Increased BST2 expression during simian immunodeficiency virus infection is not a determinant of disease progression in rhesus monkeys

BACKGROUND: Bone marrow stromal cell antigen 2 (BST2), also known as tetherin, HM1.24 or CD317 represents a type 2 integral membrane protein, which has been described to restrict the production of some enveloped viruses by inhibiting the virus release from the cell surface. This innate antiviral mechanism is counteracted by the HIV-1 viral factor Vpu, targeting BST2 for cellular degradation. Since antiviral BST2 activity has been mainly confirmed by in vitro data, we investigated its role in vivo on the disease progression using the SIV/macaque model for AIDS. We determined BST2 expression in PBMC and leukocyte subsets of uninfected and SIV-infected rhesus macaques by real-time PCR and flow cytometry and correlated it with disease progression and viral load. RESULTS: Compared to pre-infection levels, we found increased BST2 expression in PBMC, purified CD4 + lymphocytes and CD14 + monocytes of SIV-infected animals, which correlated with viral load. Highest BST2 levels were found in progressors and lowest levels comparable to uninfected macaques were observed in long-term non-progressors (LTNPs). During acute viremia, BST2 mRNA increased in parallel with MX1, a prototype interferon-stimulated gene. This association was maintained during the whole disease course. CONCLUSION: The detected relationship between BST2 expression and viral load as well as with MX1 indicate a common regulation by the interferon response and suggest rather limited influence of BST2 in vivo on the disease outcome

Human APOBEC3A isoforms translocate to the nucleus and induce DNA double strand breaks leading to cell stress and death.

Human APOBEC3 enzymes deaminate single stranded DNA. At least five can deaminate mitochondrial DNA in the cytoplasm, while three can deaminate viral DNA in the nucleus. However, only one, APOBEC3A, can hypermutate genomic DNA. We analysed the distribution and function of the two APOBEC3A isoforms p1 and p2 in transfected cell lines. Both can translocate to the nucleus and hypermutate CMYC DNA and induce DNA double strand breaks as visualized by the detection of ©H2AX or Chk2. APOBEC3A induced G1 phase cell cycle arrest and triggered several members of the intrinsic apoptosis pathway. Activation of purified human CD4+ T lymphocytes with PHA, IL2 and interferon α resulted in C->T hypermutation of genomic DNA and double stranded breaks suggesting a role for APOBEC3A in pro-inflammatory conditions. As chronic inflammation underlies many diseases including numerous cancers, it is possible that APOBEC3A induction may generate many of the lesions typical of a cancer genome

MOESM1 of Increased BST2 expression during simian immunodeficiency virus infection is not a determinant of disease progression in rhesus monkeys

Additional file 1: Figure S1.  BST2 protein expression in uninfected and SIVmac251-infected macaques. Comparison of BST2 surface expression, displayed as median fluorescence intensity (MFI) on different leukocyte populations from uninfected monkeys (A). Comparison of BST2 surface expression between uninfected animals and infected animals for granulocytes (C), on B cells (E), CD8 + T cells cells (G) and NK cells (J). Horizontal lines depict median and quartiles. Group comparisons were calculated using Kruskal–Wallis test with Dunn’s multiple comparison analysis (A) and two-tailed Mann–Whitney’s U test (C, E, G, I). Correlations of plasma viral load with BST2 surface expression on granulocytes (D), B cells (F), CD8 + T cells (H) and NK cells (J). Each data point represents one individual animal. Regression lines are depicted; r, Spearman’s correlation coefficient; p, P value

Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage

International audienceForeign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA ago-nists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers inter-feron and production through the RNA poly-merase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromo-somal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer

A3A expression leads to DSBs and requires UNG.

<p>(A) (B) A3A induces DSBs in the quail QT6 cell line at 24 and 48 h post-transfection respectively. Mean and SEM are shown for 4-5 independent experiments. Group comparisons to APOBEC2 at 24 and 48 h were calculated using the Mann-Whitney test (*p < 0.05; **p < 0.01). (C) DSBs originate from <i>de novo</i> genomic DNA damage. HeLa cells transfected with TOPO3.1 and HindIII cleaved TOPO3.1, which cleaves the vector just once were fixed. Mean and SEM are shown at 48 h post-transfection. (D) A3A induced DSBs require UNG cleavage of uracil. HeLa cells were transfected with APOBEC2, p1S and p1S-NLS alone and in the absence or presence of the UNG inhibitor (UGI) expressing plasmid. Mean and SEM are shown for 4-5 independent transfections at 24 h post-transfection. Group comparisons and differences to APOBEC2 were calculated using the Mann-Whitney test (*p < 0.05).</p

A3A expression induces DNA damage response and cell cycle arrest.

<p>(A) (B) Results illustrating percentage of P-Chk2 in V5 expressing cells at 24 and 48 h post-transfection. Mean and SEM are shown. Group comparisons to APOBEC2 at 24 and 48 h were calculated using the Mann-Whitney test (*p < 0.05). (C) (D) Linear relationship of ©H2AX and P-Chk2 at 24 and 48 h post-transfection respectively. r, Spearman’s correlation coefficient; line shows nonlinear regression; p, P value. (E) Twenty-four hours post-transfection RNA was removed with RNase A and DNA was stained with propidium iodide (PI) prior to analysis by flow cytometry. Graph shows percentage of cell cycle distribution from three independent experiments. Cellular aggregates and debris were excluded from analysis by proper gating. Data were fitted to define the G1, S, G2/M phases by using the Dean-Jett-Fox mathematical model of the FlowJo software. The data for 100 µM actinomycin D and etoposide (positive controls) were taken at 16 h. Mean and SEM are shown. Differences in G1 phases were compared to APOBEC2 and were calculated by using the Mann-Whitney test (*p < 0.05).</p

A3A over-expression triggers intrinsic apoptotic pathway.

<p>(A) FACS analysis of cytochrome c release (striped) in HeLa cells 24 h post-transfection. Treatment by 100 µM of actinomycin D and 100 µM etoposide served as positive controls and were measured at 16 h. Means and SEM are given for three independent transfections. Differences in mitochondrial cytochrome c content were compared to APOBEC2 and calculated by using the Mann-Whitney test (*p < 0.05). (B) Western blot analysis of cleaved caspase-3 levels at 24 h post transfection. Beta-tubulin was used as loading control. (C) FACS analysis of cleaved PARP in V5 expressing cells. Mean and SEM are shown for 2-3 independent experiments. Group comparisons to APOBEC2 were calculated using the Mann-Whitney test (*p < 0.05). (D) FACS analysis of cleaved PARP in total cells. Mean and SEM are shown for 2-5 independent experiments. Group comparisons to TOPO3.1 were calculated using the Mann-Whitney test (*p < 0.05). (E) FACS analysis of early apoptosis (Annexin V positive, PI negative cells - white) and late apoptosis/necrosis (Annexin V, PI double positive - patterned) 24 h post-transfection. Means and SEM are given from five independent experiments. Differences in early and late apoptosis were compared to TOPO3.1 and calculated by using the Mann-Whitney test (*p < 0.05; ***p< 0.001).</p