Seeing the portal in herpes simplex virus type 1 B capsids.
ABSTRACT Resolving the nonicosahedral components in large icosahedral viruses remains a technical challenge in structural virology. We have used the emerging technique of Zernike phase-contrast electron cryomicroscopy to enhance the image contrast of ice-embedded herpes simplex virus type 1 capsids. Image reconstruction enabled us to retrieve the structure of the unique portal vertex in the context of the icosahedral capsid and, for the first time, show the subunit organization of a portal in a virus infecting eukaryotes. Our map unequivocally resolves the 12-subunit portal situated beneath one of the pentameric vertices, thus removing uncertainty over the location and stoichiometry of the herpesvirus portal.
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ABSTRACT: With just one eighth the size of the major capsid protein (MCP), the smallest capsid protein (SCP) of human tumor herpesviruses-Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV)-is vital to capsid assembly, yet its mechanism of action is unknown. Here, by cryoEM of KSHV at 6-Å resolution, we show that SCP forms a crown on each hexon and uses a kinked helix to cross-link neighboring MCP subunits. SCP-null mutation decreased viral titer by 1,000 times and impaired but did not fully abolish capsid assembly, indicating an important but nonessential role of SCP. By truncating the C-terminal half of SCP and performing cryoEM reconstruction, we demonstrate that SCP's N-terminal half is responsible for the observed structure and function whereas the C-terminal half is flexible and dispensable. Serial truncations further highlight the critical importance of the N-terminal 10 aa, and cryoEM reconstruction of the one with six residues truncated localizes the N terminus of SCP in the cryoEM density map and enables us to construct a pseudoatomic model of SCP. Fitting of this SCP model and a homology model for the MCP upper domain into the cryoEM map reveals that SCP binds MCP largely via hydrophobic interactions and the kinked helix of SCP bridges over neighboring MCPs to form noncovalent cross-links. These data support a mechanistic model that tumor herpesvirus SCP reinforces the capsid for genome packaging, thus acting as a cementing protein similar to those found in many bacteriophages.Proceedings of the National Academy of Sciences 02/2015; DOI:10.1073/pnas.1420317112 · 9.81 Impact Factor
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ABSTRACT: Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.PLoS Biology 12/2014; 12(12):e1002024. DOI:10.1371/journal.pbio.1002024 · 11.77 Impact Factor
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ABSTRACT: Oncolytic virus (OV) is a genetically engineered virus that can selectively replicate in and kill tumor cells while not harming normal cells. OV therapy has been explored as a treatment for numerous cancers including glioblastoma, an aggressive and devastating brain tumor. Experiments show that extracellular matrix protein CCN1 limits OV therapy of glioma by orchestrating an antiviral response and enhancing the proinflammatory activation and migration of macrophages. Neutralizing CCN1 by antibody has been demonstrated to improve OV spread and tends to increase the time to disease progression. In this paper, we develop a mathematical model to investigate the effects of CCN1 on the treatment of glioma with oncolytic herpes simplex virus. We show that numerical simulations of the model are in agreement with the experimental results and then use the model to explore the anti-tumor effects of combining antibodies with OV therapy. Model simulations suggest that the macrophage content of the tumor is a critical factor to the success of OV therapy and to the reduction in tumor volume gained with the CCN1 antibody.Bulletin of Mathematical Biology 03/2015; DOI:10.1007/s11538-015-0074-8 · 1.29 Impact Factor