GFP-VP26 Co-Localizes with the Actin Motor Myosin V

<div><p>(A) Anti-myosin V antibody was used to stain for presence of myosin V. A single focal plane through the nucleus is shown. White arrows highlight areas of co-localization between myosin V and GFP-VP26 foci. Yellow arrow indicates nuclear actin filament associating with both myosin V and GFP-VP26 foci. Scale bar = 10 μm.</p><p>(B) Profile plots of the GFP-VP26 (green) and anti-myosin V (blue) signal intensities along a straight line intersecting GFP-VP26 foci through the nucleus. Inset shows the trajectory of the straight line on a merged image of the nucleus. Signal intensity profile plots were obtained using ImageJ and corrected for background noise by subtracting the average intensity of the approximate nuclear area from the profile data. Curve points below zero correspond approximately with points outside the nucleus. Correlation coefficients between GFP-VP26 and myosin V for plots shown: (a) 0.87 and (b) 0.72.</p></div

Conservation of Formation of Nuclear Actin Filaments

<div><p>(A) PK15s infected with PRV expressing GFP-VP26, fixed at 9 hpi. Asterisks show cells that are infected and show short actin filaments that appear to associate with nuclear membrane. Each image is a 2-D projection from four consecutive layers in an image stack, taken 0.1 μm apart. Scale bar = 20 μm.</p><p>(B) Enlarged image of nucleus labeled with yellow asterisk in (A) Arrowhead indicates nuclear actin filaments. Scale bar = 10 μm.</p><p>(C) SCG neurons infected with HSV-1 (KOS), fixed at 15 hpi. Cells were stained for the presence of viral capsid with anti-VP5 (capsid protein) antibody. Arrowheads indicate nuclear actin filaments. Each image is a 2-D projection from four consecutive layers in an image stack, taken 0.5 μm apart. Scale bar = 20 μm.</p></div

GFP-VP26 Co-Localizes with Nuclear Actin Filaments

<div><p>(A) Neurons were infected with PRV expressing GFP-VP26 and were fixed at time points shown. A single focal plane through the nucleus is shown, which can result in actin filaments appearing “discontinous” due to filaments weaving in and out of the plane of focus. An enlarged image of the nucleus (inset) is shown for clarity. Merged image was created in ImageJ and color adjusted linearly to appear yellow. Scale bar = 10 μm.</p><p>(B) Histogram shows percentage of cells with co-localized GFP-VP26 and nuclear actin filaments in cells positive for nuclear actin at time points indicated. This histogram represents the percentage of cells within a population that show co-localization (this histogram does not show the degree to which GFP-VP26 and nuclear actin filaments are co-localized within a given cell).</p></div

Time Course of Actin Filament Formation and Capsid Assembly in the Nucleus

<div><p>(A) SCG neurons were infected with PRV expressing GFP-VP26 at fixed times shown. The inset at 6 hpi is an enlargement of the nucleus from the cell on the right, which has small nuclear actin filaments. The brightness of the inset image has been enhanced in order to more clearly visualize the filaments. The arrowheads indicate actin filaments that appear to emanate from the nuclear envelope at 9 hpi. A single focal plane through the nucleus is shown. Scale bar = 20 μm.</p><p>(B) Chart showing the relative formation of nuclear actin filaments, the presence of GFP-VP26 fluorescence, and emergence of GFP-VP26 foci over the course of infection.</p></div

Polarity of Nuclear Actin Filaments Reflect the Overall Polarity of the Cell

<p>Neurons were stained with AF568-phalloidin, anti-GM130 to stain the Golgi. GFP-VP26 is visualized by direct fluorescence. Each image is a 2-D projection from four consecutive layers in a confocal image stack, taken 0.5 μm apart. Scale bar = 20 μm. Top two rows show polarized SCG neurons with one axon. Bottom row shows a single SCG neuron with two axons emanating from opposite sides of the cell body.</p

Actin Filaments Form in the Nuclei of PRV-Infected Neurons

<div><p>(A) Confocal images are 2-D projections from five consecutive layers in an image stack, taken 0.5 μm apart. GFP-VP26 is visualized by direct fluorescence. Scale bar = 20 μm. An enlargement of one of the nuclei is shown for clarity. Scale bar for enlargement = 10 μm.</p><p>(B) Quantitation of actin filament formation within nuclei (infected versus mock-infected neurons).</p></div

Host and Viral Requirements for Nuclear Actin Filament Formation

<p>Cells were treated with cycloheximide or PAA, as indicated. Alternatively, viral stocks were treated with UV irradiation. Each image is a 2-D projection from four consecutive layers in an image stack, taken 0.5 μm apart. Scale bar = 20 μm.</p

Drug Effects on Actin Filament Formation and Capsid Assembly Organization in the Nucleus

<div><p>Infected cells were treated with latA, jasp, or DMSO as indicated.</p><p>(A) Each image is a 2-D projection from four consecutive layers in an image stack, taken 0.5 μm apart. Scale bar = 20 μm. The contrast of the α-LAP2 signal has been enhanced in jasp-treated cells.</p><p>(B) TEM of infected cells treated with with latA, jasp, or DMSO as indicated. Asterisks indicate capsid assemblies; arrows point out individual capsids. Inset shows filaments from a different jasp-treated cell; arrows point to filaments. Scale bar = 2 μm.</p></div

A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

<div><p>Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004060#ppat.1004060-Bratcher1" target="_blank">[1]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004060#ppat.1004060-Chatterjee1" target="_blank">[3]</a>. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.</p></div

Viral kinetics of GPCMV during congenital infection.

A<p>SG, salivary glands from pregnant dams.</p>B<p>NAb, dam's neutralizing antibody titer, expressed as serum dilution determined by qPCR.</p>C<p>virus detected by qPCR.</p>D<p>virus detected by nested PCR.</p>E<p>AF, amniotic fluid.</p>F<p>ND, not done.</p