scholarly journals Proof-of-concept for the yadokari nature: a capsidless replicase-encoding but replication-dependent (+)ssRNA virus hosted by an unrelated dsRNA virus

2021 ◽  
Author(s):  
Subha Das ◽  
Md Mahfuz Alam ◽  
Rui Zhang ◽  
Sakae Hisano ◽  
Nobuhiro Suzuki
Keyword(s):  
Density Gradient ◽  
Reverse Genetics ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Expression System ◽  
Cesium Chloride ◽  
Dsrna Virus ◽  
Equilibrium Density ◽  
Proof Of Concept ◽  
Ssrna Virus

We have previously proposed a new virus lifestyle or yadokari/yadonushi nature exhibited by a positive-sense ssRNA virus, yadokari virus 1 (YkV1), and an unrelated dsRNA virus, yadonushi virus 1 (YnV1) in a phytopathogenic ascomycete, Rosellinia necatrix . We have proposed that YkV1 diverts the YnV1 capsid to trans-encapsidate YkV1 RNA and RNA-dependent RNA polymerase (RdRp) and replicate in the heterocapsid. However, it remains uncertain whether YkV1 replicates using its own RdRp, and whether YnV1 capsid co-packages both YkV1 and YnV1 components. To address these questions, we first took advantage of the reverse genetics tools available for YkV1. Mutations in the GDD RdRp motif, one of the two identifiable functional motifs on the YkV1 polyprotein, abolished its replication competency. Mutations were also introduced in the conserved 2A-like peptide motif, hypothesized to cleave the YkV1 polyprotein co-translationally. Interestingly, the replication proficiency of YkV1 mutants in the host fungus agreed with the cleavage activity of the 2A-like peptide tested using a baculovirus expression system. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with a subset of YnV1 capsid solely packaging YkV1 dsRNA and RdRp. These results provide proof-of-concept that a capsidless (+)ssRNA virus is hosted by an unrelated dsRNA virus. Importance Viruses typically encode their own capsids that encase their genomes. However, a capsidless (+)ssRNA virus, YkV1, depends on an unrelated dsRNA virus, YnV1, for encapsidation and replication. We have previously shown that YkV1 highjacks the capsid of YnV1 for trans-encapsidation of its own RNA and RdRp. YkV1 was hypothesized to divert the hetero-capsid as the replication site, as is commonly observed for dsRNA viruses. Herein, mutational analyses showed that the RdRp and 2A-like domains on the YkV1 polyprotein are important for its replication. The active RdRp must be cleaved by a 2A-like peptide from the C-proximal protein. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with YnV1 capsid solely packaging YkV1 dsRNA and RdRp. This study provides proof-of-concept of a virus neo-lifestyle where a (+)ssRNA virus snatches capsids from an unrelated dsRNA virus to replicate with its own RdRp, thereby mimicking the typical dsRNA virus lifestyle.

Abstract 437: Optimizing a Prokaryotic Cell Free Expression System for the Generation of synthetic ApoB-Containing Lipoprotein Particles

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Humra Athar ◽  
Zhenghui G Jiang ◽  
Christopher J McKnight
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Expression System ◽  
Low Density Lipoproteins ◽  
Free Expression ◽  
Low Density ◽  
Prokaryotic Cell ◽  
Lipoprotein Particles ◽  
Cell Free Expression System

High serum levels of low density lipoproteins (LDL) is associated with increased risk of atherosclerosis. Apolipoprotein B (ApoB) is required for the assembly and secretion of chylomicrons and very low density lipoproteins (VLDL), the precursor of low density lipoproteins (LDL). Despite its clinical significance, the mechanism of the assembly of these ApoB containing lipoproteins is poorly understood. The assembly process is an interplay of several key components including but not limited to nascent ApoB, lipids, ER resident chaperones and importantly, microsomal triglyceride transfer protein (MTP). In the current study, we are trying to understand several unanswered questions in the mechanism of the lipoprotein assembly. We have used a novel prokaryotic cell-free expression system and lipids mimicking the ER membrane to produce particles that represent the early dense initiation particles formed in the ER. After optimizing several different conditions, we were able to make “synthetic” lipoproteins by cotranslational expression of constructs from the first 22% of ApoB tagged with a 6-histidine tag at the C-terminus (ApoB 22-His) with small unilamellar phosphatidylcholine (PC) vesicles and phosphatidylcholine:triolein (PC:TO) emulsions. After cotranslational interaction with lipids, these constructs migrate to a lower density in potassium bromide (KBr) density gradient centrifugation. Here we report a new ApoB 22 construct with a FLAG tag at the N-terminus in addition to the C-terminal His tag. The construct makes significant amount of soluble protein that is soluble in the cell free reaction. The two N- and C-terminal tags allow us to purify full length construct from any truncation products. In addition, the dual-tag approach will allow us to purify the synthetic lipoproteins directly from the cell free system, and thereby avoid the requirement for KBr density gradient centrifugation. This new strategy will provide far more efficient generation and purification of synthetic ApoB containing lipoprotein particles.

scholarly journals Proteoglycans of the intervertebral disc. Absence of degradation during the isolation of proteoglycans from the intervertebral disc

1979 ◽  
Vol 179 (3) ◽  
pp. 573-578 ◽  
Author(s):  
R L Stevens ◽  
P G Dondi ◽  
H Muir
Keyword(s):  
Intervertebral Disc ◽  
Density Gradient ◽  
Core Protein ◽  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Hydrodynamic Size ◽  
Laryngeal Cartilage ◽  
Cartilage Proteoglycans

Proteoglycans extracted with 4M-guanidinium chloride from pig intervetebral discs, and purified by equilibrium density-gradient centrifugation in CsCl, were of smaller hydrodynamic size than those extracted and purified in the same way from the laryngeal cartilage of the same animal. Whether this difference in size arose from degradation during the extraction and purification of the proteoglycans of the disc was investigated. Purified proteoglycans labelled either in the chondroitin sulphate chains or in the core protein were obtained from laryngeal cartilage by short-term organ culture. These labelled proteoglycans were added at the beginning of the extraction of the disc proteoglycans, and labelled cartilage and unlabelled disc proteoglycans were isolated and purified together. There was no appreciable loss of radioactivity after density-gradient centrifugation nor decrease in hydrodynamic size of the labelled cartilage proteoglycans on chromatography on Sepharose 2B, when these were present during the extraction of disc proteoglycans. It is concluded that disc proteoglycans are intrinsically of smaller size than cartilage proteoglycans and this difference in size does not arise from degradation during the extraction.

scholarly journals Biosynthesis of proteoglycans in cartilage slices. Fractionation by gel chromatography and equilibrium density-gradient centrifugation

1972 ◽  
Vol 126 (4) ◽  
pp. 791-803 ◽  
Author(s):  
T. E. Hardingham ◽  
Helen Muir
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Specific Radioactivity ◽  
Equilibrium Density ◽  
Gel Chromatography ◽  
Sulphate Content

The kinetics of incorporation of [35S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ‘pulse–chase’ radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ‘pulse–chase’ experiments over 5h did not demonstrate any precursor–product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ‘pulse–chase’ experiments there was again no evidence for precursor–product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

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