Figure 1.
Generation of VZV deletion and rescue clones.
A. Generation of the ORFX deletion mutant clone. 1. The E. coli DY380 strain provides a highly efficient homologous recombination system, which allows recombination of homologous sequences as short as 40bp. The homologous recombination system is strictly regulated by a temperature-sensitive repressor, which permits transient switching-on by incubation at 42°C for 15min. VZVluc BAC DNA is introduced into DY380 by electroporation. Electro-competent cells are prepared with homologous recombination system activation. 2. Amplification of the kanR expression cassette by PCR using a primer pair adding 40-bp homologies flanking ORFX. 3. About 200ng of above PCR product are transformed into DY380 carrying the VZVluc BAC via electroporation. 4 and 5. Homologous recombination between upstream and downstream homologies of ORFX replaces ORFX with the KanR cassette, creating the ORFX deletion VZV clone. The recombinants are selected on LB agar plates containing kanamycin at 32°C. 6. The deletion of ORFX DNA is isolated and confirmed by testing antibiotic sensitivity and PCR analysis. The integrity of the viral genome after homologous recombination is examined by restriction enzyme digestion. 7. Purified BAC DNA is transfected into MeWo cells. 8. 3–5 days after transfection the infected cells are visualized by fluorescence microscopy. B. Generation of ORFX rescue virus. 1. To generate the ORFX clone, ORFX was amplified by PCR from the wild-type VZV BAC DNA. 2. ORFX was directionally cloned into plasmid pGEM-lox-zeo to form pGEM-zeo-ORFX. 3. Amplfication of the ORFX-ZeoR cassette by PCR using a primer pair adding 40 bp homologies flanking ORFX. 4. The PCR product was transformed into DY380 carrying the VZVLuc ORFX deletion via electroporation. 5 and 6. Homologous recombination between upstream and downstream homologies of ORFX replaced KanR with the ORFX-ZeoR cassette, creating the ORFX rescue clone. 7. ZeoR and BAC vector sequences were removed while generating virus from BAC DNA (by co-transfecting a Cre recombinase-expressing plasmid).
Figure 2.
VZV genome-wide functional profiling based on analysis of viral mutants with single open reading frame deletion mutants.
Genomic organization and ORFs arrangement are based on the viral sequence of the VZV pOka strain. Each VZV ORF is color-coded according to the growth properties of its corresponding virus gene-deletion mutant in cultured MeWo cells and human fetal skin organ cultures. The grey lines for ORF42 represent a splicing junction. For all growth curves, wild-type infections served as positive controls and mock infections served as negative controls.
Table 1.
A list of VZV pOka strain ORFs categorized by the growth properties of their respective deletion mutants in MeWo cells and human fetal skin organ cultures (SOC).
Figure 3.
Distribution of functional annotations for essential and non-essential genes.
A. Distribution of functional annotations for essential genes. Essential genes are significantly enriched for DNA replication (Bonferroni corrected p-value <10−4) and DNA packing (corrected p-value <10−4) functional categories. B. Distribution of functional annotations for non-essential genes. Non-essential genes are significantly enriched for other (corrected p-value <10−3) and unknown (corrected p-value <0.01) functional categories. Statistical significance was determined by a hypergeometric test.
Figure 4.
Growth curve analysis of some VZV deletion mutants.
A. Eight VZV ORF deletion mutants showing slow growth kinetics in cultured MeWo cells. One hundred PFU of each deletion mutant and VZVLuc (WT) were infected with MeWo cells in 6-well dishes in triplicate. Bioluminescence was measured using the IVIS system every day for 7 days after D-luciferin was applied to the cultured media. Total Photon Count in each well (photons/sec/cm2/steradian) was measured, and the values from the triplicate results were averaged. The growth curves were generated when averaged photon counts for each day were plotted. Error bars represent standard deviation for three replicates. B. In vitro growth curve analysis of VZV ORF7, ORF10, and ORF47 mutant viruses. One hundred PFU of ORF7 and ORF10 mutant and rescue (ORF7D, ORF7S, ORF7R, ORF10D, and ORF10R) and ORF47 deletion virus (ORF47D) from infected MeWo cells were used to infect 50% confluent MeWos seeded in 6-well dishes. Experiments were performed in triplicates. The growth curves were generated as described in A. C. Growth curve analysis of VZV ORF7, ORF10, and ORF47 mutants in human fetal skin organ cultures. Skin tissues were inoculated with 5×103 PFU VZVLuc or other VZV variants, as indicated. VZV replication was monitored daily by IVIS for one week as bioluminescence emitting from each skin culture was measured. Each line represents an average of the data from 3 different skin tissue samples, all infected with the same virus. D-luciferin was also applied to three uninfected skin tissue samples (injected with PBS) as mock infection.