A pathogen's ability to engage host receptors is a critical determinant of its host range and interspecies transmissibility, key issues for understanding emerging diseases. However, the identification of host receptors, which are also attractive drug targets, remains a major challenge. Our structural bioinformatics studies reveal that both bacterial and viral pathogens have evolved to structurally mimic native host ligands (ligand mimicry), thus enabling engagement of their cognate host receptors. In contrast to the structural homology, amino acid sequence similarity between pathogen molecules and the mimicked host ligands was low. We illustrate the utility of this concept to identify pathogen receptors by delineating receptor tyrosine kinase Axl as a candidate receptor for the polyomavirus SV40. The SV40-Axl interaction was validated, and its participation in the infection process was verified. Our results suggest that ligand mimicry is widespread, and we present a quick tool to screen for pathogen-host receptor interactions.

Crystallization is a fundamental and ubiquitous process that is well understood in the case of atoms or small molecules, but its outcome is still hard to predict in the case of nanoparticles or macromolecular complexes. Controlling the organization of virus nanoparticles into a variety of 3D supramolecular architectures is often done by multivalent ions and is of great interest for biomedical applications such as drug or gene delivery and biosensing, as well as for bionanomaterials and catalysis. In this paper, we show that slow dialysis, over several hours, of wild-type Simian Virus 40 (wt SV40) nanoparticle solution against salt solutions containing MgCl, with or without added NaCl, results in wt SV40 nanoparticles arranged in a body cubic center crystal structure with Im3m space group, as a thermodynamic product, in coexistence with soluble wt SV40 nanoparticles. The nanoparticle crystals formed above a critical MgCl concentrations. Reentrant melting and resolubilization of the virus nanoparticles took place when the MgCl concentrations passed a second threshold. Using synchrotron solution X-ray scattering we determined the structures and the mass fraction of the soluble and crystal phases as a function of MgCl and NaCl concentrations. A thermodynamic model, which balances the chemical potentials of the Mg ions in each of the possible states, explains our observations. The model reveals the mechanism of both the crystallization and the reentrant melting and resolubilization and shows that counterion entropy is the main driving force for both processes.

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.

The canonical gate of viruses and viral genomes into the nucleus in non-dividing cells is the nuclear pore, embedded within the nuclear envelope. However, we found that for SV40, the nuclear envelope poses a major hurdle to infection: FISH analysis revealed that the majority of viral DNA remains trapped in the ER; silencing of Lamin A/C rendered the cells more susceptible to infection; and proliferating cells are more susceptible to infection than quiescent cells. Surprisingly, we observed that following SV40 infection the nuclear envelope, including lamins A/C, B1, B2 and the nuclear pore complex, was dramatically deformed, as seen by immunohistochemistry. The infection induced fluctuations in the level of lamin A/C, dephosphorylation of an unknown epitope and leakage to the cytoplasm just prior to and during nuclear entry. Deformations were transient, and the spherical structure of the nuclear envelope was restored subsequent to nuclear entry. Nuclear envelope deformations and lamin A/C dephosphorylation depended on caspase-6 cleavage of lamin A/C. Notably, we have previously reported that inhibition of caspase-6 abolishes SV40 infection. Taken together the results suggest that alterations of the nuclear lamina, induced by the infecting virus, are involved in the nuclear entry of the SV40 genome. We propose that SV40 utilize this unique, previously unknown mechanism for direct trafficking of its genome from the ER to the nucleus. As SV40 serves as a paradigm for the pathogenic human BK, JC and Merkel cell polyomavirus, this study suggests nuclear entry as a novel drug target for these infections.

Simian virus 40 (SV40) capsid assembly occurs in the nucleus. All three capsid proteins bind DNA nonspecifically, raising the dilemma of how they attain specificity to the SV40 minichromosome in the presence of a large excess of genomic DNA. The SV40 packaging signal, ses, which is required for assembly, is composed of multiple DNA elements that bind transcription factor Sp1. Our previous studies showed that Sp1 participates in SV40 assembly and that it cooperates in DNA binding with VP2/3. We hypothesized that Sp1 recruits the capsid proteins to the viral minichromosome, conferring upon them specific DNA recognition. Here, we have tested the hypothesis. Computer analysis showed that the combination of six tandem GC boxes at ses is not found at cellular promoters and therefore is unique to SV40. Cooperativity in DNA binding between Sp1 and VP2/3 was not abolished at even a 1,000-fold excess of cellular DNA, providing strong support for the recruitment hypothesis. Sp1 also binds VP1 and cooperates with VP1 in DNA binding. VP1 pentamers (VP1 5 ) avidly interact with VP2/3, utilizing the same VP2/3 domain as described for polyomavirus. We conclude that VP1 5 -VP2/3 building blocks are recruited by Sp1 to ses, where they form the nucleation center for capsid assembly. By this mechanism the virus ensures that capsid formation is initiated at a single site around its minichromosome.