Targeted deletion of liver-expressed Choriogenin L results in the production of soft eggs and infertility in medaka, Oryzias latipes

Egg envelopes (chorions) in medaka, Oryzias latipes, are composed of three major glycoproteins: ZI-1, − 2, and − 3. These gene-encoded chorion glycoproteins are expressed in the liver and/or ovarian oocytes of sexually mature female fish. In medaka, the glycoproteins produced in the female liver are induced by estrogen as Choriogenin (Chg.) H and Chg. H minor (m), which correspond to the zona pellucida (ZP) B (ZPB) protein in mammals, and Chg. L, which corresponds to ZPC in mammals. Chg. H, Chg. Hm, and Chg. L, are then converted to ZI-1, − 2, and − 3, respectively, during oogenesis in medaka ovaries.In the present study, we established a medaka line in which the chg.l gene was inactivated using the transcription activator-like effector nuclease (TALEN) technique. Neither intact chg.l transcripts nor Chg. L proteins were detected in livers of sexually mature female homozygotes for the mutation (homozygous chg.l knockout: chg.l−/−). The chg.l−/− females spawned string-like materials containing “smashed eggs.” Closer examination revealed the oocytes in the ovaries of chg.l−/− females had thin chorions, particularly at the inner layer, despite a normal growth rate. In comparing chorions from normal (chg.l+/+) and chg.l−/− oocytes, the latter exhibited abnormal architecture in the chorion pore canals through which the oocyte microvilli pass. These microvilli mediate the nutritional exchange between the oocyte and surrounding spaces and promote sperm-egg interactions during fertilization. Thus, following in vitro fertilization, no embryos developed in the artificially inseminated oocytes isolated from chg.l−/− ovaries. These results demonstrated that medaka ZI-3 (Chg.L) is the major component of the inner layer of the chorion, as it supports and maintains the oocyte’s structural shape, enabling it to withstand the pressures exerted against the chorion during spawning, and is essential for successful fertilization. Therefore, gene products of oocyte-specific ZP genes that may be expressed in medaka oocytes cannot compensate for the loss Chg. L function to produce offspring for this species.

The Influence of Newcastle Disease Virus Major Proteins on Virulence

The Newcastle disease virus (NDV) negative-strand RNA genome containssix genes. These genes encode nucleoprotein (NP), phosphoprotein (P),matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN),and RNA-dependent RNA polymerase (L) proteins. The six proteins affectthe virulence of NDV in different ways, but available information on thesix proteins is disparate and scattered across many databases and sources.A comprehensive overview of the proteins determining NDV virulence islacking. This review summarizes the virulence of NDV as a complex traitdetermined by these six different proteins.

Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection

Liquid–liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infections is largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host casein kinase 1 (CK1)-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted virus replication through concentrating the N, L proteins, and genome RNA into viroplasms. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state leading to enhanced virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of viral P protein and virus infection.

Is the Synthetic Fungicide Fosetyl-Al Safe for the Ecotoxicological Models Danio rerio and Enchytraeus crypticus?

Worldwide, pesticides have contaminated the environment, affecting non-target species. The aim of this work was to evaluate the effects of fosetyl-Al (FOS) on model organisms. Based on the 3 Rs for animal research and described guidelines, the OECD 236 and 220 were applied with some modifications. The FOS test concentrations were 0.02–0.2–2–20–200 mg/L for Danio rerio and 250–500–750–1000–1250 mg/kg for Enchytraeus crypticus. Besides the standard endpoints, additional endpoints were evaluated (D. rerio: behavior and biochemical responses; E. crypticus: extension of exposure duration (28 d (days) + 28 d) and organisms’ sizes). For D. rerio, after 96 h (h), hatching was inhibited (200 mg/L), proteins’ content increased (2 and 20 mg/L), lipids’ content decreased (2 mg/L), glutathione S-transferase activity increased (2 mg/L), and, after 120 h, larvae distance swam increased (20 mg/L). For E. crypticus, after 28 d, almost all the tested concentrations enlarged the organisms’ sizes and, after 56 d, 1250 mg/kg decreased the reproduction. In general, alterations in the organisms’ biochemical responses, behavior, and growth occurred at lower concentrations than the effects observed at the standard endpoints. This ecotoxicological assessment showed that FOS may not be considered safe for the tested species, only at higher concentrations than the predicted environmental concentrations (PECs). This research highlighted the importance of a multi-endpoint approach to assess the (eco)toxic effects of the contaminants.

Structural basis for recognition and regulation of arenavirus polymerase L by Z protein

Junin virus (JUNV) causes Argentine hemorrhagic fever, a debilitating human disease of high mortality rates and a great risk to public health worldwide. Studying the L protein that replicates and transcribes the genome of JUNV, and its regulator Z protein should provide critical clues to identify therapeutic targets for disrupting the life cycle of JUNV. Here we report the 3.54 Å cryo-EM structure of the JUNV L protein complexed with regulator Z protein. JUNV L structure reveals a conserved architecture containing signature motifs found in other L proteins. Structural analysis shows that L protein is regulated by binding of Z protein at the RNA product exit site. Based on these findings, we propose a model for the role of Z protein as a switch to turn on/off the viral RNA synthesis via its interaction with L protein. Our work unveils the mechanism of JUNV transcription, replication and regulation, which provides a framework for the rational design of antivirals for combating viral infections.

