Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5′ cyclization sequence (5′CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5′CS, and the presence of DCS-PK facilitates the formation of 5′-3′ RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.     

MOSAIC: A Multiscale Model of Osteogenesis and Sprouting Angiogenesis with Lateral Inhibition of Endothelial Cells

The healing of a fracture depends largely on the development of a new blood vessel network (angiogenesis) in the callus. During angiogenesis tip cells lead the developing sprout in response to extracellular signals, amongst which vascular endothelial growth factor (VEGF) is critical. In order to ensure a correct development of the vasculature, the balance between stalk and tip cell phenotypes must be tightly controlled, which is primarily achieved by the Dll4-Notch1 signaling pathway. This study presents a novel multiscale model of osteogenesis and sprouting angiogenesis, incorporating lateral inhibition of endothelial cells (further denoted MOSAIC model) through Dll4-Notch1 signaling, and applies it to fracture healing. The MOSAIC model correctly predicted the bone regeneration process and recapitulated many experimentally observed aspects of tip cell selection: the salt and pepper pattern seen for cell fates, an increased tip cell density due to the loss of Dll4 and an excessive number of tip cells in high VEGF environments. When VEGF concentration was even further increased, the MOSAIC model predicted the absence of a vascular network and fracture healing, thereby leading to a non-union, which is a direct consequence of the mutual inhibition of neighboring cells through Dll4-Notch1 signaling. This result was not retrieved for a more phenomenological model that only considers extracellular signals for tip cell migration, which illustrates the importance of implementing the actual signaling pathway rather than phenomenological rules. Finally, the MOSAIC model demonstrated the importance of a proper criterion for tip cell selection and the need for experimental data to further explore this. In conclusion, this study demonstrates that the MOSAIC model creates enhanced capabilities for investigating the influence of molecular mechanisms on angiogenesis and its relation to bone formation in a more mechanistic way and across different time and spatial scales.     

BRIZ1 and BRIZ2 Proteins Form a Heteromeric E3 Ligase Complex Required for Seed Germination and Post-germination Growth in Arabidopsis thaliana

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growth-arrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form hetero-oligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.     

Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System

Long INterspersed Element-1 (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers in vitro. While valuable for understanding ORF1p function, these in vitro approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H) system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction in trans leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p cis-preference in L1 mobilization is established, the impact of ability of ORF1pto interact in trans on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu) RNP nuclear access in the host cell.     

MMS21/HPY2 and SIZ1, Two Arabidopsis SUMO E3 Ligases,Have Distinct Functions in Development

The small ubiquitin related modifier (SUMO)-mediated posttranslational protein modification is widely conserved among eukaryotes. Similar to ubiquitination, SUMO modifications are attached to the substrate protein through three reaction steps by the E1, E2 and E3 enzymes. To date, multiple families of SUMO E3 ligases have been reported in yeast and animals, but only two types of E3 ligases have been identified in Arabidopsis: SAP and Miz 1 (SIZ1) and Methyl Methanesulfonate-Sensitivity protein 21 (MMS21)/HIGH PLOIDY 2 (HPY2), hereafter referred to as HPY2. Both proteins possess characteristic motifs termed Siz/PIAS RING (SP-RING) domains, and these motifs are conserved throughout the plant kingdom. Previous studies have shown that loss-of-function mutations in HPY2 or SIZ1 cause dwarf phenotypes and that the phenotype of siz1-2 is caused by the accumulation of salicylic acid (SA). However, we demonstrate here that the phenotype of hpy2-1 does not depend on SA accumulation. Consistently, the expression of SIZ1 driven by the HPY2 promoter does not complement the hpy2-1 phenotypes, indicating that they are not functional homologs. Lastly, we show that the siz1-2 and hpy2-1 double mutant results in embryonic lethality, supporting the hypothesis that they have non-overlapping roles during embryogenesis. Together, these results suggest that SIZ1 and HPY2 function independently and that their combined SUMOylation is essential for plant development.     

Drosophila terminalia as an appendage-like structure.

In Drosophila, the homeotic gene Distal-less (Dll) has a fundamental role in the establishment of the identity of ventral appendages such as the leg and antenna. This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). During the development of the two components of the anal primordium - the hindgut and the analia - only the latter is dependent on Dll and hedgehog (hh) functions. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream to Dpp. These results suggest that the terminalia show similar behaviour to the leg disc or the antennal part of the eye-antennal disc consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage.     

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.     

Bioengineered Chinese Hamster Ovary Cells with Golgi-targeted 3-O-Sulfotransferase-1 Biosynthesize Heparan Sulfate with an Antithrombin-binding Site

HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.     

The Linker for Activation of B Cells (LAB)/Non-T Cell Activation Linker (NTAL) Regulates Triggering Receptor Expressed on Myeloid Cells (TREM)-2 Signaling and Macrophage Inflammatory Responses Independently of the Linker for Activation of T Cells

Triggering receptor expressed on myeloid cells-2 (TREM-2) is rapidly emerging as a key regulator of the innate immune response via its regulation of macrophage inflammatory responses. Here we demonstrate that proximal TREM-2 signaling parallels other DAP12-based receptor systems in its use of Syk and Src-family kinases. However, we find that the linker for activation of T cells (LAT) is severely reduced as monocytes differentiate into macrophages and that TREM-2 exclusively uses the linker for activation of B cells (LAB encoded by the gene Lat2^−/−) to mediate downstream signaling. LAB is required for TREM-2-mediated activation of Erk1/2 and dampens proximal TREM-2 signals through a novel LAT-independent mechanism resulting in macrophages with proinflammatory properties. Thus, Lat2^−/− macrophages have increased TREM-2-induced proximal phosphorylation, and lipopolysaccharide stimulation of these cells leads to increased interleukin-10 (IL-10) and decreased IL-12p40 production relative to wild type cells. Together these data identify LAB as a critical, LAT-independent regulator of TREM-2 signaling and macrophage development capable of controlling subsequent inflammatory responses.