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

Intermittent bulk release of human cytomegalovirus

Human Cytomegalovirus (HCMV) can infect a variety of cell types by using virions of varying glycoprotein compositions. It is still unclear how this diversity is generated, but spatio-temporally separated envelopment and egress pathways might play a role. So far, one egress pathway has been described in which HCMV particles are individually enveloped into small vesicles and are subsequently exocytosed continuously. However, some studies have also found enveloped virus particles inside multivesicular structures but could not link them to productive egress or degradation pathways. We used a novel 3D-CLEM workflow allowing us to investigate these structures in HCMV morphogenesis and egress at high spatio-temporal resolution. We found that multiple envelopment events occurred at individual vesicles leading to multiviral bodies (MViBs), which subsequently traversed the cytoplasm to release virions as intermittent bulk pulses at the plasma membrane to form extracellular virus accumulations (EVAs). Our data support the existence of a novel bona fide HCMV egress pathway, which opens the gate to evaluate divergent egress pathways in generating virion diversity

Herpesvirus Replication Compartments: Dynamic Biomolecular Condensates?

Recent progress has provided clear evidence that many RNA-viruses form cytoplasmic biomolecular condensates mediated by liquid–liquid phase separation to facilitate their replication. In contrast, seemingly contradictory data exist for herpesviruses, which replicate their DNA genomes in nuclear membrane-less replication compartments (RCs). Here, we review the current literature and comment on nuclear condensate formation by herpesviruses, specifically with regard to RC formation. Based on data obtained with human cytomegalovirus (human herpesvirus 5), we propose that liquid and homogenous early RCs convert into more heterogeneous RCs with complex properties over the course of infection. We highlight how the advent of DNA replication leads to the maturation of these biomolecular condensates, likely by adding an additional DNA scaffold

A Modified Screening System for Loss-of-Function and Dominant Negative Alleles of Essential MCMV Genes

Inactivation of gene products by dominant negative mutants is a valuable tool to assign functions to yet uncharacterized proteins, to map protein-protein interactions or to dissect physiological pathways. Detailed functional and structural knowledge about the target protein would allow the construction of inhibitory mutants by targeted mutagenesis. Yet, such data are limited for the majority of viral proteins, so that the target gene needs to be subjected to random mutagenesis to identify suitable mutants. However, for cytomegaloviruses this requires a two-step screening approach, which is time-consuming and labor-intensive. Here, we report the establishment of a high-throughput suitable screening system for the identification of inhibitory alleles of essential genes of the murine cytomegalovirus (MCMV). In this screen, the site-specific recombination of a specifically modified MCMV genome was transferred from the bacterial background to permissive host cells, thereby combining the genetic engineering and the rescue test in one step. Using a reference set of characterized pM53 mutants it was shown that the novel system is applicable to identify non-complementing as well as inhibitory mutants in a high-throughput suitable setup. The new cis-complementation assay was also applied to a basic genetic characterization of pM99, which was identified as essential for MCMV growth. We believe that the here described novel genetic screening approach can be adapted for the genetic characterization of essential genes of any large DNA viruses

Open LED Illuminator: A Simple and Inexpensive LED Illuminator for Fast Multicolor Particle Tracking in Neurons

<div><p>Dual-color live cell fluorescence microscopy of fast intracellular trafficking processes, such as axonal transport, requires rapid switching of illumination channels. Typical broad-spectrum sources necessitate the use of mechanical filter switching, which introduces delays between acquisition of different fluorescence channels, impeding the interpretation and quantification of highly dynamic processes. Light Emitting Diodes (LEDs), however, allow modulation of excitation light in microseconds. Here we provide a step-by-step protocol to enable any scientist to build a research-grade LED illuminator for live cell microscopy, even without prior experience with electronics or optics. We quantify and compare components, discuss our design considerations, and demonstrate the performance of our LED illuminator by imaging axonal transport of herpes virus particles with high temporal resolution.</p></div

Fluorescent protein tagging of adenoviral proteins pV and pIX reveals 'late virion accumulation compartment'.

The human adenovirus type 5 (HAdV5) causes disease of the upper and lower respiratory tract. The early steps of HAdV5 entry up to genome replication in the host nucleus have been extensively studied. However, late stages of infection remain poorly understood. Here, we set out to elucidate the spatiotemporal orchestration of late adenovirus nuclear remodeling in living cells. We generated virus mutants expressing fluorescently tagged protein IX (pIX) and protein V (pV), a capsid and viral genome associated protein, respectively. We found that during progeny virion production both proteins localize to a membrane-less, nuclear compartment, which is highly impermeable such that in immunofluorescence microscopy antibodies can hardly penetrate it. We termed this compartment 'late virion accumulation compartment' (LVAC). Correlation between light- and electron microscopy revealed that the LVAC contains paracrystalline arrays of viral capsids that arrange tightly packed within a honeycomb-like organization of viral DNA. Live-cell microscopy as well as FRAP measurements showed that the LVAC is rigid and restricts diffusion of larger molecules, indicating that capsids are trapped inside

Triggering allows high-speed multi-channel live-imaging of axonal transport.

