Herpesviruses: An In-Depth View

Abstract

Transmission electron microscopy of thin sections is the primary means for visualizing structures within cells. In this issue of Structure, Peng et al. extend this approach by using electron tomography to examine the life cycle of a herpesvirus and provide fresh insights into the processes of DNA packaging and release. Transmission electron microscopy of thin sections is the primary means for visualizing structures within cells. In this issue of Structure, Peng et al. extend this approach by using electron tomography to examine the life cycle of a herpesvirus and provide fresh insights into the processes of DNA packaging and release. Transmission electron microscopy (TEM) of sectioned, embedded material remains the most widely used high-resolution method for visualizing structures within cells and is the method of choice for examining virus-infected cells. The basic technique has been essentially unchanged since its inception and despite its proven value, is less than ideal for biological analysis, as it gives only restricted views of single time points of heavily fixed and processed samples. Consequently, the resulting pictures need to be interpreted imaginatively but cautiously. In many respects, TEM has lagged behind the advances in other visualization techniques, such as confocal fluorescent light microscopy, which can now provide multicolored, real-time images of living cells in 3D. The difficulty in placing observed structures in their correct spatial context is one of the major limitations of TEM. Conventional TEM produces images that are two-dimensional projections of the specimen being observed, in which all features along the illumination axis are superimposed to form the final image. This is a particular problem in thick samples; to minimize this effect, TEM is typically carried out on very thin sections. However, the improved clarity is achieved at the expense of the information on the overall architecture and spatial relatedness of structures in the specimen. Electron tomography (ET), where different angular views are combined to produce a 3D map of a subject, has long been recognized as offering a way around this problem by allowing detailed reconstruction of relatively thick sections. Increasing access to modern microscopes with high tilt capability and digital imaging systems means that this approach is now being applied to an ever-expanding variety of questions. In their paper in this issue of Structure, Peng et al., 2010Peng L. Ryazantsev S. Sun R. Zhou Z.H. Structure. 2010; 18 (this issue): 47-58Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar have used ET to examine herpesvirus-infected cells, and their images reveal previously unseen aspects of the virus life cycle. Herpesviruses provide good models for an ET study because they form large distinctive viral particles and their infectious cycles have been well-characterized using conventional TEM (Mettenleiter et al., 2009Mettenleiter T.C. Klupp B.G. Granzow H. Virus Research. 2009; 143: 222-234Crossref PubMed Scopus (301) Google Scholar). The study by Peng et al., 2010Peng L. Ryazantsev S. Sun R. Zhou Z.H. Structure. 2010; 18 (this issue): 47-58Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar examines all stages of the virus life cycle, and provides informative insights into several aspects of the infectious process. Perhaps their most notable observations concern the processes of DNA packaging and release. One of their most striking images is of DNA leaving the capsid and passing into the nucleus through a nuclear pore. This process (Figure 1A) has been assumed to occur (Batterson et al., 1983Batterson W. Furlong D. Roizman B. J. Virol. 1983; 45: 397-407Crossref PubMed Google Scholar) but this image appears to be the first time the DNA has been visualized directly and is a clear example of the advantage of using ET in comparison to conventional TEM. Although it feels counterintuitive, an ET of relatively thick sections produces better discrimination of detail than a TEM of thinner sections, since the 3D model produced can be digitally sliced to any desired thickness. In this case, 1 nm digital slices were used, and the ability to track DNA molecules by examining successive views allows them to be traced throughout the volume of the 3D density. This is clearly illustrated by the animation provided in the supplemental information for this study, and in the example shown, the virus genome can be traced from inside the capsid through the nuclear pore to which the capsid is bound. Interestingly, the capsid docks 40 nm from the surface of the pore, requiring the seemingly naked DNA to traverse the intervening space. The observed binding of pore filaments to capsid vertices suggests a receptor-recognition type of interaction, but how release of the DNA is initiated and what drives its translocation will require further analysis. The behavior of the virus DNA during packaging has also been examined, although the findings in this case are less definitive. The internal scaffold is a transient structure that is required during assembly of the capsid shell but is lost at some stage during the process of DNA packaging. One unresolved question is whether removal of the scaffold precedes or is concurrent with entry of the DNA. The images recorded by Peng et al., 2010Peng L. Ryazantsev S. Sun R. Zhou Z.H. Structure. 