Binding of hnRNP I-vRNA Regulates Sindbis Virus Structural Protein Expression to Promote Particle Infectivity.

Alphaviruses cause significant outbreaks of febrile illness and debilitating multi-joint arthritis for prolonged periods after initial infection. We have previously reported that several host hnRNP proteins bind to the Sindbis virus (SINV) RNAs, and disrupting the sites of these RNA-protein interactions results in decreased viral titers in tissue culture models of infection. Intriguingly, the primary molecular defect associated with the disruption of the hnRNP interactions is enhanced viral structural protein expression; however, the precise underlying mechanisms spurring the enhanced gene expression remain unknown. Moreover, our previous efforts were unable to functionally dissect whether the observed phenotypes were due to the loss of hnRNP binding or the incorporation of polymorphisms into the primary nucleotide sequence of SINV. To determine if the loss of hnRNP binding was the primary cause of attenuation or if the disruption of the RNA sequence itself was responsible for the observed phenotypes, we utilized an innovative protein tethering approach to restore the binding of the hnRNP proteins in the absence of the native interaction site. Specifically, we reconstituted the hnRNP I interaction by incorporating the 20nt bovine immunodeficiency virus transactivation RNA response (BIV-TAR) at the site of the native hnRNP I interaction sequence, which will bind with high specificity to proteins tagged with a TAT peptide. The reestablishment of the hnRNP I-vRNA interaction via the BIV-TAR/TAT tethering approach restored the phenotype back to wild-type levels. This included an apparent decrease in structural protein expression in the absence of the native primary nucleotide sequences corresponding to the hnRNP I interaction site. Collectively, the characterization of the hnRNP I interaction site elucidated the role of hnRNPs during viral infection.

Mutations at the Alphavirus E1'-E2 Interdimer Interface Have Host-Specific Phenotypes.

Alphaviruses are enveloped viruses transmitted by arthropod vectors to vertebrate hosts. The surface of the virion contains 80 glycoprotein spikes embedded in the membrane, and these spikes mediate attachment to the host cell and initiate viral fusion. Each spike consists of a trimer of E2-E1 heterodimers. These heterodimers interact at the following two interfaces: (i) the intradimer interactions between E2 and E1 of the same heterodimer and (ii) the interdimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (E1'). We hypothesized that the interdimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus (chikungunya piggyback [CPB]) where we replaced six interdimeric residues in the E2 protein of Sindbis virus (wild-type [WT] SINV) with those from the E2 protein from chikungunya virus and studied its effect in both mammalian and mosquito cell lines. CPB produced fewer infectious particles in mammalian cells than in mosquito cells, relative to WT SINV. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to the capsid protein and contained defects in particle morphology compared with virus derived from mosquito cells. Using cryo-electron microscopy (cryo-EM), we determined that the spikes of CPB had a different conformation than WT SINV. Last, we identified two revertants, E2-H333N and E1-S247L, that restored particle growth and assembly to different degrees. We conclude the interdimer interface is critical for spike trimerization and is a novel target for potential antiviral drug design. IMPORTANCE Alphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as emerging pathogens, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This finding is important because it identifies a region in the spike that could be a target for antiviral drug design.

Revisiting an old friend: new findings in alphavirus structure and assembly.

Alphaviruses are transmitted by an arthropod vector to a vertebrate host. The disease pathologies, cellular environments, immune responses, and host factors are very different in these organisms. Yet, the virus is able to infect, replicate, and assemble into new particles in these two animals using one set of genetic instructions. The balance between conserved mechanisms and unique strategies during virus assembly is critical for fitness of the virus. In this review, we discuss new findings in receptor binding, polyprotein topology, nucleocapsid core formation, and particle budding that have emerged in the last five years and share opinions on how these new findings might answer some questions regarding alphavirus structure and assembly.

Virion-Associated Polyamines Transmit with Bunyaviruses to Maintain Infectivity and Promote Entry.