<p>Embryonic superior cervical ganglion (SCG) neurons were cultured in tri-chambers [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.ref004" target="_blank">4</a>] and neurons in the soma-compartment were infected with PRV 137 expressing gM-EGFP (gM, green) and mRFP-VP26 (VP26, red). 10 hours post infection viral particle trafficking was imaged in axons penetrating into the middle and neuron compartment using non-triggered Arc lamp illumination (Arc lamp–no trigger) or triggered LED illumination (LED–trigger) (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#sec002" target="_blank">Materials and Methods</a>), a) Three consecutive stills of a representative track for each mode with roughly the same length (≈14 sec) and average particle speed of ≈1.5μm/sec are shown. The time between consecutive dual-color images is ~106 ms for LED illumination and 625 ms for Arc lamp illumination. b) The same tracks as in a) shown as a Kymograph. As triggered LED illumination allows much higher frame rates, particle positions with LED illumination almost overlap in both channels while they do not for non-triggered Arc lamp illumination. c) Signal-to-noise ratio (SNR) calculated for each imaging condition. Scale bar indicates 5 μm.</p

Koehler illumination results in superior illumination evenness.

<p>a) Schematic of critical (top) and Koehler modes. Images were inspired by [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.ref015" target="_blank">15</a>]. In critical illumination, the LED light (green) is collimated using a collimation lens (blue) and projected onto the objective. The objective focuses the light, resulting in an image of the LED at the sample plane. In Koehler illumination mode, the collimated light gets focused onto the back focal plane (BFP) of the objective using a second lens (right most lens, blue). This way, the objective projects a collimated cone of light onto the sample plane, resulting in better illumination flatness. A set of two more lenses can be used to generate conjugate planes that than can be regulated by diaphragms to adjust the brightness, contrast, resolution and depth of field of the image (Aperture diaphragm) and the illuminated field of view (Field diaphragm) (grey box). In critical illumination with infinite conjugate plate, an Aperture diaphragm can be used to regulate brightness, contrast, resolution and depth of field. b) Brightness and illumination flatness in critical and Koehler illumination modes were measured on a custom-built epifluorescence microscope (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#sec002" target="_blank">Materials and Methods</a> for details). Representative images are shown and intensities are coded as colors as indicated in the legend and can be compared between critical and Koehler modes but not between LEDs as different exposure times were used. Koehler illumination showed a better field flatness and slightly increase in intensity (≈ 1.3–29.9% depending on image position for the 470 nm LED and about 2.8–12.96% for the 595 nm LED (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.s005" target="_blank">S5 Table</a>)). c) Comparison of fluorescence intensity distributions. At least three images were taken and the fluorescent plastic slide was moved between images. Images were analyzed in Fiji [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.ref003" target="_blank">3</a>] by drawing a diagonal line ROI and intensities along that line were measured using the “plot profile” tool (black arrow). Resulting intensity values were imported into Matlab (Mathworks), the mean was calculated for three images each and means were normalized to their individual maxima and plotted as a line plot depicting the degree of illumination differences over the measured diagonal. Critical illumination with directly emitting LEDs (470 nm LED) lead to uneven illumination, while the evenness with the phosphor-converted LED (595 nm LED) was only slightly worse than with Koehler illumination.</p

Dimming circuitry allows dimming through μManager.

<p>If brightness modulation through μManager is needed, an alternative circuit can be used. Here, a digital to analog converter (DAC) is used to generate voltage levels between 0 to 5 V. As the voltage sensing circuitry in the BuckPuck driver is reversed, 0 V corresponds to LED on and 5V to LED off. The dimming response curve is however not linear and can be found in the BuckPuck data sheet [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.ref016" target="_blank">16</a>]. As the DAC board does not allow triggering through μManager, an Arduino is used instead and its signal is used to switch the DAC signal on and off through a simple 2N3904 NPN transistor. A 330 ohm resistor is used to protect the transistors gate (middle pin) and a 3.3 kohm or higher resistor is used as a pull-up resistor making sure that the LED shuts off when the Arduino signal is off. Blue lines show signal connections and black lines ground connections. Red lines show a 5 V line used for the pull-up resistors. A 12V power supply (12V DC) of at least 3A is needed. The schematic was partially made with Fritzing [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.ref017" target="_blank">17</a>].</p