Functional activity of HIV-1 nef alleles in in vitro assays. (A) Jurkat T (lanes a and b) or HeLa CIITA (lane c) cells were transduced with eGFP-expressing HIV-1 NL4-3 particles containing a disrupted nef gene (nef-) or the indicated nef alleles pseudotyped with the vesicular stomatitis virus G glycoprotein and analyzed by flow cytometric analysis. The ranges of no expression and low, medium, and high levels of eGFP expression used to calculate receptor modulation are indicated in the upper panel. (B) Quantitative effect of HIV-1 Nef proteins on CD4, MHC-I, and Ii surface expression. Values were determined as described in Materials and Methods. Shown are data derived from the transduced cells whose results are presented in panel A. Similar results were obtained in two independent experiments. The ranges of no expression (N) and low (L), medium (M), and high (H) levels of eGFP expression used to calculate receptor modulation are indicated on the x axes. (C) Quantitative presentation of CD4 down-modulation in Jurkat T cells transiently transfected with bicistronic vectors expressing GFP alone (nef-) or together with Nef. Symbols and abbreviations are as shown in panel B. (D) Infectivity of HIV-1 IRES-eGFP variants containing the indicated HIV-1 nef alleles. TZM-bl or P4-CCR5 indicator cells were infected in triplicate with 293T cell-derived virus stocks containing 1 ng p24 antigen. Infectivity is shown relative to that of the recombinant virus containing the NL4-3 nef allele. Similar results were obtained in another independent experiment.

Expression of HIV-1 Nef in CD4C/HIV-Nef^allele Tg mice. Thymuses from different founder lines with each of the CD4C/HIV-Nef^allele transgenes were analyzed. (A) Northern blot analysis of HIV-1 RNA. Total RNAs from thymuses were hybridized with a ³²P-labeled HIV-1-specific probe. The blots were stripped and rehybridized with a ³²P-labeled actin DNA probe. Also shown below are the semiquantitative values for the levels of expression of each Nef allele relative to Nef^NL4-3(WT) (value of 1), measured in Northern blot or quantitative RT-PCR (Q-RT-PCR) analysis, as described in Materials and Methods. (B) Western blot analysis of protein extracts (100 μg) from thymuses of 1-month-old Tg and non-Tg littermates, using the following anti-Nef antibodies: rabbit polyclonal antibody produced in our laboratory (Lab) or monoclonal EH1 or 6.2 antibody. Nef^NL4-3(WT) represents the gene carried by the CD4C/HIV^MutG (F27367) Tg mice previously reported (34) and used as a positive control. The membranes were stripped and reanalyzed with anti-actin antibody.

Interaction of various Nef alleles with NAK in Tg mice. IP, immunoprecipitation. (A) Total protein extracts from thymocytes (250 μg) and macrophages (100 μg) from CD4C/HIV-Nef Tg mice expressing the indicated Nef alleles and from their non-Tg littermates were incubated with rabbit anti-Nef polyclonal antibodies overnight and subjected to an IVKA using 10 μCi of [γ-³²P]ATP for 5 min at room temperature. The phosphorylated proteins were next separated by SDS-PAGE and visualized by autoradiography. (B) Thymocytes from CD4C/HIV-Nef^JR-CSF, CD3C/HIV-Nef⁰³⁹, and CD4C/HIV-Nef^NL4-3(WT) Tg and non-Tg mice were metabolically labeled with [³⁵S]methionine. Lysates were immunoprecipitated with rabbit polyclonal anti-Nef antibody produced in our laboratory. The ³⁵S-labeled proteins were separated by SDS-PAGE and visualized by autoradiography. Note the ability of the polyclonal anti-Nef antibody used to immunoprecipitate the Nef^JR-CSF proteins effectively.

Alignment of Nef amino acid sequences. The NL4-3 sequence is shown in the upper panel for comparison. Some conserved sequence elements in Nef, the position of some binding (bdg) domains, and the position of the polypurine tract (PPT), as well as the start of the 3′ long terminal repeat (LTR) are indicated schematically. Dots indicate identity with the consensus sequence, and dashes indicate gaps introduced to optimize the alignment.

Structure of the CD4C/HIV transgenes expressing various Nef alleles. Nef allele fragments Nef^AD-93, Nef^032an, Nef^039nm, Nef^JR-CSF, Nef^SF2, and Nef^YU10x, as well as the mutant Nef^NL4-3(T71R) were inserted within the same CD4C/HIV^MutG backbone sequences replacing Nef^NL4-3(WT). The transgenes were constructed as described in Materials and Methods. Abbreviations: mCD4enh, mouse CD4 enhancer; hCD4 prom, human CD4 promoter; SV40, polyadenylation sequences from simian virus 40; Ex1, CD4 gene exon 1; X, interruption of the ORF of the indicated HIV-1 genes; LTR, long terminal repeat; A, restriction site AatII; Bs, restriction site BssHII; S, restriction site SstI.

