In addition to vaccines, noninfectious virus-like particles (VLPs) that mimic the viral capsid show an attractive possibility of presenting immunogenic epitopes or targeting molecules on their surface.Here, functionalization of norovirus-derived VLPs by simple non-covalent conjugation of various molecules is shown. By using the affinity between a surface-exposed polyhistidine-tag and multivalent tris-nitrilotriacetic acid (trisNTA), fluorescent dye molecules and streptavidin-biotin conjugated to trisNTA are displayed on the VLPs to demonstrate the use of these VLPs as easily modifiable nanocarriers as well as a versatile vaccine platform. The VLPs are able to enter and deliver surface-displayed fluorescent dye into HEK293T cells via a surface-attached cell internalization peptide (VSV-G). The ease of manufacturing, the robust structure of these VLPs, and the straightforward conjugation provide a technology, which can be adapted to various applications in biomedicine. Manuscript Click here to view linked References

Aim To design a theranostic capsule using the virus-like nanoparticle of the hepatitis E virus modified to display breast cancer cell targeting functional group (LXY30). Methods Five surface-exposed residues were mutated to cysteine to allow conjugation to maleimide-linked chemical groups via thiol-selective linkages. Engineered virus-like nanoparticles were then covalently conjugated to a breast cancer recognized ligand, LXY30 and an amine-coupled near-infrared fluorescence dye. Results LXY30-HEV VLP was checked for its binding and entry to a breast cancer cell line and for tumor targeting in vivo to breast cancer tissue in mice. The engineered virus-like nanoparticle not only targeted cancer cells, but also appeared immune silent to native hepatitis E virus antibodies due to epitope disruption at the antibody-binding site. Conclusion These results demonstrate the production of a theranostic capsule suitable for cancer diagnostics and therapeutics based on surface modification of a highly stable virus-like nanoparticle.

Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au102 pMBA44 (Au102) was an ideal conjugate given its small 2.5 nm size and detectability in cryoEM. Au102 was bound directly to nanocapsid surface cysteines via direct ligand exchange. In addition, Au102 was functionalized with a maleimide linker (Au102_C6MI) for maleimide-thiol conjugation to nanocapsid cysteines. The AuNC-bound nanocapsid constructs were conjugated in various conditions. We found Au102_C6MI to bind nanocapsid more efficiently, while Au102 remained more soluble over time. Nanocapsids conjugated to Au102_C6MI were imaged in cryoEM for single particle reconstruction to localize AuNC position on the nanocapsid surface. We resolved five unique high intensity volumes that formed a ring-shaped density at the 5-fold symmetry center. This finding was further supported by independent rigid modeling.

Nanoparticle diagnostics and therapeutics (nanotheranostics) have significantly advanced cancer detection and treatment. However, many nanotheranostics are ineffective due to defects in tumor localization and bioavailability. An engineered Hepatitis E Virus (HEV) nanocapsid is a proposed platform for targeted cancer-cell delivery. Self-assembling from HEV capsid subunits, nanocapsids retain the capacity to enter cells and resist proteolytic/acidic conditions, but lack infectious viral elements. The nanocapsid surface was modified for chemical activation to confer tumor-specific targeting and detection, immune-response manipulation and controlled theranostic delivery. Nanotheranostic molecules can be packaged in the hollow nanocapsid shell during in vitro assembly. Complementing the adapted stability and cell-entry characteristics of the HEV capsid, a modified nanocapsid serves as a tunable tumor-targeting platform for nanotheronostic delivery.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly. Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

Enterovirus B species typically cause a rapid cytolytic infection leading to efficient release of progeny viruses. However, they are also capable of persistent infections in tissues, which are suggested to contribute to severe chronic states such as myocardial inflammation and type 1 diabetes. In order to understand the factors contributing to differential infection strategies, we constructed a chimera by combining the capsid proteins from fast-cytolysis-causing echovirus 1 (EV1) with nonstructural proteins from coxsackievirus B5 (CVB5), which shows persistent infection in RD cells. The results showed that the chimera behaved similarly to parental EV1, leading to efficient cytolysis in both permissive A549 and semipermissive RD cells. In contrast to EV1 and the chimera, CVB5 replicated slowly in permissive cells and showed persistent infection in semipermissive cells. However, there was no difference in the efficiency of uptake of CVB5 in A549 or RD cells in comparison to the chimera or EV1. CVB5 batches constantly contained significant amounts of empty capsids, also in comparison to CVB5's close relative CVB3. During successive passaging of batches containing only intact CVB5, increasing amounts of empty and decreasing amounts of infective capsids were produced. Our results demonstrate that the increase in the amount of empty particles and the lowering of the amount of infective particles are dictated by the CVB5 structural proteins, leading to slowing down of the infection between passages. Furthermore, the key factor for persistent infection is the small amount of infective particles produced, not the high number of empty particles that accumulate. IMPORTANCE Enteroviruses cause several severe diseases, with lytic infections that lead to rapid cell death but also persistent infections that are more silent and lead to chronic states of infection. Our study compared a cytolytic echovirus 1 infection to persistent coxsackievirus B5 infection by making a chimera with the structural proteins of echovirus 1 and the nonstructural proteins of coxsackievirus B5. Coxsackievirus B5 infection was found to lead to the production of a high number of empty viruses (empty capsids) that do not contain genetic material and are unable to continue the infection. Coinciding with the high number of empty capsids, the amount of infective virions decreased. This characteristic property was not observed in the constructed chimera virus, suggesting that structural proteins are in charge of these phenomena. These results shed light on the mechanisms that may cause persistent infections. Understanding events leading to efficient or inefficient infections is essential in understanding virus-caused pathologies.

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