A tail-like assembly at the portal vertex in intact herpes simplex type-1 virions

Herpes viruses are prevalent and well characterized human pathogens. Despite extensive study, much remains to be learned about the structure of the genome packaging and release machinery in the capsids of these large and complex double-stranded DNA viruses. However, such machinery is well characterized in tailed bacteriophage, which share a common evolutionary origin with herpesvirus. In tailed bacteriophage, the genome exits from the virus particle through a portal and is transferred into the host cell by a complex apparatus (i.e. the tail) located at the portal vertex. Here we use electron cryo-tomography of human herpes simplex type-1 (HSV-1) virions to reveal a previously unsuspected feature at the portal vertex, which extends across the HSV-1 tegument layer to form a connection between the capsid and the viral membrane. The location of this assembly suggests that it plays a role in genome release into the nucleus and is also important for virion architecture

Viral MicroRNA Effects on Pathogenesis of Polyomavirus SV40 Infections in Syrian Golden Hamsters

Shaojie Zhang, Vojtech Sroller, Preeti Zanwar, Steven J. Halvorson, Nadim J. Ajami, Corey W. Hecksel, Jody L. Swain, Connie Wong, Janet S. Butel, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of AmericaChun Jung Chen, Christopher S. Sullivan, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of AmericaJody L. Swain, Center for Comparative Medicine, Baylor College of Medicine, Houston, Texas, United States of AmericaEffects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×107 plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.This work was supported in part by research grants R01 CA134524 (JSB) and R01 AI077746 (CSS) from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Molecular BiosciencesEmail: [email protected]

Comparison of portal and pentonal vertices.

<p>(<b>A</b>) Central section through an MSA-guided virion average. The left-hand panel shows a grey-scale density map. In the right-hand panel, the capsid shell is radially colored, with internal DNA density shown in cream, and portal density in purple. (<b>B–F</b>) Individual panels of the capsid shell cut away orthogonal to the axis running through the portal and opposing vertex, at the radii indicated by arrows in (<b>A</b>). Views are from the outside of the portal vertex (bottom) and its opposing pentonal vertex (top).</p

Visualization of a distinctive tegument structure associated with the portal vertex in HSV-1 virions.

<p>(<b>A</b>) Projection image through a tomogram of HSV-1 virions embedded in vitreous ice (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002961#ppat.1002961.s002" target="_blank">Video S2</a>). (<b>B–C</b>) The symmetry-free virion averages generated from 213 subtomograms using MSA-guided classification and melon ball alignment methods, respectively. The maps are shown radially colored, with non-capsid densities trimmed to reveal the underlying capsid. Views are shown looking down a 2-fold axis of symmetry with the portal vertex at the bottom.</p

Workflow for melon ball alignment.

<p>(<b>A</b>) Projected slices from a representative tomogram of HSV-1 virions. (<b>B</b>) 12 spherical masks (magenta), in an icosahedral arrangement, superimposed on a map of the capsid (grey), showing the relationship of the masks to the capsid surface. (<b>C</b>) Density extracted by the masking procedure in (<b>B</b>). After generating an initial model, the model may be updated iteratively by realigning the particles (red lines). (<b>D</b>) Final melon ball aligned average, radially colored. (<b>E</b>) Flow chart of the procedure (described in detail within the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002961#s4" target="_blank">Materials and Methods</a>).</p

Workflow for MSA-guided classification.

<p>(<b>A</b>) Projected slices from a representative tomogram of HSV-1 virions. (<b>B</b>) 12 MSA-derived class averages, top, side and lower views. The class average representing a putative portal vertex is shown in yellow. (<b>C</b>) The putative portal vertex (top), the average of all other vertices (middle) and the difference map calculated from them (bottom), with the positive difference densities in blue, and negative difference density in red. (<b>D</b>) Final MSA-guided average, radially colored. (<b>E</b>) Flow chart of the procedure (described in detail within the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002961#s4" target="_blank">Materials and Methods</a>).</p

Multi-scale 3D Cryo-Correlative Microscopy for Vitrified Cells

Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labeled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments

Frequent persistence of SV40 in hamster tissues following intracardiac inoculation.

<p>ND, not done; p.i., postinoculation; SV40, simian virus 40.</p>a<p>A tissue was considered positive if viral DNA copies were detected by real-time quantitative polymerase chain reaction (described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003912#s4" target="_blank">Materials and Methods</a>).</p

Effect of simian virus 40 microRNA on viral loads in hamster liver and kidney over time.

<p>(A) Strain 776, wild-type virus and microRNA-negative mutant SM1. (B) Strain SVCPC, wild-type virus and microRNA-negative mutant SM2. Viral loads are presented as the geometric mean titers of normalized (adjusted) viral DNA copies (natural log,) per 10,000 cells. The number of different animals analyzed per virus at each time point (usually n = 4) is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003912#ppat-1003912-t001" target="_blank">Table 1</a>. Viral DNA levels were higher for the two microRNA mutants than for wild-type viruses in both liver and kidney through day 45. The differences between WT and mutant viruses were statistically significant (<i>p</i>≤0.05) for both viral systems in both tissues (see text for details). Error bars represent the standard deviation.</p

Chimeric O1K foot-and-mouth disease virus with SAT2 outer capsid as an FMD vaccine candidate

Foot-and-mouth disease virus (FMDV) is highly contagious and infects cloven-hoofed domestic livestock leading to foot-and-mouth disease (FMD). FMD outbreaks have severe economic impact due to production losses and associated control measures. FMDV is found as seven distinct serotypes, but there are numerous subtypes within each serotype, and effective vaccines must match the subtypes circulating in the field. In addition, the O and Southern African Territories (SAT) serotypes, are relatively more thermolabile and their viral capsids readily dissociate into non-immunogenic pentameric subunits, which can compromise the effectiveness of FMD vaccines. Here we report the construction of a chimeric clone between the SAT2 and O serotypes, designed to have SAT2 antigenicity. Characterisation of the chimeric virus showed growth kinetics equal to that of the wild type SAT2 virus with better thermostability, attributable to changes in the VP4 structural protein. Sequence and structural analyses confirmed that no changes from SAT2 were present elsewhere in the capsid as a consequence of the VP4 changes. Following exposure to an elevated temperature the thermostable SAT2-O1K chimera induced higher neutralizing-antibody titres in comparison to wild type SAT2 virus.</p