Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening

Sarah Straschewski,Giada Frascaroli,Michael Winkler,Martin Warmer,Thomas Mertens,Heinrich Hohenberg,Peter Sommer

doi:10.1371/journal.pone.0009174

Abstract

Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity). We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus–infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture.

Highlights

The human cytomegalovirus (HCMV) is a herpesvirus belonging to the Betaherpesvirinae subfamily
We could show that all recombinant viruses grew similar to wild-type and observed no disturbance of protein expression and intracellular localization
To generate the recombinant viruses we developed a vector, where enhanced yellow fluorescent protein (EYFP) was placed next to a kanamycin resistance marker, to be able to select for transfer

Summary

Introduction

The human cytomegalovirus (HCMV) is a herpesvirus belonging to the Betaherpesvirinae subfamily. The replication cycle of HCMV in the host can be divided into three different phases, the IE-phase, where mainly genes with regulatory functions are expressed, the early-phase for the expression of enzymatic proteins and the late-phase for expression of the structural components [3,4]. The lower matrix protein ppUL83 is a non-essential protein for replication in fibroblasts but important for replication in macrophages [9,10]. It first accumulates in the nucleus of infected cells but is translocated to the cytoplasm in the late phase in a process which depends on cyclindependent kinases and the Crm exporter [11]. This basic phosphoprotein is a major component of the tegument and is able to bind to the viral capsid [12,15]

Methods

Results

Conclusion