A highly efficient protocol of generating and analyzing VZV ORF deletion mutants based on a newly developed luciferase VZV BAC system
Introduction
Varicella Zoster Virus (VZV) is a human alpha-herpesvirus. More than 90% of the US population is infected with VZV (Abendroth and Arvin, 1999). Primary infection of VZV causes varicella (chicken pox) usually during childhood, and reactivation of VZV infection later in life leads to herpes zoster (shingles). VZV has a 125 kb-long DNA genome, which encodes at least 70 unique open reading frames (ORFs). However, the functions of a majority of these ORFs remain uncharacterized.
The prevailing method in VZV genetic studies involves a four-cosmid system containing the entire viral genome (Cohen and Seidel, 1993, Niizuma et al., 2003). Using the cosmid system to generate recombinant VZV variants inevitably requires several technically challenging steps, including co-transfection of four large cosmids into permissive mammalian cells and multiple homologous recombination events within a single mammalian cell to create the full-length viral genome. Additionally, VZV has a narrow host range and is highly cell-associated in vitro, which makes it more complicated to be mutagenized and isolated than other alpha herpesviruses (Cohen et al., 2007).
To solve this problem, the full-length VZV (P-Oka strain, a cloned clinical isolate of VZV) genome has been cloned as a VZV bacteria artificial chromosome (BAC) (Nagaike et al., 2004, Zhang et al., 2007). Recently, in our laboratory a firefly luciferase cassette was inserted into the VZV BAC to generate a novel luciferase-expressing VZV (Zhang et al., 2007). A highly efficient protocol is reported in this study for generating VZV ORF deletion mutants and carrying out subsequent growth kinetic studies in cultured cells.
Section snippets
Cells, VZVluc plasmids and Escherichia coli strain
Human melanoma (MeWo) cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U penicillin-streptomycin/ml and 2.5 μg amphotericin B/ml at 37 °C in a humidified incubator with 5% CO2. VZVluc was recently developed in the laboratory (Zhang et al., 2007). It contains a full-length VZV P-Oka genome with a firefly luciferase cassette. This cassette, driven by a SV40 early promoter, was inserted between VZV ORF65 and ORF66. The BAC vector was inserted between VZV ORF60 and ORF61, which
Generation of VZV ORF deletion mutants
In order to test this new VZVluc system for genetic studies, 12 single ORF deletion mutants from ORF0 to ORF11 were generated. We took advantage of an efficient recombination system for chromosome engineering in E. coli DY380 strain (Yu et al., 2000). A defective lambda prophage supplies the function that protects and recombines linear DNA. This system is highly efficient and allows recombination between homologies as short as 40 bp.
The first step in making each VZV ORF deletion was to amplify
Conclusions
Despite the fact that VZV has the smallest genome among human herpesviruses, less than 20% of the VZV genome has been functionally characterized. In recent years, a cosmid-based mutagenesis approach has been developed (Cohen and Seidel, 1993, Niizuma et al., 2003) in order to facilitate studies of VZV genome function. In order to generate recombinant VZV genomes using a four-cosmid system, four complementary cosmids need to be co-transfected into a single permissive mammalian cell and 4
Acknowledgements
We are grateful to A. Chu and C. Patterson for critically reading the manuscript. This work was supported by NIH grant AI050709-01 (H.Z.) and American Cancer Association grant RSG-05-076-01-MBC (H.Z.).
References (11)
- et al.
Cloning of the varicella-zoster virus genome as an infectious bacterial artificial chromosome in Escherichia coli
Vaccine
(2004) - et al.
Coupling generation of cytomegalovirus deletion mutants and amplification of viral BAC clones
J. Virol. Methods
(2004) - et al.
Varicella-zoster virus immune evasion
Immunol. Rev.
(1999) - et al.
Generation of varicella-zoster virus (VZV) and viral mutants from cosmid DNAs: VZV thymidylate synthetase is not essential for replication in vitro
Proc. Natl. Acad. Sci. U.S.A.
(1993) - et al.
Varicella-zoster virus ORF4 latency-associated protein is important for establishment of latency
J. Virol.
(2005)
Cited by (40)
Productive vs non-productive infection by cell-free varicella zoster virus of human neurons derived from embryonic stem cells is dependent upon infectious viral dose
2013, VirologyCitation Excerpt :In order to assess the infectivity of virus produced by neurons infected with cell-free virus, we used two approaches. In the first approach, neurons were infected with free-virus prepared from VZV-GFP (Zhang et al., 2008). Infection by this virus diffusely fills infected cells with GFP, which allows ease of visualization in living cultures (Fig. 4A–C).
Simple generation of neurons from human embryonic stem cells using agarose multiwell dishes
2013, Journal of Neuroscience MethodsCitation Excerpt :Coverslips were then rinsed and mounted on slides in 90% glycerol/10% PBS/1% n-propyl-gallate and sealed with nail polish. VZV-GFP (Zhang et al., 2008) was grown in human MeWo melanoma or ARPE (human retinal pigmented epithelium line) cells as previously described (Markus et al., 2011). Cells infected with the virus were seeded on top of neurons and living cultures were monitored and photographed with a fluorescence-equipped inverted microscope.
Varicella-zoster virus: Molecular controls of cell fusion-dependent pathogenesis
2020, Biochemical Society TransactionsAnalysis of Virus and Host Proteomes During Productive HSV-1 and VZV Infection in Human Epithelial Cells
2020, Frontiers in Microbiology