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

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.

Figure 1

BLT humanized mice exhibited a profound loss of human CD4⁺T cells/thymocytes following HIV-1_LAIinfection. (A-B) Three cohorts of BLT humanized mice were exposed intravenously to increasing doses of HIV-1_LAI (LAI). LAI inoculations included a low dose of 3000 TCIU (triangles), an intermediate dose of 30,000 TCIU (circles), and a high dose of 600,000 TCIU (diamonds). A fourth cohort consisted of naïve BLT humanized mice that were not exposed to virus (squares). Longitudinal analysis of plasma viral load revealed efficient viral replication in each of the three groups of LAI infected mice regardless of the inoculation dose, and no virus replication was observed in the naïve animals (A). In the absence of infection in naïve BLT humanized mice, the percentage of human CD4⁺ T cells in the peripheral blood remains constant. In contrast, LAI infected mice showed an inoculation dose dependent difference in the kinetics of CD4⁺ T cell loss. Asterisks represent the absence of detectable levels of peripheral blood human CD4⁺ T cells in the BLT humanized mice infected with 600,000 TCIU of LAI at the time point indicated (B). (C) Regardless of the inoculation dose, systemic loss of CD4⁺ T cells was observed in infected BLT humanized mice when compared to naïve animals. Shown are the percentages of human CD4⁺ T cells present in peripheral blood, lymph nodes, and spleen, 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. “—” indicates naïve mice (n = 5 in PB, spleen and HTO or n = 4 in LN). Triangles indicate the increasing doses: 3000 TCIU (n = 3), 30,000 TCIU (n = 3), 600,000 TCIU (n = 3). Unpaired two-tailed t tests were performed to compare the naïve mice to the mice in each LAI infection dose group within the same 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 < 0.05; **p < 0.01).

Figure 2

Schematic of WT LAI and LAINefdd. Three bars represent nef coding sequence and the U3 sequence for LAI, LAINefdd and patient SG1 clone 27 sequence [35]. SG1 clone 27 reflects the tendency of patients with defective nefs to lose all remaining coding sequence [5,7,35,36]. The coding sequence of WT type LAI nef (NCBI Accession, K02013) is presented in the top bar (nucleotides 8390–9010). PPT, polypurine tract is nucleotides 8662–8678. U3 consists of nucleotides 8678 to 9132. The core U3 promoter elements include two NF-κB binding sites, three SP1 sites and the TFIID binding site or TATA box. In the lower bar the 5’ deletion incorporated into LAINefdd is 114 nucleotides (8495–8608). Within the deletion, a four base insertion was added to create a frame shift (See Methods). The 3′ gap in LAINefdd is a frame-shifting deletion of 290 bases (8692–8981). In the middle bar the deletions in SG1 isolate 27 are aligned with LAI. The gaps are not numbered for simplicity of presentation. The 5′ deletion in SG1 is 217 nucleotides and the 3′ deletion is 292 nucleotides.

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).

Figure 4

Analysis of BLT humanized mice inoculated with an intermediate dose ofnef(−) or wild-type LAI. (A) Each line depicts longitudinal plasma viral load data from individual BLT humanized mice infected with 30,000 TCIU of LAINefdd (closed symbols) or LAI (open symbols). Two LAINefdd infected mice and all three LAI infected mice showed detectable viremia by three weeks post-exposure while one LAINefdd infected mouse exhibited delayed detection of viremia. (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 an intermediate dose of LAINefdd showed no changes in CD4⁺ T cell percentages whereas BLT humanized mice inoculated with an equal dose of LAI exhibited a decline of these cells. (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 30,000 TCIU of LAINefdd (n = 3) 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).

Figure 5

Analysis of BLT humanized mice inoculated with a high dose ofnef(−) or wild-type LAI. (A) Each line depicts longitudinal plasma viral load data from individual BLT humanized mice infected with 600,000 TCIU of LAINefdd (closed symbols) or LAI (open symbols). All LAINefdd and LAI infected mice showed detectable viremia by two weeks post-exposure. (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 high dose of LAINefdd showed minimal changes in CD4⁺ T cell percentages. In contrast, BLT humanized mice inoculated with an equal dose of LAI exhibited a very rapid decline of CD4⁺ T cells. Asterisks indicate that essentially all of these cells are eventually depleted in BLT humanized mice infected with 600,000 TCIU of LAI. (C) Naïve BLT mice (n = 5 in PB, spleen and HTO or n = 4 in LN) and BLT mice inoculated with 600,000 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 and spleen, 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).

Figure 6

Distinct phenotypes in BLT humanized mice during long term infection with a high dose of LAINefdd. (A-B) Each line depicts longitudinal plasma viral load data (A), or the CD4⁺ T cell percentages (B) from individual BLT humanized mice infected with 600,000 TCIU of LAINefdd where each animal in (A) and (B) is symbol matched. Persistent viral replication and loss of CD4⁺ T cells in the peripheral blood were observed in each of these mice. (C-D) Each line depicts longitudinal plasma viral load data (C) or the CD4⁺ T cell percentages (D) from individual BLT humanized mice infected with 600,000 TCIU of LAINefdd where each animal in (C) and (D) is symbol matched. Viral replication and minimal loss of CD4⁺ T cells in the blood were observed in these mice over this extended experimental analysis.

Figure 7

Impact of long-term infection with a high dose of LAINefddin BLT humanized mice. The six BLT humanized mice infected with 600,000 TCIU of LAINefdd presented in Figure 6 were harvested for multiple tissue analyses at the last time points depicted in that figure. These mice are referred to here by the panels in which they appear in that figure (6AB or 6CD). Data from these mice are presented alongside data from 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 600,000 TCIU of LAI (n = 3) to reveal that the CD4⁺ T cell loss patterns observed in the peripheral blood are mirrored by the multiple organ analyses performed. 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. Delta symbols serve to indicate no data are available from bone marrow, lung or liver for LAI at 600,000 TCIU. 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 six 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 < 0.05; **p < 0.01).