Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp’s) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation.

BackgroundThe concept of innate immunity is well recognized within the spectrum of atherosclerosis, which is primarily dictated by macrophages. Although current insights to this process are largely based on murine models, there are fundamental differences in the atherosclerotic microenvironment and associated inflammatory response relative to humans. In this light, we characterized the cellular aspects of innate immune response in normal, nonprogressive, and progressive human atherosclerotic plaques.Methods and ResultsA systematic analysis of innate immune response was performed on 110 well‐characterized human perirenal aortic plaques with immunostaining for specific macrophage subtypes (M1 and M2 lineage) and their activation markers, neopterin and human leukocyte antigen–antigen D related (HLA‐DR), together with dendritic cells (DCs), natural killer (NK) cells, mast cells, neutrophils, and eosinophils. Normal aortae were devoid of low‐density lipoprotein, macrophages, DCs, NK cells, mast cells, eosinophils, and neutrophils. Early, atherosclerotic lesions exhibited heterogeneous populations of (CD68+) macrophages, whereby 25% were double positive “M1” (CD68+/ inducible nitric oxide synthase [iNOS]+/CD163−), 13% “M2” double positive (CD68+/iNOS −/CD163+), and 17% triple positive for (M1) iNOS (M2)/CD163 and CD68, with the remaining (≈40%) only stained for CD68. Progressive fibroatheromatous lesions, including vulnerable plaques, showed increasing numbers of NK cells and fascin‐positive cells mainly localized to the media and adventitia whereas the M1/M2 ratio and level of macrophage activation (HLA‐DR and neopterin) remained unchanged. On the contrary, stabilized (fibrotic) plaques showed a marked reduction in macrophages and cell activation with a concomitant decrease in NK cells, DCs, and neutrophils.ConclusionsMacrophage “M1” and “M2” subsets, together with fascin‐positive DCs, are strongly associated with progressive and vulnerable atherosclerotic disease of human aorta. The observations here support a more complex theory of macrophage heterogeneity than the existing paradigm predicated on murine data and further indicate the involvement of (poorly defined) macrophage subtypes or greater dynamic range of macrophage plasticity than previously considered.

Adult B‐lymphoblastic leukemia (B‐ALL) is a hematological malignancy characterized by genetic heterogeneity. Despite successful remission induction with classical chemotherapeutics and novel targeted agents, enduring remission is often hampered by disease relapse due to outgrowth of a pre‐existing subclone resistant against the treatment. In this study, we show that small glycophosphatidylinositol (GPI)‐anchor deficient CD52‐negative B‐cell populations are frequently present already at diagnosis in B‐ALL patients, but not in patients suffering from other B‐cell malignancies. We demonstrate that the GPI‐anchor negative phenotype results from loss of mRNA expression of the PIGH gene, which is involved in the first step of GPI‐anchor synthesis. Loss of PIGH mRNA expression within these B‐ALL cells follows epigenetic silencing rather than gene mutation or deletion. The coinciding loss of CD52 membrane expression may contribute to the development of resistance to alemtuzumab (ALM) treatment in B‐ALL patients resulting in the outgrowth of CD52‐negative escape variants. Additional treatment with 5‐aza‐2′‐deoxycytidine may restore expression of CD52 and revert ALM resistance.

To improve treatment outcome of patients with B cell acute lymphoblastic leukemia (B-ALL), several immunotherapeutic approaches have been developed in recent years. E.g., direct targeting of CD19 or CD20 by (bispecific) antibodies or chimeric antigen receptors result in effective control of the disease. In contrast, introduction of alemtuzumab which targets the glycophosphatidylinositol (GPI) anchored CD52 protein has been less successful. Despite its profound lymphodepleting potential, the clinical efficacy was unsatisfactory. Similar to development of escape variants following anti-CD19 and anti-CD20 treatment, this might have been due to the outgrowth of CD52 negative B-ALL escape variants. Indeed, in previous studies (Nijmeijer et al, 2010) we have found outgrowth of CD52 negative escape variants after alemtuzumab treatment in a mouse model engrafted with human B-ALL. Further analysis showed that these variants expressed normal CD52 mRNA levels, but lacked CD52 membrane expression which was found to be due to the loss of GPI anchor expression, as confirmed by loss of staining with the GPI-specific aearolysin FLAER. The aim of the current study was to further unravel the mechanism underlying loss of CD52 membrane expression in adult B-ALL. To study whether the relatively frequent development of these CD52 negative/GPI anchor deficient escape variants during alemtuzumab treatment was the result of outgrowth of pre-existing GPI negative cells, we analyzed 10 Peripheral Blood (PB) and 8 Bone Marrow (BM) samples from B-ALL patients at the moment of diagnosis. GPI negative cells were present in 7 out of 10 (70%) PB and 5 out of 8 (63%) BM samples, and comprised between 0.01% and 4.98% of the B-ALL population as analyzed by flow cytometry using FLAER and CD52 counterstaining (detection limit 100 cells per million). Interestingly, these obvious GPI negative populations were not found in other B cell malignancies such as chronic lymphocytic leukemia (n=5), mantle cell lymphoma (n=5), hairy cell leukemia (n=6), or in healthy donors (n=5). To investigate the mechanism of GPI loss, gene expression analysis was performed for the 26 genes that comprise the GPI anchor biosynthesis pathway. GPI positive and GPI negative B-ALL populations (n=7) were purified by fluorescent activated cell sorting (FACS). Recurrent loss of PIGH mRNA expression, but of none of the other genes involved in GPI anchor biosynthesis pathway, was found in GPI negative cells but not in GPI+ cells in all cases. To validate the relevance of this finding, GPI negative and GPI+ B-ALL cell cultures were generated from diagnosis material (n=2) and subsequently transduced with a retroviral construct encoding PIGH, PIGA (another member of the GPI-N-acetylglucosaminyl-transferase complex, which catalyzes the first step in the GPI synthesis) or an empty vector. Restored GPI anchor expression and coinciding CD52 membrane expression was observed in the GPI negative B-ALL cultures upon transduction with the PIGH, but not PIGA or empty vector. To explore the mechanism underlying the recurrent loss of PIGH mRNA expression, we performed extensive DNA screening of the GPI negative B-ALL cultures to discover possible mutations or indels in the promoter region, gene body, or at splice sites and compared this to GPI+ B-ALL cultures from the same individual. These analyses revealed that both alleles of the PIGH gene were present, unmutated and intact. To investigate whether epigenetic regulation could be the cause of PIGH deficiency, we measured promoter CpG methylation by bisulfite sequencing, comparing GPI negative and GPI+ B-ALL cultures (n=2). This analysis revealed that the region surrounding the transcription start site (-200bp up to +100bp) was heavier methylated in the GPI negative B-ALL cultures compared to the GPI+ counterparts. Also, a 14 day treatment of GPI negative B-ALL cultures with the demethylating agent 5-Azacitidine resulted in re-expression of the GPI anchor. In conclusion, the majority of B-ALL patients presented a CD52/GPI negative, alemtuzumab resistant, B-ALL population in samples taken at diagnosis. These cells lost PIGH expression, a key component in GPI anchor synthesis. This is not due to genomic instability, but rather to epigenetic down regulation. Combining epigenetic modulatory drugs with alemtuzumab might be a promising therapeutic strategy to prevent outgrowth of CD52/GPI negative escape variants in B-ALL. Disclosures No relevant conflicts of interest to declare.