Journal: Journal of Virology
Article Title: The DNase Activity of KaposiÃ¢ÂÂs Sarcoma-Associated Herpesvirus SOX Protein Serves an Important Role in Viral Genome Processing during Lytic Replication
Figure Lengend Snippet: Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) DNA sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) GFP expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ÃÂÃÂ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
Article Snippet: The ChIP-Seq libraries were validated using a Bioanalyzer 2100 with a high-sensitivity DNA ChIP (Agilent) and quantified by qPCR (Kappa kit).
Techniques: Recombinant, Mutagenesis, Homologous Recombination, Hybridization, Staining, Southern Blot, Labeling, DNA Sequencing, BAC Assay, Next-Generation Sequencing, Expressing, Stable Transfection, Transfection, Selection, Fluorescence, Microscopy, Western Blot, Activity Assay, Northern Blot, Immunofluorescence, Real-time Polymerase Chain Reaction