Journal: Journal of Virology
Article Title: Glycoprotein B Cleavage Is Important for Murid Herpesvirus 4 To Infect Myeloid Cells
Figure Lengend Snippet: Mutation of the furin cleavage site (FCS) of MuHV-4 gB. (A) The location of the FCS within a structure model of the MuHV-4 gB ectodomain (amino acids 60 to 680) is shown in purple. The amino acid and nucleotide sequences of the wild-type (WT) FCS and the ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutants are shown. A diagnostic KpnI restriction site was introduced in the ΔFCSv2 and ΔFCSv3 mutants. The site at which furin is expected to cleave gB is indicated by an arrow. I to V, domains I to V; N, amino terminus; C, carboxy terminus; FL, fusion loops. (B) Schematic drawing of the MuHV-4 ORF8 encoding gB. HindIII and KpnI restriction sites, the HindIII fragment used as probe for Southern blot hybridization, and the position of primers P1 and P2 used for PCR analysis are shown. (C) Restriction enzyme analysis of mutant MuHV-4 BACs. The WT BAC and ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutant and revertant BACs were digested with HindIII and separated by agarose gel electrophoresis. (D) Southern blot analysis of mutant viruses. Viral DNA was extracted, digested with KpnI, and analyzed by Southern blotting hybridization with the radioactively labeled MuHV-4 HindIII N fragment. In the mutant viruses ΔFCSv2 and ΔFCSv3, the 5.6-kb KpnI fragment is cleaved into 3.2-kb and 2.4-kb bands due to the introduced KpnI site. (E) PCR analysis of mutant viruses. Viral DNA was subjected to PCR with primers P1 and P2 spanning the sequence encoding the FCS, followed by agarose gel electrophoresis. The observed PCR products correspond to the expected sizes shown below the gel image (in bp).
Article Snippet: For the latter, viral DNA was digested with KpnI (Roche), separated on a 1% agarose gel, transferred and UV-cross-linked to a positively charged nylon membrane (Roche), hybridized with a P32-labeled probe consisting of the MuHV-4 genomic HindIII N fragment (genomic coordinates 16239 to 19870), and exposed to X-ray films (Fujifilm).
Techniques: Mutagenesis, Diagnostic Assay, Southern Blot, Hybridization, Polymerase Chain Reaction, BAC Assay, Agarose Gel Electrophoresis, Labeling, Sequencing