2010; 18 (this issue): 47-58Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar seem to support both possibilities, with DNA visualized in the process of entering both empty and scaffold-containing capsids. Several images appear to show DNA wound around a distorted scaffold. Interestingly, low pH has been shown to destabilize a herpesvirus scaffold structure (McClelland et al., 2002McClelland D.A. Aitken J.D. Bhella D. McNab D. Mitchell J. Kelly S.M. Price N.C. Rixon F.J. J. Virol. 2002; 76: 7407-7417Crossref PubMed Scopus (33) Google Scholar), and these pictures, which demonstrate direct contact with the DNA, may illustrate the means by which scaffold disruption is achieved within individual particles (Figure 1B). Despite the new insights provided by these images, it is important to consider the limitations of this approach. A major concern is the need for harsh methods of specimen preparation inherent in most TEM studies. The samples used here have been chemically fixed, dehydrated, embedded in resin, and stained with heavy metals before being viewed. The potential for producing artifacts under these conditions is well known and is acknowledged by the authors. These concerns might have been somewhat mitigated if they had used high-pressure vitrification and freeze substitution, but the underlying problem would have remained. Indeed, some of the images shown here are reminiscent of the now discredited toroidal model, which proposed that the packaged viral genome was wound around a central protein core. This was later shown to be caused by the preparation methods used, and the true nature of the packaged genome was eventually revealed by examining virus particles in their native state as vitrified specimens (Booy et al., 1991Booy F.P. Newcomb W.W. Trus B.L. Brown J.C. Baker T.S. Steven A.C. Cell. 1991; 64: 1007-1015Abstract Full Text PDF PubMed Scopus (220) Google Scholar, Zhou et al., 1999Zhou Z.H. Chen D.H. Jakana J. Rixon F.J. Chiu W. J. Virol. 1999; 73: 3210-3218Crossref PubMed Google Scholar). The ability to carry out ET studies on vitrified eukaryotic cells would remove concerns regarding preparation artifacts and is being vigorously pursued by a number of research groups. Unfortunately, although pioneering studies have shown the potential of this approach (Maurer et al., 2008Maurer U.E. Sodeik B. Grunewald K. Proc. Natl. Acad. Sci. USA. 2008; 105: 10559-10564Crossref PubMed Scopus (133) Google Scholar, Leis et al., 2009Leis A. Rockel B. Andrees L. Baumeister W. Trends Biochem. Sci. 2009; 34: 60-70Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), its general use will remain limited until the technical challenges of sectioning vitrified material lessen, and the methods become more tractable. While awaiting such developments, or until X-ray microscopy is further advanced, the tomographic analysis of resin sections is likely to grow in importance as a general method for high-resolution 3D analysis. Peng et al., 2010Peng L. Ryazantsev S. Sun R. Zhou Z.H. Structure. 2010; 18 (this issue): 47-58Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar are to be congratulated for again demonstrating its potential in this interesting study. Three-Dimensional Visualization of Gammaherpesvirus Life Cycle in Host Cells by Electron TomographyPeng et al.StructureJanuary 13, 2010In BriefGammaherpesviruses are etiologically associated with human tumors. A three-dimensional (3D) examination of their life cycle in the host is lacking, significantly limiting our understanding of the structural and molecular basis of virus-host interactions. Here, we report the first 3D visualization of key stages of the murine gammaherpesvirus 68 life cycle in NIH 3T3 cells, including viral attachment, entry, assembly, and egress, by dual-axis electron tomography. In particular, we revealed the transient processes of incoming capsids injecting viral DNA through nuclear pore complexes and nascent DNA being packaged into progeny capsids in vivo as a spool coaxial with the putative portal vertex. Full-Text PDF Open Archive