Viral Chromosome Conformation Capture (V3C) Assays for Identifying Trans-interaction Sites between Lytic Viruses and the Cellular Genome.

Kinjal Majumder,David Pintel,Maria Boftsi

doi:10.21769/bioprotoc.3198

Abstract

The folding mechanisms of the mammalian genome package our genetic material into the nucleus, and in doing so, dictate its appropriate replication and expression. Chromosome conformation capture technology has enabled the dissection of the folding principles of the cellular genome. This has led to a better understanding of the role played by architectural proteins in forming and dissolving 3D-chromatin-structure. These assays are based on the principle of crosslinking distant cellular sites that are proximal to each other in 3D space using formaldehyde followed by digestion of formed hybrid DNA junctions. Invading viruses, such as the lytic parvovirus Minute Virus of Mice (MVM), establish distinct replication centers within the nuclear environment at cellular sites that preferentially undergo DNA damage, but do not integrate into the cellular DNA. We have adapted chromosome conformation capture technology to study the trans-interaction between MVM and the cellular genome, which we have dubbed V3C, which can be extended to a whole-genome analysis we term V3C-seq. This protocol describes the procedure for performing, as well as analyzing V3C-seq assays, and can be adapted for mapping the cellular interaction sites of any non-integrating DNA virus.

Highlights

[Background] Chromosome conformation capture technologies have helped us gain significant insights into the folding principles of the mammalian genome, helping identify the short-range promoterenhancer loops that underlie epigenetic regulation of gene expression, as well as long-range multi-loop domains that form sub-chromosomal chromatin compartments (Dekker et al, 2013; Denker and de Laat, 2016)
While these technologies have helped define important rules related to the patterns of cis-folding of the cellular genome, their implementation in studying the trans-interaction between the genome of an invading DNA virus and its target cell remained largely unstudied
We describe the procedure for studying the interactome of the parvovirus Minute Virus of Mice (Figure 1)

Summary

Introduction

[Background] Chromosome conformation capture technologies have helped us gain significant insights into the folding principles of the mammalian genome, helping identify the short-range promoterenhancer loops that underlie epigenetic regulation of gene expression, as well as long-range multi-loop domains that form sub-chromosomal chromatin compartments (Dekker et al, 2013; Denker and de Laat, 2016). 50 ml Falcon tubes (Thermo Scientific, catalog number: 339652) 4. Eppendorf tubes (Fisher, catalog number: 05-408-129) 5. Disposable Cell Scrapers (Fisher, catalog number: 08-100-241) 7.

Results

Conclusion