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The spread of Zika virus (ZIKV) has caused an international health emergency due to its ability to cause microcephaly in infants. Yet, our knowledge of how ZIKV replicates at the molecular level is limited. For example, how the non-structural protein 5 (NS5) performs replication, and in particular whether the N-terminal methytransferase (MTase) domain is essential for the function of the C-terminal RNA-dependent RNA polymerase (RdRp) remains unclear. In contrast to previous reports, we find that MTase is absolutely essential for all activities of RdRp in vitro. For instance, the MTase domain confers stability onto the RdRp elongation complex (EC) and and is required for de novo RNA synthesis and nucleotide incorporation by RdRp. Finally, structure function analyses identify key conserved residues at the MTase-RdRp interface that specifically activate RdRp elongation and are essential for ZIKV replication in Huh-7.5 cells. These data demonstrate the requirement for the MTase-RdRp interface in ZIKV replication and identify a specific site within this region as a potential site for therapeutic development.

Many hurdles have plagued the development of an effective vaccine for hepatitis C virus. In this issue of Cell Host & Microbe, Kinchen et al. (2018) and Flyak et al. (2018) report on the characterization of neutralizing antibodies from individuals that spontaneously cleared infection, providing insights that promise to propel vaccine design forward.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A beta) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A beta, which forms neurotoxic amyloid aggregates in Alzheimer's disease (AD), has been shown to interact with fibrinogen. This A beta-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A beta-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A beta-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A beta and A beta-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A beta-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A beta-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A beta-fibrinogen interaction inhibitor.

The N-glycans attached to the Fab and Fc domains play distinct roles in modulating the functions of antibodies. However, post-translational site-selective modifications of glycans in antibodies and other multiply glycosylated proteins remain a challenging task. Here, we report a chemoenzymatic method that permits independent manipulation of the Fab and Fc N-glycans, using cetuximab as a model therapeutic monoclonal antibody. Taking advantage of the substrate specificity of three endoglycosidases (Endo-S, Endo-S2, and Endo-F3) and their glycosynthase mutants, together with an unexpected substrate site-selectivity of a bacterial alpha 1,6-fucosidase from Lactobacillus casei (AlfC), we were able to synthesize an optimal homogeneous glycoform of cetuximab in which the heterogeneous and immunogenic Fab N-glycans were replaced with a single sialylated N-glycan, and the core-fucosylated Fc N-glycans were remodeled with a nonfucosylated and fully galactosylated N-glycan. The glycoengineered cetuximab demonstrated increased affinity for the Fc gamma IIIa receptor and significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

Activation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease.

Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by clinical disease caused by weakly virulent mycobacteria, such as environmental mycobacteria and Bacillus Calmette-Guerin vaccines, in otherwise healthy individuals. All known genetic etiologies disrupt interferon (IFN)-gamma immunity. Germline bi-allelic mutations of IFNGR2 can underlie partial or complete forms of IFN-gamma receptor 2 (IFN-gamma R2) deficiency. Patients with partial IFN-gamma R2 deficiency express a dysfunctional molecule on the cell surface. We studied three patients with MSMD from two unrelated kindreds from Turkey (P1, P2) and India (P3), by whole-exome sequencing. P1 and P2 are homozygous for a mutation of the initiation codon (c.1A>G) of IFNGR2, whereas P3 is homozygous for a mutation of the second codon (c.4delC). Overexpressed mutant alleles produce small amounts of full-length IFN-gamma R2 resulting in an impaired, but not abolished, response to IFN-gamma. Moreover, SV40-fibroblasts of P1 and P2 responded weakly to IFN-gamma, and Epstein Barr virus-transformed B cells had a barely detectable response to IFN-gamma. Studies in patients' primary T cells and monocyte-derived macrophages yielded similar results. The residual expression of IFN-gamma R2 protein of normal molecular weight and function is due to the initiation of translation between the second and ninth non-AUG codons. We thus describe mutations of the first and second codons of IFNGR2, which define a new form of partial recessive IFN-gamma R2 deficiency. Residual levels of IFN-gamma signaling were very low, accounting for the more severe clinical phenotype of these patients with residual expression levels of normally functional surface receptors than of patients with partial recessive IFN-gamma R2 deficiency due to surface-expressed dysfunctional receptors, whose residual levels of IFN-gamma signaling were higher.

Athukoralage et al. (2018) identify a new class of nuclease that degrades cyclic oligoadenylate (cOA), a second messenger that activates non-specific RNA degradation by the type III CRISPR-Cas accessory RNase Csm6/Csx1. This discovery provides a mechanism for regulating the degradation of foreign transcripts during the type III CRISPR immune response.