Immunophenotypic analysis of thymic and peripheral T lymphocytes from CD4C/HIV-Nef^allele Tg mice. Thymus (A) and peripheral LN (pLN) (B) cells from a representative Tg mouse with each allele (CD4C/HIV-Nef^AD93 [F115820], CD4C/HIV-Nef^032an [F95582], CD4C/HIV-Nef^039nm [F75581], CD4C/HIV-Nef^JR-CSF [F104995], CD4C/HIV-Nef^YU10x [F92751], CD4C/HIV-Nef^SF2 [F92748], CD4C/HIV-Nef^NL4-3(T71R) [F101376], and CD4C/HIV-Nef^NL4-3(WT) [F27367]) and from a non-Tg littermate were analyzed the same day by flow cytometry for the expression of CD4 and CD8. The percentage of cells found in the relevant quadrant is indicated on the left of the colon, while the underlined number on the right refers to the mean fluorescence intensity. Note the presence of two subsets of CD4⁺ T cells (CD4^high and CD4^low) in pLN cells expressing Nef^039nm and to a lesser extent in those expressing Nef^SF2 and Nef^JR-CSF. These correspond, respectively, to cells expressing low and high levels of Nef, as previously reported for Tg mice expressing Nef^NL4-3(WT) (103). These data are representative of at least three independent experiments with 4 to 10 mice. (C) Three-color FACS analysis (CD4-allophycocyanin, CD8-fluorescein isothiocyanate, and CD44-PE) was performed on pLN cells from a representative mouse from CD4C/HIV-Nef^AD-93 (F115820), CD4C/HIV-Nef^032an (F95582 and F95581 [not shown]), CD4C/HIV-Nef^039nm (F75581 and F115817 [not shown]), CD4C/HIV-Nef^YU10x (F92751), CD4C/HIV-Nef^JR-CSF (F104995), CD4C/HIV-Nef^SF2 (F92748), or CD4C/HIV-Nef^NL4-3(T71R) (F101376 and F101374 [not shown]) Tg lines and from a non-Tg littermate. Isotype control antibody was used as a negative control. Data are shown only for the CD4⁺ T-cell population. The results shown are representative of at least two or three independent experiments. (D) Table representing ratios (Tg/non-Tg) of the percentages of cells expressing (high for CD25, CD44, and CD69) or not expressing (negative/low for CD62L and CD45RB) the indicated cell surface marker, as shown in panel C. Four to six Tg mice of each allelic line along with their respective non-Tg littermates lines were used. (E) Quantitation of apoptotic/dead peripheral CD4⁺ T cells from the same CD4C/HIV-Nef^allele Tg mice whose analysis is shown in panel D. LN cells of Tg and non-Tg littermates were analyzed by FACS after staining with anti-CD4 monoclonal antibody and 7AAD. The data were pooled from results from Tg and non-Tg mice of each line as described for panel D and represent ratios (Tg/non-Tg) of the percentage of apoptotic/dead cells among the CD4⁺ T cells. Statistical analysis was performed with the Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Survival and histopathology of CD4/HIV-Nef^allele Tg mice. (A) Nef-expressing strains showing shorter survival and/or pathological lesions. (Upper panel) Cumulative incidence of mortality. Mice from the indicated Tg lines were observed for a period of up to 12 months. The cumulative incidence of mortality of the different Tg founder lines from each indicated Tg mouse strain, as well as that of their non-Tg littermates, was plotted as the percentage of surviving mice. Nef^NL4-3(WT) represents the CD4C/HIV-Nef^NL4-3(WT) (F27367) Tg mice used as positive controls. The number of animals observed (n) in each indicated Tg line is shown. The number of non-Tg mice represents the sum of control mice for all founders in these lines. (Lower panel) Kidney histology of a representative Tg mouse from corresponding Tg lines. All these Tg mice exhibit the typical pathology previously observed for CD4C/HIV-Nef^NL4-3(WT) Tg mice. Note the tubular dilatation and atrophy, with some cystic changes, tubulointerstitial nephritis as well as focal segmental glomerulosclerosis in all Tg kidneys. (B) No or minimal lung or kidney organ disease in CD4C/HIV-Nef^YU10x, CD4C/HIV-Nef^AD-93, and CD4C/HIV-Nef^JR-CSF Tg mice. (Upper panel) Absence or very low mortality. Mice were observed for up to 12 months. The data are presented as in the upper panel of panel A. (Lower panel) Light microscopic analysis of kidneys from Tg mice. The histological appearance of the kidney from these Tg animals was indistinguishable (Nef^YU10x and Nef^AD-93) or showed only minimal lesions (Nef^JR-CSF) relative to that of non-Tg mice. (C) Semiquantitative assessment of the kidney disease in Tg mice. A score 1 to 4 was assigned to each mouse, and the average score for each group is shown.