In vivo analysis of Nef's role in HIV-1 replication, systemic T cell activation and CD4(+) T cell loss

Abstract

Background: Nef is a multifunctional HIV-1 protein critical for progression to AIDS. Humans infected with nef(-) HIV-1 have greatly delayed or no disease consequences. We have contrasted nef(-) and nef(+) infection of BLT humanized mice to better characterize Nef's pathogenic effects.

Results: Mice were inoculated with CCR5-tropic HIV-1JRCSF (JRCSF) or JRCSF with an irreversibly inactivated nef (JRCSFNefdd). In peripheral blood (PB), JRCSF exhibited high levels of viral RNA (peak viral loads of 4.71 × 10(6) ± 1.23 × 10(6) copies/ml) and a progressive, 75% loss of CD4(+) T cells over 17 weeks. Similar losses were observed in CD4(+) T cells from bone marrow, spleen, lymph node, lung and liver but thymocytes were not significantly decreased. JRCSFNefdd also had high peak viral loads (2.31 × 10(6) ± 1.67 × 10(6)) but induced no loss of PB CD4(+) T cells. In organs, JRCSFNefdd produced small, but significant, reductions in CD4(+) T cell levels and did not affect the level of thymocytes. Uninfected mice have low levels of HLA-DR(+)CD38(+)CD8(+) T cells in blood (1-2%). Six weeks post inoculation, JRCSF infection resulted in significantly elevated levels of activated CD8(+) T cells (6.37 ± 1.07%). T cell activation coincided with PB CD4(+) T cell loss which suggests a common Nef-dependent mechanism. At 12 weeks, in JRCSF infected animals PB T cell activation sharply increased to 19.7 ± 2.9% then subsided to 5.4 ± 1.4% at 14 weeks. HLA-DR(+)CD38(+)CD8(+) T cell levels in JRCSFNefdd infected mice did not rise above 1-2% despite sustained high levels of viremia. Interestingly, we also noted that in mice engrafted with human tissue expressing a putative protective HLA-B allele (B42:01), JRCSFNefdd exhibited a substantial (200-fold) reduced viral load compared to JRCSF.

Conclusions: Nef expression was necessary for both systemic T cell activation and substantial CD4(+) T cell loss from blood and tissues. JRCSFNefdd infection did not activate CD8(+) T cells or reduce the level of CD4(+) T cells in blood but did result in a small Nef-independent decrease in CD4(+) T cells in organs. These observations strongly support the conclusion that viral pathogenicity is mostly driven by Nef. We also observed for the first time substantial host-specific suppression of HIV-1 replication in a small animal infection model.

Figure 1

HIV-1_JRCSF with a truncated nef. a Upper panel schematic representation of wild type JRCSF nef (WT JRCSF) is presented. Nucleotides 8784–9434 in NCBI accession number, M38429, represent the nef coding sequence. PPT polypurine tract. Lower panel, a schematic presentation of nef with two deletions (JRCSFNefdd). A 114 bp deletion 5′ of the PPT from 8919 to 9032 inclusive was introduced into nef. The downstream sequence was shifted to the +1 reading frame by cutting with XhoI and filling in with Klenow (“Methods”). The 293 bp deletion 3′ of the PPT shifted downstream nef sequence to reading frame to +2. b The proviral clones for JRCSF and JRCSFNefdd were transfected into 293T cells and after 2 days Nef and Env expressions assessed by Western blots, GADPH is a loading control. c Replication competence of JRCSFNefdd was not diminished by loss of Nef as determined by p24^gag production in CEM cells expressing CCR5. Cells were infected at 1 × 10⁵ TCIU at an MOI 0.01 and the production of p24^gag was followed for 21 days.

Figure 2

Viral load analysis and PB CD4⁺ T cell loss in mice infected with JRCSF and JRCSFNefdd. a Viral loads (copies of viral RNA per ml of plasma) were plotted for BLT mice that were exposed to 90,000 TCIU. JRCSF, JRCSFNefdd and uninfected mice were followed for 17 weeks. b The percent of CD4⁺ T cells out of total T cells in peripheral blood are plotted for the three groups of mice in a.

Figure 3

Systemic CD4⁺ T cell but not thymocyte loss in mice infected with JRCSF and JRCSFNefdd. a CD4⁺ T cell analysis was performed on five organs from un-exposed BLT mice (uninfected, n = 4), JRCSF infected BLT mice (n = 5) and JRCSFNefdd infected BLT mice (n = 6). Significant differences are indicated by lines and arrows above respective bars (*p < 0.05). b The same analysis as in a is presented for CD4⁺CD8⁺ double positive thymocytes relative to total thymocytes. No statistical differences were observed. Error bars are the mean ± SEM.

Figure 4

Systemic T cell activation during infection with JRCSF and JRCSFNefdd. Systemic T cell activation was assessed by the levels of HLA-DR⁺CD38⁺CD8⁺ relative to total CD8⁺ T cells in blood. a Representative dot plots for the presence of activated CD8⁺ T cells in blood of uninfected, JRCSF-infected (JRCSF 1) and JRCSFNefdd-infected (JRCSFNefdd 6) mice. Gating protocol was performed as indicated in “Methods”. Data shown are from 12 wpi. b Aggregate data of CD8⁺ T cell activation over the course of 17 weeks of infection is shown for uninfected (n = 4), JRCSF infected (n = 6) and JRCSFNefdd infected (n = 5) mice.

Figure 5

Systemic T cell activation is compared to PB CD4⁺ T cell loss in individual mice. Plots for individual mice of CD4⁺ T cells (left y axis) and of HLA-DR⁺CD38⁺CD8⁺ T cells (right y axis) over the infection time course are shown. Top row all six JRCSF infected mice show increases in CD8⁺ T cell activation concomitant to PB CD4⁺ T cell loss. Middle row JRCSFNefdd infected BLT mice show very low T cell activation and no CD4⁺ T cell loss except for JRCSFNefdd 6 which had a drop in CD4⁺ at week 17. Bottom row uninfected mice show very low T cell activation and no CD4⁺ T cell loss.

Figure 6

Viral load time courses for JRCSFNefdd infected mice and JRCSF infected mice with Cohort 1 tissue. a The data in Figure 2a is re-plotted to separate the four Cohort 1 mice from the other eight mice. Two Cohort 1 mice were infected with JRCSF and two were infected with JRCSFNefdd. b The remaining eight mice were plotted. These mice were from Cohorts 2–5; see Table 1).