Dual Myxovirus Screen Identifies a Small-Molecule Agonist of the Host Antiviral Response

As we are confronted with an increasing number of emerging and reemerging viral pathogens, the identification of novel pathogen-specific and broad-spectrum antivirals has become a major developmental objective. Targeting of host factors required for virus replication presents a tangible approach toward obtaining novel hits with a broadened indication range. However, the identification of developable host-directed antiviral candidates remains challenging. We describe a novel screening protocol that interrogates the myxovirus host-pathogen interactome for broad-spectrum drug candidates and simultaneously probes for conventional, pathogen-directed hits. With resource efficiency and pan-myxovirus activity as the central developmental parameters, we explored coscreening against two distinct, independently traceable myxoviruses in a single-well setting. Having identified a pair of unrelated pathogenic myxoviruses (influenza A virus and measles virus) with comparable replication kinetics, we observed unimpaired coreplication of both viruses, generated suitable firefly and Renilla luciferase reporter constructs, respectively, and validated the protocol for up to a 384-well plate format. Combined with an independent counterscreen using a recombinant respiratory syncytial virus luciferase reporter, implementation of the protocol identified candidates with a broadened antimyxovirus profile, in addition to pathogen-specific hits. Mechanistic characterization revealed a newly discovered broad-spectrum lead that does not block viral entry but stimulates effector pathways of the innate cellular antiviral response. In summary, we provide proof of concept for the efficient discovery of broad-spectrum myxovirus inhibitors in parallel to para- and orthomyxovirus-specific hit candidates in a single screening campaign. The newly identified compound provides a basis for the development of a novel broad-spectrum small-molecule antiviral class.     

Eukaryotic translation initiation factor 4E-mediated recessive resistance to plant viruses and its utility in crop improvement

The use of genetic resistance is considered to be the most effective and sustainable approach to the control of plant pathogens. Although most of the known natural resistance genes are monogenic dominant R genes that are predominant against fungi and bacteria, more and more recessive resistance genes against viruses have been cloned in the last decade. Interestingly, of the 14 natural recessive resistance genes against plant viruses that have been cloned from diverse plant species thus far, 12 encode the eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. This review is intended to summarize the current state of knowledge about eIF4E and the possible mechanisms underlying its essential role in virus infection, and to discuss recent progress and the potential of eIF4E as a target gene in the development of genetic resistance to viruses for crop improvement.     

Effects of carnitine palmitoyltransferases on cancer cellular senescence

The carnitine palmitoyltransferase (CPT) family is essential for fatty acid oxidation. Recently, we found that CPT1C, one of the CPT1 isoforms, plays a vital role in cancer cellular senescence. However, it is unclear whether other isoforms (CPT1A, CPT1B, and CPT2) have the same effect on cellular senescence. This study illustrates the different effects of CPT knockdown on PANC-1 cell proliferation and senescence and MDA-MB-231 cell proliferation and senescence, as demonstrated by cell cycle kinetics, Bromodeoxyuridine incorporation, senescence-associated β-galactosidase activity, colony formation, and messenger RNA (mRNA) expression of key senescence-associated secretory phenotype factors. CPT1C exhibits the most substantial effect on cell senescence. Lipidomics analysis was performed to further reveal that the knockdown of CPTs changed the contents of lipids involved in mitochondrial function, and lipid accumulation was induced. Moreover, the different effects of the isoform deficiencies on mitochondrial function were measured and compared by the level of radical oxygen species, mitochondrial transmembrane potential, and the respiratory capacity, and the expression of the genes involved in mitochondrial function were determined at the mRNA level. In summary, CPT1C exerts the most significant effect on mitochondrial dysfunction-associated tumor cellular senescence among the members of the CPT family, which further supports the crucial role of CPT1C in cellular senescence and suggests that inhibition of CPT1C may represent as a new strategy for cancer treatment through the induction of tumor senescence.     

Turnip mosaic virus co-opts the vacuolar sorting receptor VSR4 to promote viral genome replication in plants by targeting viral replication vesicles to the endosome

