The Use of Chromatin Immunoprecipitation (ChIP) to Study the Binding of Viral Proteins to the Adenovirus Genome In Vivo - Adenovirus: Methods and Protocols - page 78

Chapter 6

The Use of Chromatin Immunoprecipitation (ChIP)
to Study the Binding of Viral Proteins to the Adenovirus
Genome In Vivo

Yueting Zheng and Patrick Hearing

Abstract

The encapsidation of adenovirus (Ad) DNA into virus particles depends on cis -acting sequences located at
the left end of the viral genome. Repeated DNA sequences in the packaging domain contribute to viral
DNA encapsidation and several viral proteins bind to these repeats when analyzed using in vitro DNA-
protein binding assays. In this chapter, we describe a chromatin immunoprecipitation (ChIP) approach to
study the binding of viral proteins to packaging sequences in vivo. This assay permits accurate quantifi ca-
tion over a wide range of DNA concentrations. The use of formaldehyde cross-linking to stabilize DNA-
protein and protein-protein complexes formed in vivo allows the identifi cation of macromolecular
complexes found in living cells.

Key words Chromatin immunoprecipitation, ChIP, DNA-protein complex, Adenovirus, Real-time
PCR, qPCR

1 Introduction

The encapsidation of adenovirus (Ad) DNA into virus particles
depends on cis -acting sequences located at the left end of the genome
(Ad5 nucleotides 230-380) [ 1 ]. Seven repeated sequences, termed A
repeats due to their AT-rich content, are located within this domain
that contribute to viral DNA packaging. A repeats A1, A2, A5, and A6
are the most important repeats for packaging activity. A repeats con-
tain a bipartite consensus motif (5 ). Both the fi rst

and the second half-site of the consensus motif, as well as the eight
base pair spacing between the half sites, are critical for viral DNA pack-
aging [ 2 , 3 ]. Two viral proteins, L1 52/55 K and IVa2, have been
found to play important roles in Ad packaging and virus assembly,
although their exact roles in this process remain unclear [ 4 - 7 ]. The
Ad L1-52/55 K protein is found within immature virus particles and
this protein forms a physical complex with the Ad IVa2 protein [ 4 ].

′ -TTTG N 8 CG-3 ′