Cryo-Electron Tomography of the Herpesvirus Procapsid Reveals Interactions of the Portal with the Scaffold and a Shift on Maturation.

Herpes simplex virus 1 (HSV-1) requires seven proteins to package its genome through a vertex in its capsid, one of which is the portal protein, pUL6. The portal protein is also thought to facilitate assembly of the procapsid. While the portal has been visualized in mature capsids, we aimed to elucidate its role in the assembly and maturation of procapsids using cryo-electron tomography (cryoET). We identified the portal vertex in individual procapsids, calculated a subtomogram average, and compared that with the portal vertex in empty mature capsids (A-capsids). The resulting maps show the portal on the interior surface with its narrower end facing outwards, while maintaining close contact with the capsid shell. In the procapsid, the portal is embedded in the underlying scaffold, suggesting that assembly involves a portal-scaffold complex. During maturation, the capsid shell angularizes with a corresponding outward movement of the vertices. We found that in A-capsids, the portal translocates outward further than the adjacent capsomers and strengthens its contacts with the capsid shell. Our methodology also allowed us to determine the number of portal vertices in each capsid, with most having one per capsid, but some none or two, and rarely three. The predominance of a single portal per capsid supports facilitation of the assembly of the procapsid.IMPORTANCE Herpes simplex virus 1 (HSV-1) infects a majority of humans, causing mostly mild disease but in some cases progressing toward life-threatening encephalitis. Understanding the life cycle of the virus is important to devise countermeasures. Production of the virion starts with the assembly of an icosahedral procapsid, which includes DNA packaging proteins at a vertex, one of which is the dodecameric portal protein. The procapsid then undergoes maturation and DNA packaging through the portal, driven by a terminase complex. We used cryo-electron tomography to visualize the portal in procapsids and compare them to mature empty capsids. We found the portal located inside one vertex interacting with the scaffold protein in the procapsid. On maturation, the scaffold is cleaved and dissociates, the capsid angularizes, and the portal moves outward, interacting closely with the capsid shell. These transformations may provide a basis for the development of drugs to prevent HSV-1 infections.

Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity.

Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

Structure analysis of the major capsid proteins of human polyomaviruses 6 and 7 reveals an obstructed sialic acid binding site.

UNLABELLED: Human polyomavirus 6 (HPyV6) and HPyV7 are commonly found on human skin. We have determined the X-ray structures of their major capsid protein, VP1, at resolutions of 1.8 and 1.7 Å, respectively. In polyomaviruses, VP1 commonly determines antigenicity as well as cell-surface receptor specificity, and the protein is therefore linked to attachment, tropism, and ultimately, viral pathogenicity. The structures of HPyV6 and HPyV7 VP1 reveal uniquely elongated loops that cover the bulk of the outer virion surfaces, obstructing a groove that binds sialylated glycan receptors in many other polyomaviruses. In support of this structural observation, interactions of VP1 with α2,3- and α2,6-linked sialic acids could not be detected in solution by nuclear magnetic resonance spectroscopy. Single-cell binding studies indicate that sialylated glycans are likely not required for initial attachment to cultured human cells. Our findings establish distinct antigenic properties of HPyV6 and HPyV7 capsids and indicate that these two viruses engage nonsialylated receptors. IMPORTANCE: Eleven new human polyomaviruses, including the skin viruses HPyV6 and HPyV7, have been identified during the last decade. In contrast to better-studied polyomaviruses, the routes of infection, cell tropism, and entry pathways of many of these new viruses remain largely mysterious. Our high-resolution X-ray structures of major capsid proteins VP1 from HPyV6 and from HPyV7 reveal critical differences in surface morphology from those of all other known polyomavirus structures. A groove that engages specific sialic acid-containing glycan receptors in related polyomaviruses is obstructed, and VP1 of HPyV6 and HPyV7 does not interact with sialylated compounds in solution or on cultured human cells. A comprehensive comparison with other structurally characterized polyomavirus VP1 proteins enhances our understanding of molecular determinants that underlie receptor specificity, antigenicity, and, ultimately, pathogenicity within the polyomavirus family and highlight the need for structure-based analysis to better define phylogenetic relationships within the growing polyomavirus family and perhaps also for other viruses.

Mutations in the GM1 binding site of simian virus 40 VP1 alter receptor usage and cell tropism.

Polyomaviruses are nonenveloped viruses with capsids composed primarily of 72 pentamers of the viral VP1 protein, which forms the outer shell of the capsid and binds to cell surface oligosaccharide receptors. Highly conserved VP1 proteins from closely related polyomaviruses recognize different oligosaccharides. To determine whether amino acid changes restricted to the oligosaccharide binding site are sufficient to determine receptor specificity and how changes in receptor usage affect tropism, we studied the primate polyomavirus simian virus 40 (SV40), which uses the ganglioside GM1 as a receptor that mediates cell binding and entry. Here, we used two sequential genetic screens to isolate and characterize viable SV40 mutants with mutations in the VP1 GM1 binding site. Two of these mutants were completely resistant to GM1 neutralization, were no longer stimulated by incorporation of GM1 into cell membranes, and were unable to bind to GM1 on the cell surface. In addition, these mutant viruses displayed an infection defect in monkey cells with high levels of cell surface GM1. Interestingly, one mutant infected cells with low cell surface GM1 more efficiently than wild-type virus, apparently by utilizing a different ganglioside receptor. Our results indicate that a small number of mutations in the GM1 binding site are sufficient to alter ganglioside usage and change tropism, and they suggest that VP1 divergence is driven primarily by a requirement to accommodate specific receptors. In addition, our results suggest that GM1 binding is required for vacuole formation in permissive monkey CV-1 cells. Further study of these mutants will provide new insight into polyomavirus entry, pathogenesis, and evolution.