Bromodeoxyuridine Labelling to Determine Viral DNA Localization in Fluorescence and Electron Microscopy: The Case of Adenovirus.

Gabriela N Condezo,Carmen San Martín

doi:10.3390/v13091863

Abstract

The localization of viral nucleic acids in the cell is essential for understanding the infectious cycle. One of the strategies developed for this purpose is the use of nucleotide analogs such as bromodeoxyuridine (BrdU, analog to thymine) or bromouridine (BrU, analog of uridine), which are incorporated into the nucleic acids during replication or transcription. In adenovirus infections, BrdU has been used to localize newly synthesized viral genomes in the nucleus, where it is key to distinguish between host and viral DNA. Here, we describe our experience with methodological variations of BrdU labeling to localize adenovirus genomes in fluorescence and electron microscopy. We illustrate the need to define conditions in which most of the newly synthesized DNA corresponds to the virus and not the host, and the amount of BrdU provided is enough to incorporate to the new DNA molecules without hampering the cell metabolism. We hope that our discussion of problems encountered and solutions implemented will help other researches interested in viral genome localization in infected cells.

Highlights

The localization of viral nucleic acids in the cell is essential for understanding the infectious cycle
As early as 1990, when low temperature dehydration and embedding protocols facilitated the development of immunoelectron microscopy, BrdU was used to follow different DNA species in adenovirus (AdV)-infected cells [8,9]
For our AdV genome localization assays, we used infected cells at late times postinfection (36 or 48 hpi), because at this point the AdV factory is established, cell modifications induced by the virus are clearly visible, and differences between Ad5GL and

Summary

Introduction

The localization of viral nucleic acids in the cell is essential for understanding the infectious cycle. The need to localize viral nucleic acids in infected cells has driven the development of different strategies: stains, antibodies against nucleic acids, radioactive labeling of nucleotides, in situ hybridization, or labeling of nucleotide analogs [1]. Each one of these methods has its pros and cons. In situ hybridization uses DNA or RNA probes labeled with haptens such as biotin, digoxigenin or acetoxyacetylaminofluorene, or enzymatic labels such as biotin-streptavidin This methodology allows accurate localization of specific segments of viral genomes, it takes time to design probes and standardize hybridization conditions [2]. Virions with BrdU labeled genomes are generated by maintaining BrdU in the medium throughout the infection [14]

Methods

Results

Conclusion