AIDS. 2013 Jul 31;27(12):1847-56. doi: 10.1097/QAD.0b013e328361d059.
Functional polarization of human monocyte-derived macrophages (MDMs) into M1 cells leads to inhibition of R5 HIV-1 replication and viral DNA synthesis in comparison to control, unpolarized cells together with CD4 downregulation from the cell surface and upregulation of CCR5-binding chemokine secretion. We here investigated whether a postentry restriction of virus replication is also induced by M1 polarization of MDM.
MDM were first polarized to M1 cells by 18 h stimulation with interferon-[gamma] and tumor necrosis factor-[alpha]; the cytokines were then removed and the cells were infected with vesicular stomatitis virus G-protein pseudotyped enhanced green fluorescence protein HIV-1 (HIV-GFP) generating a single-round infection cycle.
HIV-1 expression was monitored in terms of eGFP expression by fluorescence activated cell sorter (FACS) analysis and real-time PCR analysis of total HIV-1 gag DNA, 2-long terminal repeat DNA, proviral DNA, and multiply spliced RNA transcripts. Expression of apolipopoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G), and APOBEC3A was tested by western blotting and FACS analysis.
Inhibition of HIV-GFP expression was observed in M1-MDM along with impaired viral DNA synthesis, delayed proviral integration, and reduced proviral transcription. Although APOBEC3G levels were similar in M1 and unpolarized MDM, APOBEC 3A was selectively expressed only by M1 cells.
M1 polarization of in-vitro differentiated primary MDM determines a transient, but profound restriction of HIV-1 replication affecting multiple (entry and postentry) steps in the virus life cycle likely involving the upregulated expression of APOBEC3A.
与对照（未极化）细胞相比，人单核细胞来源的巨噬细胞（MDM）分化为 M1 细胞后，会导致 R5 型 HIV-1 复制受到抑制，病毒 DNA 合成受到抑制，同时细胞表面 CD4 下调和 CCR5 结合趋化因子分泌上调。在此，我们研究了 M1 极化是否也会导致 MDM 中的病毒复制后限制。
首先通过 18 小时干扰素-γ和肿瘤坏死因子-α刺激将 MDM 极化至 M1 细胞；然后去除细胞因子并感染水疱性口炎病毒 G 蛋白假型增强型绿色荧光蛋白 HIV-1（HIV-GFP），从而产生单轮感染周期。
通过荧光激活细胞分选（FACS）分析和实时 PCR 分析总 HIV-1 gag DNA、2 长末端重复 DNA、前病毒 DNA 和多剪接 RNA 转录本，监测 HIV-1 表达。通过 Western blot 和 FACS 分析检测载脂蛋白 B mRNA 编辑、酶催化多肽样 3G（APOBEC3G）和 APOBEC3A 的表达。
在 M1-MDM 中观察到 HIV-GFP 表达受到抑制，同时病毒 DNA 合成受损、前病毒整合延迟和前病毒转录减少。尽管 M1 和未极化 MDM 中的 APOBEC3G 水平相似，但 APOBEC3A 仅选择性表达于 M1 细胞。
体外分化的原代 MDM 的 M1 极化决定了 HIV-1 复制的短暂但深刻的限制，影响病毒生命周期中的多个（进入和进入后）步骤，可能涉及上调 APOBEC3A 的表达。
