1. Myeloid differentiation and susceptibility to HIV-1 are linked to APOBEC3 expression
by Gang Peng, Teresa Greenwell-Wild, Salvador Nares, Wenwen Jin, Ke Jian Lei, Zoila G. Rangel, Peter J. Munson, and Sharon M. Wahl
Blood
Volume 110(1):
July 1, 2007
©2007 by American Society of Hematology

2. Differential HIV infection of monocytes, DCs, and macrophages.
Differential HIV infection of monocytes, DCs, and macrophages. Peripheral-blood monocytes were processed immediately or cultured as described (“Materials and methods, Monocute isolation and differentiation to macrophages and dendritic cells”) to generate immature DCs and differentiated macrophages. (A) By flow-cytometry analysis using antibodies specific to CD14 and DC-SIGN, the 3 populations were phenotypically distinct. (B) Cultures of monocytes, DCs, and macrophages were incubated with HIV-1BaL for 90 minutes, washed, and incubated for 12 to 14 days. Media aliquots were removed every third day for p24 ELISA and replaced with fresh DMEM containing antibiotics and serum. Representative of 4 experiments (day 14 after infection; *P < .001, #P < .05) (SEM). (C) Transmission electron microscopy (EM) of macrophage infected with HIV-1 for 10 days, demonstrating intracellular budding and accumulation of virions (original magnification, × 20,000) using a Zeiss EM10 Microscope (LEO Electron Microscopy, Oberkochen, Germany). (D) Dual-color flow-cytometry analysis of CD4 and CCR5 expression and single-color annexin II staining on monocytes, macrophages, and DCs.
Gang Peng et al. Blood 2007;110:
©2007 by American Society of Hematology

3. HIV susceptibility and resistance gene expression.
HIV susceptibility and resistance gene expression. (A) Monocytes, DCs, and macrophages were prepared and RNA was extracted and processed for microarray analysis using the Affymetrix system. Relative transformed expression intensity values were calculated within probe set (row) and hierarchically clustered (“Materials and methods, Oligonucleotide microarray data processing and analysis”), and the corresponding heat map and dendrogram are shown here (red indicates above-average expression; black, average expression; and green, below-average expression for genes associated with HIV resistance and susceptibility). Some genes were represented by multiple probe sets that often fall within the same cluster. Those that show distinct expression behavior may suggest the presence of splice variants (eg, Dicer 1 in clusters 2 and 4). (B) Using the clusters from panel A, parallel line plots by cluster-compare gene-expression profiles in monocytes, DCs, and macrophages from 3 donors. (C) Monocytes, DCs, and macrophages were unstimulated or treated with LPS for 1 hour and RNA was extracted and processed for microarray analysis using the Affymetrix system. Data were treated as in panel A. Data shown are for all 11 probe sets corresponding to the APOBEC cytidine deaminase gene superfamily, represented on the U133Plus2 chip. (D) Parallel line plot comparing APOBEC3 family gene-expression profiles in monocytes, DCs, and macrophages from 3 donors for the 6 probe sets having more than 50% present calls (206632_s_at - 3B, _at - 3G, _x_at - 3C, _x_at - 3A, _s_at - 3G/ 3F, _at - 3F).
Gang Peng et al. Blood 2007;110:
©2007 by American Society of Hematology

4. Expression of APOBEC3 family genes during monocyte differentiation to macrophages.
Expression of APOBEC3 family genes during monocyte differentiation to macrophages. (A) Mean (± SEM) gene-expression level in S10 units (similar to log base 10 units) over 3 donors for 3 APOBEC3 family genes (A,G,F) in monocytes (Mono), dendritic cells (imDC), and macrophages (Mac). *P < .001 between monocyte hA3A and DCs or macrophage hA3A. (B) By RT-PCR, monocyte, DC, and macrophage expression of APOBEC3G and 3A in a representative donor. GAPDH was used as a control gene. (C) Protein levels of APOBEC3G and APOBEC3A were monitored in monocytes, DCs, and macrophages by Western blot using specific antibodies with GAPDH as the loading control.
Gang Peng et al. Blood 2007;110:
©2007 by American Society of Hematology

5. Kinetics of APOBEC3 family gene expression during monocyte differentiation to macrophages.
Kinetics of APOBEC3 family gene expression during monocyte differentiation to macrophages. Peripheral-blood monocytes were harvested immediately after isolation or cultured for indicated days as adherent cells and their RNA were analyzed by RT-PCR for the expression of hA3A, hA3F, hA3G, PKR, and GAPDH (inset). Graph represents the APOBEC-to-GAPDH ratio (representative kinetics, n = 2).
Gang Peng et al. Blood 2007;110:
©2007 by American Society of Hematology

6. IFN coordinately enhances macrophage APOBEC3 expression and decreases susceptibility to HIV-1.
IFN coordinately enhances macrophage APOBEC3 expression and decreases susceptibility to HIV-1. (A-B) Macrophages (n = 3 donors) were cultured in the presence or absence of 10 ng/mL IFNα or IFNγ for 4 hours or 100 ng/mL LPS for 1 hour and the RNA was processed for microarray analysis. The bar graph shows the fold change for hA3G (A) and hA3A (B) across treatments. (Insets) IFNα dose-response (0, 0.1, 1, 10 ng/mL; 4 hours) induction of hA3G (A) and hA3A (B) by RT-PCR. (C) Macrophages were infected or not with HIV-1BaL for 90 minutes, washed, and IFNα or IFNγ added once after infection at the indicated concentrations (0.1–10 ng/mL). HIV-1 infection was monitored by p24 levels in the supernatants (day 14 shown; *P < .005) (mean ± SEM).
Gang Peng et al. Blood 2007;110:
©2007 by American Society of Hematology

7. Silencing of APOBEC3A blocks monocyte resistance to HIV
Silencing of APOBEC3A blocks monocyte resistance to HIV. (A) Monocytes were transfected with siRNA sequences specific for hA3A or a control siRNA (siC) or mock transfected (electroporation control [EPC]) before infection with HIV. Two to 3 days after HIV-1 ...
Silencing of APOBEC3A blocks monocyte resistance to HIV. (A) Monocytes were transfected with siRNA sequences specific for hA3A or a control siRNA (siC) or mock transfected (electroporation control [EPC]) before infection with HIV. Two to 3 days after HIV-1 infection, cells were processed and tested for viral DNA synthesis by nested PCR. (B) Transfected monocytes were adhered and infected with HIV-1. Supernatants were harvested every third day for analysis of p24 levels by ELISA (day 13 shown; mean ± SE). Differences between electroporation control (EPC) and siRNA for hA3A were significant (*P < .05). (Inset) Monocytes transfected as described in (A) with siRNA specific for hA3G or control sequences (siC) were infected with HIV-1 and supernatants were tested for HIV p24. (C) Transfected cells were cultured for 48 hours and treated with IFNα (10 ng/mL) overnight before monitoring of hA3A and α tubulin protein levels by Western-blot analysis.
Gang Peng et al. Blood 2007;110:
©2007 by American Society of Hematology