD4+ T cells or thymocytes. Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared <1% for uninfected mice. Three exceptional LAINefdd-infected mice that lost CD4+ T cells received 600,000 TCIU. All three exhibited peak viral loads over 3,000,000 copies of LAINefdd RNA/ml. Over an extended time course, substantial systemic CD4+ T cell loss was observed for the three mice, but there was no loss of CD4+CD8+ or CD4+CD8- thymocytes.

Conclusion: We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing. Further, Nef was not necessary for the activation of peripheral blood CD8+ T cells following infection. However, CD4+CD8+ thymocyte killing was dependent on Nef even in cases of elevated LAINefdd replication and T cell loss. This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.

Show MeSH
Related in: MedlinePlus