Development of a New Reverse Genetics System for Ebola Virus

ABSTRACT Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are available for studying live viruses under biosafety level 4 (BSL-4) or subviral particles under BSL-2 conditions. However, these systems all require cotransfection of multiple plasmids expressing viral genome and viral proteins essential for EBOV replication, which is technically challenging and unable to naturally mimic virus propagation using the subviral particle. Here, we established a new EBOV reverse genetics system only requiring transfection of a single viral RNA genome into an engineered cell line that stably expresses viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and large (L) proteins and has been fine-tuned for its superior permissiveness for EBOV replication. Using this system, subviral particles expressing viral VP40, glycoprotein (GP), and VP24 could be produced and continuously propagated and eventually infect the entire cell population. We demonstrated the authentic response of the subviral system to antivirals and uncovered that the VP35 amount is critical for optimal virus replication. Furthermore, we showed that fully infectious virions can be efficiently rescued by delivering the full-length EBOV genome into the same supporting cell, and the efficiency is not affected by genome polarity or virus variant specificity. In summary, our work provides a new tool for studying EBOV under different biosafety levels. IMPORTANCE Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in Africa have revealed the lack of investigation on this notorious virus. A reverse genetics system is an important tool for studying viruses by producing mutant viruses or generating safer and convenient model systems. Here, we developed an EBOV life cycle modeling system in which subviral particles can spontaneously propagate in cell culture. In addition, this system can be employed to rescue infectious virions of homologous or heterologous EBOV isolates using either sense or antisense viral RNA genomes. In summary, we developed a new tool for EBOV research.

Polymerized Albumin Receptor of Hepatitis B Virus for Evading the Reticuloendothelial System

Various strategies, such as optimization of surface chemistry, size, shape, and charge, have been undertaken to develop nanoparticles (NPs) as DDS (drug delivery system) nanocarriers for evading the reticuloendothelial system (RES) in vivo. We previously developed a hollow NP composed of hepatitis B virus (HBV) surface antigen L proteins and lipid bilayers, hereinafter referred to as bio-nanocapsule (BNC), as a nonviral DDS nanocarrier. Such a BNC harbors the HBV-derived human hepatic cell-specific infection mechanism, and intravenously injected BNCs by themselves were shown to avoid clearance by RES-rich organs and accumulate in target tissues. In this study, since the surface modification with albumins is known to prolong the circulation time of nanomedicines, we examined whether the polymerized albumin receptor (PAR) of BNCs contributes to RES evasion in mouse liver. Our results show that NPs conjugated with peptides possessing sufficient PAR activity were captured by Kupffer cells less efficiently in vitro and were able to circulate for a longer period of time in vivo. Comparing with polyethylene glycol, PAR peptides were shown to reduce the recognition by RES to equal content. Taken together, our results strongly suggest that the PAR domain of BNCs, as well as HBV, harbors an innate RES evasion mechanism. Therefore, the surface modification with PAR peptides could be an alternative strategy for improving the pharmacodynamics and pharmacokinetics of forthcoming nanomedicines.

Ascorbate Peroxidase Neofunctionalization at the Origin of APX-R and APX-L: Evidence from Basal Archaeplastida

Ascorbate peroxidases (APX) are class I members of the Peroxidase-Catalase superfamily, a large group of evolutionarily related but rather divergent enzymes. Through mining in public databases, unusual subsets of APX homologs were identified, disclosing the existence of two yet uncharacterized families of peroxidases named ascorbate peroxidase-related (APX-R) and ascorbate peroxidase-like (APX-L). As APX, APX-R harbor all catalytic residues required for peroxidatic activity. Nevertheless, proteins of this family do not contain residues known to be critical for ascorbate binding and therefore cannot use it as an electron donor. On the other hand, APX-L proteins not only lack ascorbate-binding residues, but also every other residue known to be essential for peroxidase activity. Through a molecular phylogenetic analysis performed with sequences derived from basal Archaeplastida, the present study discloses the existence of hybrid proteins, which combine features of these three families. The results here presented show that the prevalence of hybrid proteins varies among distinct groups of organisms, accounting for up to 33% of total APX homologs in species of green algae. The analysis of this heterogeneous group of proteins sheds light on the origin of APX-R and APX-L and suggests the occurrence of a process characterized by the progressive deterioration of ascorbate-binding and catalytic sites towards neofunctionalization.

Stand-alone lipoylated H-protein of the glycine cleavage system enables glycine cleavage and the synthesis of glycine from one-carbon compounds in vitro

H-protein, one of the four component proteins (H, T, P and L) of glycine cleavage system (GCS), is generally considered a shuttle protein interacting with the other three GCS-proteins via a lipoyl swinging arm. We report that without P-, T- and L-proteins, lipoylated H-protein (Hlip) enables GCS reactions in both glycine cleavage and synthesis directions in vitro. This apparent catalytic activity is closely related to the cavity on the H-protein surface where the lipoyl arm is attached. Heating or mutation of selected residues in the cavity destroys or reduces the stand-alone activity of Hlip, which can be restored by adding the other three GCS-proteins. Systematic study of the Hlip-catalyzed overall GCS reactions and the individual reaction steps provides a first step towards understanding the stand-alone function of Hlip. The results in this work provide some inspiration for further understanding the mechanism of the GCS and give some interesting implications on the evolution of the GCS.