Dataset Information

Tracing Baculovirus AcMNPV Infection Using a Real-Time Method Based on ANCHORTM DNA Labeling Technology.

ABSTRACT: Many steps in the baculovirus life cycle, from initial ingestion to the subsequent infection of all larval cells, remain largely unknown; primarily because it has hitherto not been possible to follow individual genomes and their lineages. Use of ANCHORTM technology allows a high intensity fluorescent labelling of DNA. When applied to a virus genome, it is possible to follow individual particles, and the overall course of infection. This technology has been adapted to enable labelling of the baculovirus Autographa californica Multiple NucleoPolyhedroVirus genome, as a first step to its application to other baculoviruses. AcMNPV was modified by inserting the two components of ANCHORTM: a specific DNA-binding protein fused to a fluorescent reporter, and the corresponding DNA recognition sequence. The resulting modified virus was stable, infectious, and replicated correctly in Spodoptera frugiperda 9 (Sf9) cells and in vivo. Both budded viruses and occlusion bodies were clearly distinguishable, and infecting cells or larvae allowed the infection process to be monitored in living cells or tissues. The level of fluorescence in the culture medium of infected cells in vitro showed a good correlation with the number of infectious budded viruses. A cassette that can be used in other baculoviruses has been designed. Altogether our results introduce for the first time the generation of autofluorescent baculovirus and their application to follow infection dynamics directly in living cells or tissues.

SUBMITTER: Hinsberger A

PROVIDER: S-EPMC7019957 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Tracing Baculovirus AcMNPV Infection Using a Real-Time Method Based on ANCHOR<sup>TM</sup> DNA Labeling Technology.

Hinsberger Aurélie A Graillot Benoît B Blachère Lopez Christine C Juliant Sylvie S Cerutti Martine M King Linda A LA Possee Robert D RD Gallardo Franck F Lopez Ferber Miguel M

Viruses 20200102 1

Many steps in the baculovirus life cycle, from initial ingestion to the subsequent infection of all larval cells, remain largely unknown; primarily because it has hitherto not been possible to follow individual genomes and their lineages. Use of ANCHOR<sup>TM</sup> technology allows a high intensity fluorescent labelling of DNA. When applied to a virus genome, it is possible to follow individual particles, and the overall course of infection. This technology has been adapted to enable labelling ...[more]

PMID: 31906433

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Project description:Human cytomegalovirus (HCMV) induces latent lifelong infections in all human populations. Between 30% and nearly 100% of individuals are affected depending on the geographic area and socioeconomic conditions. The biology of the virus is difficult to explore due to its extreme sophistication and the lack of a pertinent animal model. Here, we present the first application of the ANCHOR DNA labeling system to a herpesvirus, enabling real-time imaging and direct monitoring of HCMV infection and replication in living human cells. The ANCHOR system is composed of a protein (OR) that specifically binds to a short, nonrepetitive DNA target sequence (ANCH) and spreads onto neighboring sequences by protein oligomerization. When the OR protein is fused to green fluorescent protein (GFP), its accumulation results in a site-specific fluorescent focus. We created a recombinant ANCHOR-HCMV harboring an ANCH target sequence and the gene encoding the cognate OR-GFP fusion protein. Infection of permissive cells with ANCHOR-HCMV enables visualization of nearly the complete viral cycle until cell fragmentation and death. Quantitative analysis of infection kinetics and of viral DNA replication revealed cell-type-specific HCMV behavior and sensitivity to inhibitors. Our results show that the ANCHOR technology provides an efficient tool for the study of complex DNA viruses and a new, highly promising system for the development of innovative biotechnology applications.IMPORTANCE The ANCHOR technology is currently the most powerful tool to follow and quantify the replication of HCMV in living cells and to gain new insights into its biology. The technology is applicable to virtually any DNA virus or viruses presenting a double-stranded DNA (dsDNA) phase, paving the way to imaging infection in various cell lines, or even in animal models, and opening fascinating fundamental and applied prospects. Associated with high-content automated microscopy, the technology permitted rapid, robust, and precise determination of ganciclovir 50% and 90% inhibitory concentrations (IC50 and IC90) on HCMV replication, with minimal hands-on time investment. To search for new antiviral activities, the experiment is easy to upgrade toward efficient and cost-effective screening of large chemical libraries. Simple infection of permissive cells with ANCHOR viruses in the presence of a compound of interest even provides a first estimation of the stage of the viral cycle the molecule is acting upon.

Dataset Information

Tracing Baculovirus AcMNPV Infection Using a Real-Time Method Based on ANCHORTM DNA Labeling Technology.

Publications

Tracing Baculovirus AcMNPV Infection Using a Real-Time Method Based on ANCHOR<sup>TM</sup> DNA Labeling Technology.

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