Multiple Functions for ORF75c in Murid Herpesvirus-4 Infection
Figure 5
ORF75c− MuHV-4 shows defective immediate early gene expression.
A. BHK-21 cells were infected (1000 genomes/cell) with wild-type (WT) or ORF75c−.4 (ORF75c−) virions. DNA and RNA were recovered at 6 h and 24 h post-infection. RNA was reverse transcribed using ORF50, ORF73 and 18S rRNA-specific primers and cDNAs quantitated by real-time PCR. Controls without reverse transcriptase were all negative. Each sample was run in triplicate and mean copy numbers determined by comparison with known template dilutions. Viral genome numbers were quantitated by amplificating 100 ng DNA with MuHV-4 M2-specific primers and comparing with known template dilutions. B. BHK-21 cells were infected with ORF75c+ (WT, rev.1) or ORF75c− (75c−.4, 75c−.7, 75c−.9) viruses (2 p.f.u./cell or an equivalent number of genomes), and 18 h later lysed in situ in agarose gels. UI = uninfected cells. Circular and linear genomes were distinguished by electrophoretic mobility and comparison with circular genomic BACs, which differ in size due to different numbers of terminal repeats. Viral genomes were identified by probing with a labelled terminal repeat fragment. The boxed area was exposed for a longer time to visualize ORF75c− genomes. C. BHK-21 cells were infected with BAC+ORF75c− MuHV-4 (1000 genomes/cell), then 24 h later super-infected with BAC−ORF50− MuHV-4 (1000 genomes/cell), then 24 h later analyzed for BAC-based eGFP expression by flow cytometry. Each bar shows 20,000 cells. The data are from 1 of 3 equivalent experiments. The increase in eGFP expression with ORF50− superinfection was highly significant (p<10−6 by Chi-squared test).