Accumulated experimental evidence has shown that viruses recruit the host intracellular machinery to establish infection. It has recently been shown that the potyvirus Turnip mosaic virus (TuMV) transits through the late endosome (LE) for viral genome replication, but it is still largely unknown how the viral replication vesicles labelled by the TuMV membrane protein 6K2 target LE. To further understand the underlying mechanism, we studied the involvement of the vacuolar sorting receptor (VSR) family proteins from Arabidopsis in this process. We now report the identification of VSR4 as a new host factor required for TuMV infection. VSR4 interacted specifically with TuMV 6K2 and was required for targeting of 6K2 to enlarged LE. Following overexpression of VSR4 or its recycling-defective mutant that accumulates in the early endosome (EE), 6K2 did not employ the conventional VSR-mediated EE to LE pathway, but targeted enlarged LE directly from cis-Golgi and viral replication was enhanced. In addition, VSR4 can be N-glycosylated and this is required for its stability and for monitoring 6K2 trafficking to enlarged LE. A non-glycosylated VSR4 mutant enhanced the dissociation of 6K2 from cis-Golgi, leading to the formation of punctate bodies that targeted enlarged LE and to more robust viral replication than with glycosylated VSR4. Finally, TuMV hijacks N-glycosylated VSR4 and protects VSR4 from degradation via the autophagy pathway to assist infection. Taken together, our results have identified a host factor VSR4 required for viral replication vesicles to target endosomes for optimal viral infection and shed new light on the role of N-glycosylation of a host factor in regulating viral infection.     

Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer

The invasion of peanut (Arachis hypogaea L.) pods and seeds by aflatoxin-forming species of Aspergillus is linked to injury by the lesser cornstalk borer and frequently causes a severe reduction in crop quality. The lesser cornstalk borer is susceptible to the lepidopteran-active Bacillus thuringiensis insecticidal crystal protein. We have introduced a codon-modified Bacillus thuringiensis cryIA(c) gene into peanut using microprojectile bombardment. The toxin-coding region of a Bt cryIA(c) gene was reconstructed for expression in plants and the resulting 3.4 kb gene cassette (promoter: 1.8 kb coding: 3) was directly cloned into the BglII site of plant transformation vectors. The vectors contained the hph gene, conferring resistance to the antibiotic hygromycin. Somatic embryos initiated from immature peanut cotyledons of two cultivars were used as the target for bombardment. DNA from hygromycin-resistant embryogenic cell lines, regenerated plants, and a progeny plant showed the presence and integration of hph and Bt genes by PCR and/or Southern blot analyses. ELISA immunoassay of the CryIA(c) protein from the hygromycin-selected plants showed the expression of CryIA(c) protein up to 0.18% of total soluble protein. Insect feeding bioassay of transformed plants indicated various levels of resistance to the lesser cornstalk borer, from complete larval mortality to a 66% reduction in larval weight. A negative correlation between percent survival or larval weight and the amount of Bt CryIA(c) protein was recorded indicating in general that the higher the protein level the lower the survival or larval weight of the insect. Based on leaf bioassay, transformation of peanut with vectors containing the Bt cryIA(c) gene may be effective in protecting the peanut plants from damage by lepidopteran insect larvae of lesser cornstalk borer     

Transformation of peanut with a soybean vspB promoter‐uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut ( Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi‐quantitation of transient expression. One hundred and sixty hygromycin‐resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R₁ seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.     

Biogenesis of cytoplasmic membranous vesicles for plant potyvirus replication occurs at endoplasmic reticulum exit sites in a COPI- and COPII-dependent manner

Single-stranded positive-sense RNA viruses induce the biogenesis of cytoplasmic membranous vesicles, where viral replication takes place. However, the mechanism underlying this characteristic vesicular proliferation remains poorly understood. Previously, a 6-kDa potyvirus membrane protein (6K) was shown to interact with the endoplasmic reticulum (ER) and to induce the formation of the membranous vesicles. In this study, the involvement of the early secretory pathway in the formation of the 6K-induced vesicles was investigated in planta. By means of live-cell imaging, it was found that the 6K protein was predominantly colocalized with Sar1, Sec23, and Sec24, which are known markers of ER exit sites (ERES). The localization of 6K at ERES was prevented by the coexpression of a dominant-negative mutant of Sar1 that disables the COPII activity or by the coexpression of a mutant of Arf1 that disrupts the COPI complex. The secretion of a soluble secretory marker targeting the apoplast was arrested at the level of the ER in cells overexpressing 6K or infected by a potyvirus. This blockage of protein trafficking out of the ER by 6K and the distribution of 6K toward the ERES may account for the aggregation of the 6K-bound vesicles. Finally, virus infection was reduced when the accumulation of 6K at ERES was inhibited by impairing either the COPI or COPII complex. Taken together, these results imply that the cellular COPI and COPII coating machineries are involved in the biogenesis of the potyvirus 6K vesicles at the ERES for viral-genome replication.     

莫桑鼻给非鲫滤泡闭锁中液晶形成的冰冻蚀刻研究

本文运用电镜的冰冻蚀刻术研究了莫桑鼻给非鲫滤泡闭锁中液晶形成的过程.结果表明,卵巢内的颗粒细胞吞噬大量的卵黄物质,消化后形成同心圆片层体,这是一种类脂加水以及镶嵌少量的蛋白质的溶致液晶态;细胞内的酶类参与液晶的形成;同时讨论了生物体内相变及液晶态存在的意义.