Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination

Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs. One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast, which results in vector backbone recircularization or illegitimate recombination products. To increase TAR assembly efficiency, we prepared a dual-selective TAR vector, pGFCS, by adding a P_ADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector, pGF, harboring a P_HIS3-HIS3 cassette as a positive selection marker. This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid. To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome, a highly transformable Saccharomyces cerevisiae strain, VL6-48B, was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin. A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B. The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79% indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.     

Cytosolic Phospholipase A2 Gamma Is Involved in Hepatitis C Virus Replication and Assembly

Similar to other positive-sense, single-stranded RNA viruses, hepatitis C virus (HCV) replicates its genome in a remodeled intracellular membranous structure known as the membranous web (MW). To date, the process of MW formation remains unclear. It is generally acknowledged that HCV nonstructural protein 4B (NS4B) can induce MW formation through interaction with the cytosolic endoplasmic reticulum (ER) membrane. Many host proteins, such as phosphatidylinositol 4-kinase IIIα (PI4KIIIα), have been identified as critical factors required for this process. We now report a new factor, the cytosolic phospholipase A2 gamma (PLA2G4C), which contributes to MW formation, HCV replication, and assembly. The PLA2G4C gene was identified as a host gene with upregulated expression upon HCV infection. Knockdown of PLA2G4C in HCV-infected cells or HCV replicon-containing cells by small interfering RNA (siRNA) significantly suppressed HCV replication and assembly. In addition, the chemical inhibitor methyl arachidonyl fluorophosphonate (MAFP), which specifically inhibits PLA2, reduced HCV replication and assembly. Electron microscopy demonstrated that MW structure formation was defective after PLA2G4C knockdown in HCV replicon-containing cells. Further analysis by immunostaining and immunoprecipitation assays indicated that PLA2G4C colocalized with the HCV proteins NS4B and NS5A in cells infected with JFH-1 and interacted with NS4B. In addition, PLA2G4C was able to transport the HCV nonstructural proteins from replication sites to lipid droplets, the site for HCV assembly. These data suggest that PLA2G4C plays an important role in the HCV life cycle and might represent a potential target for anti-HCV therapy.     

Repurposing of Antazoline Hydrochloride as an Inhibitor of Hepatitis B Virus DNA Secretion

Virologica Sinica - Hepatitis B virus (HBV) belongs to Hepadnaviridae family and mainly infects hepatocytes, which can cause acute or chronic hepatitis. Currently, two types of antiviral drugs are...     

Association of glucokinase regulatory protein polymorphism with type 2 diabetes and fasting plasma glucose: a meta-analysis

Glucokinase regulatory protein (GCKR) which binds to glucokinase (GCK) in the nucleus and inhibits its activity in the presence of fructose-6-phosphate is critical for glucose metabolism. In the past few years, a number of case–control studies have been carried out to investigate the relationship between the GCKR polymorphism and type 2 diabetes (T2D) since it was first identified to be associated with fasting plasma glucose levels, insulin resistance through genome-wide association approach. After that, a number of studies reported that the rs780094 polymorphism in GCKR has been implicated in T2D risk. However, these studies have yielded contradictory results. To investigate this inconsistency, we performed a meta-analysis of 19 studies involving a total of 298,977 subjects for GCKR rs780094 to evaluate its effect on genetic susceptibility for T2D. In a combined analysis, the summary per-allele odds ratio for T2D of the rs780094 polymorphism was 1.11 (95 % CI: 1.07–1.14, P < 10^?5). Significant results were also observed using dominant (OR = 1.18, 95 % CI: 1.05–1.34, P < 10^?5) or recessive genetic model (OR = 1.20, 95 % CI: 1.12–1.28, P < 10^?5). Significant results were found in Asians and Caucasians when stratified by ethnicity. Besides, the polymorphism was found to be significantly associated with increased fasting plasma glucose level. There was strong evidence of heterogeneity, which largely disappeared after stratification by ethnicity. This meta-analysis suggests that the rs780094 polymorphism in GCKR is associated with elevated T2D risk, but these associations vary in different ethnic populations.     

Dissecting endonuclease and exonuclease activities in endonuclease V from Thermotoga maritima

Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 3′-side 1 nt from a deaminated base lesion. DNA cleavage analysis using mutants defective in DNA binding and Mn²⁺ as a metal cofactor reveals a novel 3′-exonuclease activity in endonuclease V [Feng,H., Dong,L., Klutz,A.M., Aghaebrahim,N. and Cao,W. (2005) Defining amino acid residues involved in DNA-protein interactions and revelation of 3′-exonuclease activity in endonuclease V. Biochemistry, 44, 11486–11495.]. This study defines the enzymatic nature of the endonuclease and exonuclease activity in endonuclease V from Thermotoga maritima. In addition to its well-known inosine-dependent endonuclease, Tma endonuclease V also exhibits inosine-dependent 3′-exonuclease activity. The dependence on an inosine site and the exonuclease nature of the 3′-exonuclease activity was demonstrated using 5′-labeled and internally-labeled inosine-containing DNA and a H214D mutant that is defective in non-specific nuclease activity. Detailed kinetic analysis using 3′-labeled DNA indicates that Tma endonuclease V also possesses non-specific 5′-exonuclease activity. The multiplicity of the endonuclease and exonuclease activity is discussed with respect to deaminated base repair.