A beta-herpesvirus with fluorescent capsids to study transport in living cells.

Fluorescent tagging of viral particles by genetic means enables the study of virus dynamics in living cells. However, the study of beta-herpesvirus entry and morphogenesis by this method is currently limited. This is due to the lack of replication competent, capsid-tagged fluorescent viruses. Here, we report on viable recombinant MCMVs carrying ectopic insertions of the small capsid protein (SCP) fused to fluorescent proteins (FPs). The FPs were inserted into an internal position which allowed the production of viable, fluorescently labeled cytomegaloviruses, which replicated with wild type kinetics in cell culture. Fluorescent particles were readily detectable by several methods. Moreover, in a spread assay, labeled capsids accumulated around the nucleus of the newly infected cells without any detectable viral gene expression suggesting normal entry and particle trafficking. These recombinants were used to record particle dynamics by live-cell microscopy during MCMV egress with high spatial as well as temporal resolution. From the resulting tracks we obtained not only mean track velocities but also their mean square displacements and diffusion coefficients. With this key information, we were able to describe particle behavior at high detail and discriminate between particle tracks exhibiting directed movement and tracks in which particles exhibited free or anomalous diffusion

Fluorescent virus particles are spread-competent.

<p>(A) Confluent M2-10B4 cells were infected with S-GFP-SCP labeled virus on cover-slips in 24-wells with 100 PFU per well and overlaid with methyl-cellulose. 4 dpi cells were fixed and processed for immunofluorescence. MCP-specific antiserum was used to detect virus producing cells as well as single virus particles while GFP fluorescence was visualized directly. Cell nuclei were counterstained with TO-PRO-3. Inserts depict single virus particles surrounding a cell nucleus (circles) not showing any evidence for being on the late stage of infection and producing infectious particles. Scale bars indicate 20 µm in the upper row and 5 µm in the lower row.</p

Mutants carrying tagged SCP are viable.

<p>(A) WT (WT) as well as mutant viruses coding for either HA- (S-HA-SCP) or HA-GFP-tagged wt SCP (S-GFP-SCP) were titrated on MEF cells. 4 days post infection (dpi) cells were fixed with PFA and processed for immunofluorescence against the HA-epitope. The scale bars represent 100 µm. (B) Multistep growth curve of mutant viruses used in this study in comparison to WT virus. C) Comparison of plaque diameters of simultaneously titrated WT, HA-, GFP-, and mCherry-tagged virus 4 dpi on MEF cells. (D) Genome to PFU ratio of GFP-tagged and WT virus. The ratio between genome content and titer for two independently prepared and purified virus stocks per virus was determined by titration and quantitative PCR in triplicates.</p

Ultrastructural assessment of S-GFP-SCP infected cells.

<p>NIH-3T3 and M2-10B4 (upper row) or M2-10B4 cells (lower row) were infected at a MOI of 0.5 and centrifugal enhancement with WT (upper row) or S-GFP-SCP labeled virus (lower row) and incubated for 48 h. Afterwards, cells were high-pressure frozen, freeze-substituted, plastic-embedded and thin-sectioned. Depicted are representative details of two independent experiments showing non-enveloped B- and C-capsids in the nucleus (first column), primary envelopment in the nucleus (2^nd column), non-enveloped C-capsids near cellular membranes possibly during secondary envelopment (third column), as well as enveloped capsids in the cytoplasm (right column). Scale bars indicate 200 nm.</p

Viruses carrying labeled SCP mutants are genetically stable.

<p>Immunoblot of M2-10B4 cells infected with either wt, S-HA-SCP, or S-GFP-SCP virus derived from passages 0, 5 or 10 after reconstitution. Cells were infected at a MOI of 1, and harvested 48 hpi by lysis in total lysis buffer. Proteins were separated by SDS-PAGE, blotted and immunoprobed for GFP, SCP, HA and MCP as well as beta-Tubulin as loading controls. Lower case letters indicate discussed protein bands.</p

Nocodazole blocks MCMV fluorescent particle mobility.

<p>MEF cells were infected with S-mCherry-SCP for 23 h and treated with 5 µg/ml Noccodazole for 1 h or not. S-mCherry-SCP-emission in live cells recorded under environmentally controlled conditions with 5 frames per second. Fluorescence intensity is coded in false-colors from dark blue to yellow. Three frames from a time-lapse stack each recorded 6 seconds (30 frames) apart are shown for a non-treated (upper row) and Nocodazole treated cell (lower row). Lines indicate the nucleus (Nuc.) as well as the cytoplasm (Cyt.). Circles indicate the position of a fluorescent particle. The right picture depicts all quantified tracks with ellipses marking the tracks from the particles highlighted on the left.</p

Construction of SCP fusion proteins.

<p>(A) Alignment of beta-herpesvirus SCP sequences with T-Coffee <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040585#pone.0040585-Notredame1" target="_blank">[62]</a>. A naturally occurring glycin-serin-linker like sequence separates the N-terminus in MCMV. (B) Detailed representation of the S-GFP-SCP fusion protein as well as the basic genetic layout of the used mutant viruses carrying a fluorescent protein (FP) and a hemagglutinin tag (HA). GFP or mCherry were used as FPs. The FP could be removed by a simple SpeI-mediated digest resulting in a construct encoding for just a HA-tagged SCP. N marks the duplicated N-terminal region of SCP. The plasmids carrying the fusion constructs were inserted into the MCMV BAC by Flp-mediated recombination.</p