Atomic Resolution Structure of the Oncolytic Parvovirus LuIII by Electron Microscopy and 3D Image Reconstruction.

LuIII, a protoparvovirus pathogenic to rodents, replicates in human mitotic cells, making it applicable for use to kill cancer cells. This virus group includes H-1 parvovirus (H-1PV) and minute virus of mice (MVM). However, LuIII displays enhanced oncolysis compared to H-1PV and MVM, a phenotype mapped to the major capsid viral protein 2 (VP2). This suggests that within LuIII VP2 are determinants for improved tumor lysis. To investigate this, the structure of the LuIII virus-like-particle was determined using single particle cryo-electron microscopy and image reconstruction to 3.17 Å resolution, and compared to the H-1PV and MVM structures. The LuIII VP2 structure, ordered from residue 37 to 587 (C-terminal), had the conserved VP topology and capsid morphology previously reported for other protoparvoviruses. This includes a core ß-barrel and α-helix A, a depression at the icosahedral 2-fold and surrounding the 5-fold axes, and a single protrusion at the 3-fold axes. Comparative analysis identified surface loop differences among LuIII, H-1PV, and MVM at or close to the capsid 2- and 5-fold symmetry axes, and the shoulder of the 3-fold protrusions. The 2-fold differences cluster near the previously identified MVM sialic acid receptor binding pocket, and revealed potential determinants of protoparvovirus tumor tropism.

Imaging and Quantitation of a Succession of Transient Intermediates Reveal the Reversible Self-Assembly Pathway of a Simple Icosahedral Virus Capsid.

Understanding the fundamental principles underlying supramolecular self-assembly may facilitate many developments, from novel antivirals to self-organized nanodevices. Icosahedral virus particles constitute paradigms to study self-assembly using a combination of theory and experiment. Unfortunately, assembly pathways of the structurally simplest virus capsids, those more accessible to detailed theoretical studies, have been difficult to study experimentally. We have enabled the in vitro self-assembly under close to physiological conditions of one of the simplest virus particles known, the minute virus of mice (MVM) capsid, and experimentally analyzed its pathways of assembly and disassembly. A combination of electron microscopy and high-resolution atomic force microscopy was used to structurally characterize and quantify a succession of transient assembly and disassembly intermediates. The results provided an experiment-based model for the reversible self-assembly pathway of a most simple (T = 1) icosahedral protein shell. During assembly, trimeric capsid building blocks are sequentially added to the growing capsid, with pentamers of building blocks and incomplete capsids missing one building block as conspicuous intermediates. This study provided experimental verification of many features of self-assembly of a simple T = 1 capsid predicted by molecular dynamics simulations. It also demonstrated atomic force microscopy imaging and automated analysis, in combination with electron microscopy, as a powerful single-particle approach to characterize at high resolution and quantify transient intermediates during supramolecular self-assembly/disassembly reactions. Finally, the efficient in vitro self-assembly achieved for the oncotropic, cell nucleus-targeted MVM capsid may facilitate its development as a drug-encapsidating nanoparticle for anticancer targeted drug delivery.

Quantitative nanoscale electrostatics of viruses.

Electrostatics is one of the fundamental driving forces of the interaction between biomolecules in solution. In particular, the recognition events between viruses and host cells are dominated by both specific and non-specific interactions and the electric charge of viral particles determines the electrostatic force component of the latter. Here we probe the charge of individual viruses in liquid milieu by measuring the electrostatic force between a viral particle and the Atomic Force Microscope tip. The force spectroscopy data of co-adsorbed ϕ29 bacteriophage proheads and mature virions, adenovirus and minute virus of mice capsids is utilized for obtaining the corresponding density of charge for each virus. The systematic differences of the density of charge between the viral particles are consistent with the theoretical predictions obtained from X-ray structural data. Our results show that the density of charge is a distinguishing characteristic of each virus, depending crucially on the nature of the viral capsid and the presence/absence of the genetic material.

Parvoviral host range and cell entry mechanisms.

Parvoviruses elaborate rugged nonenveloped icosahedral capsids of approximately 260 A in diameter that comprise just 60 copies of a common core structural polypeptide. While serving as exceptionally durable shells, capable of protecting the single-stranded DNA genome from environmental extremes, the capsid also undergoes sequential conformational changes that allow it to translocate the genome from its initial host cell nucleus all the way into the nucleus of its subsequent host. Lacking a duplex transcription template, the virus must then wait for its host to enter S-phase before it can initiate transcription and usurp the cell's synthetic pathways. Here we review cell entry mechanisms used by parvoviruses. We explore two apparently distinct modes of host cell specificity, first that used by Minute virus of mice, where subtle glycan-specific interactions between host receptors and residues surrounding twofold symmetry axes on the virion surface mediate differentiated cell type target specificity, while the second involves novel protein interactions with the canine transferrin receptor that allow a mutant of the feline leukopenia serotype, Canine parvovirus, to bind to and infect dog cells. We then discuss conformational shifts in the virion that accompany cell entry, causing exposure of a capsid-tethered phospholipase A2 enzymatic core that acts as an endosomolytic agent to mediate virion translocation across the lipid bilayer into the cell cytoplasm. Finally, we discuss virion delivery into the nucleus, and consider the nature of transcriptionally silent DNA species that, escaping detection by the cell, might allow unhampered progress into S-phase and hence unleash the parvoviral Trojan horse.

Kinetics of assembly of a parvovirus, minute virus of mice, in synchronized rat brain cells.

The rates of assembly of the three classes of particles of minute virus of mice were examined in synchronized rat brain cells by a combination of electron microscopy and biochemical techniques. We observed a burst of virus assembly beginning about 8 h after the end of cellular S phase. Labeled thymidine incorporated into the 1.46 g/cm3 class of full virus particles was transferred almost quantitatively to the 1.42 g/cm3 class. The 1.46 g/cm3 virus appeared to be an immediate precursor to the 1.42 g/cm3 class. Conversion of the 1.46 density virus to the 1.42 density particles was observed at the time of virus assembly. The processing was rapid and occurred primarily in the nucleus. Infected cells did not contain significant pools of viral DNA in a form that could be encapsulated in the absence of DNA synthesis. The role of the empty virus capsids in the assembly process is discussed.