Viruses require host cell metabolites to productively infect, and the mechanisms by which viruses usurp these molecules are diverse. One group of cellular metabolites important in virus infection is the polyamines, small positively charged molecules involved in cell cycle, translation, and nucleic acid metabolism, among other cellular functions. Polyamines support replication of diverse viruses, and they are important for processes such as transcription, translation, and viral protein enzymatic activity. Rift Valley fever virus (RVFV) is a negative and ambisense RNA virus that requires polyamines to produce infectious particles. In polyamine depleted conditions, noninfectious particles are produced that interfere with virus replication and stimulate immune signaling. Here, we find that RVFV relies on virion-associated polyamines to maintain infectivity and enhance viral entry. We show that RVFV replication is facilitated by a limited set of polyamines and that spermidine and closely related molecules associate with purified virions and transmit from cell to cell during infection. Virion-associated spermidine maintains virion infectivity, as virions devoid of polyamines rapidly lose infectivity and are temperature sensitive. Further, virions without polyamines bind to cells but exhibit a defect in entry, requiring more acidic conditions than virions containing spermidine. These data highlight a unique role for polyamines, and spermidine particularly, to maintain virus infectivity. Further, these studies are the first to identify polyamines associated with RVFV virions. Targeting polyamines represents a promising antiviral strategy, and this work highlights a new mechanism by which we can inhibit virus replication through FDA-approved polyamine depleting pharmaceuticals.

An Assembly-Activating Site in the Hepatitis B Virus Capsid Protein Can Also Trigger Disassembly.

The Hepatitis B Virus (HBV) core protein homodimers self-assemble to form an icosahedral capsid that packages the viral genome. Disassembly occurs in the nuclear basket to release the mature genome to the nucleus. Small molecules have been developed that bind to a pocket at the interdimer interface to accelerate assembly and strengthen interactions between subunits; these are under development as antiviral agents. Here, we explore the role of the dimer-dimer interface by mutating sites in the drug-binding pocket to cysteine and examining the effect of covalently linking small molecules to them. We find that ligands bound to the pocket may trigger capsid disassembly in a dose-dependent manner. This result indicates that, at least transiently, the pocket adopts a destabilizing conformation. We speculate that this pocket also plays a role in virus disassembly and genome release by binding ligands that are incompatible with virus stability, "unwanted guests." Investigating protein-protein interactions, especially large protein polymers, offers new and unique challenges. By using an engineered addressable thiol, we provide a means to examine the effects of modifying an interface without requiring drug-like properties for the ligand.

Synthesis of Cross-Linked 2-Aminoethyl Methacrylate in P22 Viral Capsid via Atom-Transfer Radical Polymerization.

Virus-like particles (VLPs) resemble viruses, but are devoid their genetic material, rendering them as noninfectious, hollow protein shells. VLPs are ideal templates to synthesize nanoparticles because they have homogeneous size and their empty cavity can provide a confined environment for selectively directed synthesis. Atom-transfer radical polymerization (ATRP) is well suited for directed synthesis of polymers inside VLPs. In addition to being rapid, monomer-promiscuous, and resulting in products with relatively low polydispersity, the simplicity of the ATRP initiator allows it to be readily modified for amending to biomolecules. This chapter describes the polymerization of 2-aminoethyl methacrylate (AEMA) via ATRP in a viral capsid derived from the bacteriophage P22.

Changes in the stability and biomechanics of P22 bacteriophage capsid during maturation.

The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared.

Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9.

Use of protein cages as a template for confined synthesis of inorganic and organic nanoparticles.

Protein cages are hollow spherical proteins assembled from a defined number of subunits. Because they are extremely homogeneous in size and structure, their interior cavities can serve as ideal templates to encapsulate and synthesize well-defined nanoparticles. Here, we describe the exemplary synthesis of a hard and a soft material in two representative protein cages, i.e., magnetite nanoparticles in ferritin and a poly(2-aminoethyl)methacrylate inside a viral capsid derived from the bacteriophage P22.

Gadolinium-Loaded Viral Capsids as Magnetic Resonance Imaging Contrast Agents.

Polymeric nanohybrid P22 virus capsids were used as templates for high density Gd3+ loading to explore magnetic field-dependent (0.5-7.0 T) proton relaxivity. The field-dependence of relaxivity by the spatially constrained Gd3+ in the capsids was similar when either the loading of the capsids or the concentration of capsids was varied. The ionic longitudinal relaxivity, r1, decreased from 25-32 mM-1 s-1 at 0.5 T to 6-10 mM-1 s-1 at 7 T. The ionic transverse relaxivity, r2, increased from 28-37 mM-1 s-1 at 0.5 T to 39-50 mM-1 s-1 at 7 T. The r2/r1 ratio increased linearly with increasing magnetic field from about 1 at 0.5 T, which is typical of T1 contrast agents, to 5-8 at 7 T, which is approaching the ratios for T2 contrast agents. Increases in electron paramagnetic resonance line widths at 80 and 150 K and higher microwave powers required for signal saturation indicate enhanced Gd3+ electron spin relaxation rates for the Gd3+-loaded capsids than for low concentration Gd3+. The largest r2/r1 at 7 T was for the highest cage loading, which suggests that Gd3+-Gd3+ interactions within the capsid enhance r2 more than r1.

