CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4(+) T cells - PubMed (original) (raw)

CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4(+) T cells

A J Henderson et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The importance of CCAAT/enhancer binding proteins (C/EBPs) and binding sites for HIV-1 replication in primary macrophages, T cell lines and primary CD4(+) T cells was examined. When lines overexpressing the C/EBP dominant-negative protein LIP were infected with HIV-1, replication occurred in Jurkat T cells but not in U937 promonocytes, demonstrating a requirement for C/EBP activators by HIV-1 only in promonocytes. Primary macrophages did not support the replication of HIV-1 harboring mutant C/EBP binding sites in the long terminal repeat but Jurkat, H9 and primary CD4(+) T cells supported replication of wild-type and mutant HIV-1 equally well. Thus the requirement for C/EBP sites is also confined to monocyte/macrophages. The requirement for C/EBP proteins and sites identifies the first uniquely macrophage-specific regulatory mechanism for HIV-1 replication.

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Figures

Figure 1

Figure 1

C/EBP activators are required to establish HIV-1 infection in monocytic cells but not in T cell lines. (A) Western blot analysis of whole cell extracts prepared from H9 T cells (lanes 1–3) and Jurkat T cells (lanes 4–6) cultured in the absence or presence of 10 ng/ml of concanavalin A (lanes 2 and 5) or phorbol myristate (lanes 3 and 6). (B) Immunoblots of U937 and Jurkat mock-transfected cells (lanes 1 and 4) and U937-LIP overexpressing cell lines (lanes 2 and 3), and Jurkat-LIP overexpressing cell lines (lanes 5–9). (C) U937-mock-transfected cells (○) or U937-LIP overexpressing cells (solid symbols) were infected with HIV-1 HXB2 and at various times supernatants from cell cultures were assayed for RT activity. These data are from a single experiment that is representative of three independent experiments. Each data point represents the mean of three independent infections. (D) Jurkat-mock-transfected cells (○) or Jurkat-LIP-overexpressing cells (solid symbols) were infected with HIV-1 HXB2, and at various times supernatants were assayed for RT activity. This is a single experiment that is representative of three independent experiments. Each data point represents the mean of three independent infections.

Figure 2

Figure 2

C/EBP sites are not required for HIV-1 replication in T cells. (A) H9 cells or (B) Jurkat cells were infected with wild-type HIV-1 HXB2 (○) or mC2,C3 HIV-1 HXB2 (•). At various times postinfection supernatants were collected and RT activity was measured. This is a single experiment that is representative of three individual experiments. Each data point represents the mean of four independent infections.

Figure 3

Figure 3

C/EBP sites are required for HIV-1 replication in human primary macrophages. (A) Mononuclear cells isolated from peripheral blood obtained from two healthy donors (donor A, circles; donor B, squares) were infected with either wild-type HIV-1 HXB2 containing a Ba-L envelope (open symbols) or mC2,C3 HIV-1 HXB2 with Ba-L envelope (solid symbols). Supernatants from infected cells were collected at various times and RT activity was measured. These data are a single experiment that is representative of three individual experiments. Each data point represents the mean of three independent infections. (B) Detection of mC2,C3 Ba-L provirus in primary macrophages by PCR. Genomic DNA was prepared from primary macrophages 4 weeks after infection with wild-type Ba-L or mutant Ba-L. Provirus sequences were amplified by PCR as described (31). Specific HIV-1 LTR sequences were detected by Southern blots using the mC3 oligonucleotide. All amplified LTR sequences were detected following washes at 25°C whereas PCR products with C/EBP mutations were specifically detected by more stringent washes at 55°C (31).

Figure 4

Figure 4

C/EBP sites are not required for HIV-1 replication in primary CD4+ T cells. CD4+ T cells were positively selected and stimulated for 24 hr with 10 ng/ml of phytohemagglutinin prior to infection and maintained in medium with 50 units/ml IL-2. Cells were infected with equal amounts of wild-type HXB2 (open symbols) or HXB2 harboring C/EBP mutations (solid symbols). These data are representative of four individual experiments. Each data point represents the mean of four independent infections.

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