Transcriptional regulation of APOBEC3 antiviral immunity through the CBF-β/RUNX axis - PubMed (original) (raw)

Transcriptional regulation of APOBEC3 antiviral immunity through the CBF-β/RUNX axis

Brett D Anderson et al. Sci Adv. 2015.

Abstract

A diverse set of innate immune mechanisms protects cells from viral infections. The APOBEC3 family of DNA cytosine deaminases is an integral part of these defenses. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H would have the potential to destroy HIV-1 complementary DNA replication intermediates if not for neutralization by a proteasomal degradation mechanism directed by the viral protein Vif. At the core of this complex, Vif heterodimerizes with the transcription cofactor CBF-β, which results in fewer transcription complexes between CBF-β and its normal RUNX partners. Recent studies have shown that the Vif/CBF-β interaction is specific to the primate lentiviruses HIV-1 and SIV (simian immunodeficiency virus), although related nonprimate lentiviruses still require a Vif-dependent mechanism for protection from host species' APOBEC3 enzymes. We provide a molecular explanation for this evolutionary conundrum by showing that CBF-β is required for expression of the aforementioned HIV-1-restrictive APOBEC3 gene repertoire. Knockdown and knockout studies demonstrate that CBF-β is required for APOBEC3 mRNA expression in the nonpermissive T cell line H9 and in primary CD4(+) T lymphocytes. Complementation experiments using CBF-β separation-of-function alleles show that the interaction with RUNX transcription factors is required for APOBEC3 transcriptional regulation. Accordingly, the infectivity of Vif-deficient HIV-1 increases in cells lacking CBF-β, demonstrating the importance of CBF-β/RUNX-mediated transcription in establishing the APOBEC3 antiviral state. These findings demonstrate a major layer of APOBEC3 gene regulation in lymphocytes and suggest that primate lentiviruses evolved to hijack CBF-β in order to simultaneously suppress this potent antiviral defense system at both transcriptional and posttranslational levels.

Keywords: APOBEC3 restriction factors; CBF-β; HIV-1; HIV-1 pathogenesis; Host-Pathogen interaction; RUNX; Vif; antiviral state; innate immunity; lentivirus.

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Figures

Fig. 1

Fig. 1. _CBF_-β knockdown and deletion decreases expression of APOBEC3 mRNAs and proteins.

(A) _CBF_-β mRNA levels relative to TBP in H9 and knockdown derivatives by real-time quantitative polymerase chain reaction (RT-qPCR) (n = 3 with mean ± SD shown). (B) Representative immunoblots of CBF-β and APOBEC3G protein levels in the parental H9 T cell line and shRNA-transduced pools. Tubulin (TUB) is a loading control. (C) RT-qPCR of APOBEC3 mRNA levels relative to TBP in cells transduced with a control shRNA or a CBF-β–specific shRNA (n = 3 with mean ± SD shown; N.D., not detected). (D) Schematic of CRISPR/Cas9 disruption of _CBF_-β exon 2. (E) Representative immunoblots of CBF-β and APOBEC3G protein levels in H9 cells and a _CBF_-β knockout derivative. (F) RT-qPCR of APOBEC3 mRNA levels relative to TBP in H9 cells and a _CBF_-β knockout derivative (n = 3 with mean ± SD shown).

Fig. 2

Fig. 2. RUNX interaction is necessary to restore APOBEC3G expression in CBF-β–depleted cells.

(A) Schematic depicting established phenotypes of CBF-β separation-of-function mutants. Residue N104 is required for CBF-β transcription function with RUNX proteins, whereas F68 is required for Vif-E3 ligase-mediated degradation of APOBEC3 enzymes. (B) Histogram reporting FOXP3 promoter activity as measured by firefly luciferase levels relative to a Renilla luciferase cotransfection control. The indicated CBF-β expression construct, RUNX1 expression construct, and appropriate empty vector controls were cotransfected with luciferase vectors into _CBF_-β knockdown 293T cells 48 hours before luciferase activity measurement (n = 3; mean ± SD shown). Representative immunoblots from a single experimental replicate are shown below. (C) Representative immunoblots showing Vif functionality [APOBEC3G-HA (hemagglutinin) degradation activity] in the presence of the indicated FLAG–CBF-β constructs 48 hours after transfection into _CBF_-β knockdown 293T cells. (D) Immunoblots showing the results of a representative complementation experiment using _CBF_-β knockdown H9 cells and the indicated HA–CBF-β expression constructs or controls. APOBEC3G levels are low in the absence of CBF-β or in the presence of CBF-β N104K (even with higher expression levels relative to the other constructs). In contrast, APOBEC3G levels are restored by expressing wild-type CBF-β or the F68D mutant.

Fig. 3

Fig. 3. _CBF_-β knockout protects HIV-1 from APOBEC3-mediated restriction.

(A) Schematic of HIV-1 single-cycle infectivity assay. (B) Relative infectivity of Vif-proficient and Vif-deficient viruses produced in H9 cells or _CBF_-β knockout clones (n = 2; mean ± SD shown). Vif-proficient viral infectivity for each cell line is set to 100% to facilitate comparisons. (C) Representative immunoblots of relevant cellular and viral proteins from the experiment depicted in (B), as well as additional data from mock-treated cells.

Fig. 4

Fig. 4. New models for APOBEC3-mediated antiviral state and Vif function.

(A) CBF-β/RUNX drives transcription of APOBEC3 genes and maintains a robust antiviral state in the absence of HIV-1 infection in CD4+ T cells. (B) In HIV-1– or SIV-infected cells, Vif prevents CBF-β from binding RUNX transcription complexes to down-regulate APOBEC3 gene transcription and simultaneously promote APOBEC3 protein polyubiquitination and proteasomal degradation.

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