Manganese(III) porphyrins complexed with P22 virus-like particles as T1-enhanced contrast agents for magnetic resonance imaging.

Virus-like particles are powerful platforms for the development of functional hybrid materials. Here, we have grown a cross-linked polymer (cross-linked aminoethyl methacrylate) within the confines of the bacteriophage P22 capsid (P22-xAEMA) and functionalized the polymer with various loadings of paramagnetic manganese(III) protoporphyrin IX (MnPP) complexes for evaluation as a macromolecular magnetic resonance imaging contrast agent. The resulting construct (P22-xAEMA-MnPP) has r1,particle = 7,098 mM(-1) s(-1) at 298 K and 2.1 T (90 MHz) for a loading of 3,646 MnPP molecules per capsid. The Solomon-Bloembergen-Morgan theory for paramagnetic relaxivity predicts conjugating MnPP to P22, a supramolecular structure, would result in an enhancement in ionic relaxivity; however, all loadings experienced low ionic relaxivities, r 1,ionic, ranging from 1.45 to 3.66 mM(-1) s(-1), similar to the ionic relaxivity of free MnPP. We hypothesize that intermolecular interactions between neighboring MnPP molecules block access of water to the metal site, resulting in low r 1,ionic relaxivities. We investigated the effect of MnPP interactions on relaxivity further by either blocking or exposing water binding sites on MnPP. On the basis of these results, future design strategies for enhanced r 1,ionic relaxivity are suggested. The measured r 2,ionic relaxivities demonstrated an inverse relationship between loading and relaxivity. This results in a loading-dependent r 2/r 1 behavior of these materials indicating synthetic control over the relaxivity properties, making them interesting alternatives to current magnetic resonance imaging contrast agents.

P22 viral capsids as nanocomposite high-relaxivity MRI contrast agents.

Attachment of multiple chelated Gd(3+) ions to the interior of bacteriophage P22 viral capsids affords nanoscale MRI contrast agents with extremely high relaxivity values. Highly fenestrated "wiffleball" morphology is unique to P22 and assures water exchange between the environment and interior cavity of the capsid. The cavity of P22 "wiffleball" was functionalized with a branched oligomer comprising multiple DTPA-Gd complexes resulting in an impressive payload of 1,900 Gd(3+) ions inside each 64 nm capsid. High relaxivities of r(1,ionic) = 21.7 mM(-1) s(-1) and r(1,particle) = 41,300 mM(-1) s(-1) at 298 K, 0.65 T (28 MHz) are reported, with r(1)/r(2) ratio of 0.80 and optimized rotational correlation time for this system. Specific design modifications are suggested for future improvements of viral capsid-based MRI contrast agents directed toward clinical translation.

Use of the interior cavity of the P22 capsid for site-specific initiation of atom-transfer radical polymerization with high-density cargo loading.

Virus-like particles (VLPs) have emerged as important and versatile architectures for chemical manipulation in the development of functional hybrid nanostructures. Here we demonstrate a successful site-selective initiation of atom-transfer radical polymerization reactions to form an addressable polymer constrained within the interior cavity of a VLP. Potentially, this protein-polymer hybrid of P22 and cross-linked poly(2-aminoethyl methacrylate) could be useful as a new high-density delivery vehicle for the encapsulation and delivery of small-molecule cargos. In particular, the encapsulated polymer can act as a scaffold for the attachment of small functional molecules, such as fluorescein dye or the magnetic resonance imaging (MRI) contrast agent Gd-diethylenetriaminepentacetate, through reactions with its pendant primary amine groups. Using this approach, a significant increase in the labelling density of the VLP, compared to that of previous modifications of VLPs, can be achieved. These results highlight the use of multimeric protein-polymer conjugates for their potential utility in the development of VLP-based MRI contrast agents with the possibility of loading other cargos.

Functional virus-based polymer-protein nanoparticles by atom transfer radical polymerization.

Viruses and virus-like particles (VLPs) are useful tools in biomedical research. Their defined structural attributes make them attractive platforms for engineered interactions over large molecular surface areas. In this report, we describe the use of VLPs as multivalent macroinitiators for atom transfer radical polymerization. The introduction of chemically reactive monomers during polymerization provides a robust platform for post-synthetic modification via the copper-catalyzed azide-alkyne cycloaddition reaction. These results provide the basis to construct nanoparticle delivery vehicles and imaging agents using protein-polymer conjugates.