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Infection by human astrovirus (HAstV), a small, positive-strand RNA virus, is a major cause of gastroenteritis and has been implicated in an increasing number of severe, sometimes fatal, neurological diseases since 2008. Currently, there are no vaccines or antiviral treatments available to treat HAstV infection. An attractive target for antiviral therapeutics is the viral protease due to its essential functions throughout infection. However, the molecular mechanisms of the HAstV protease, nonstructural protein 1a/3 (nsp1a/3), are poorly understood. In fact, the specific residues within the cleavage junctions that are targeted by nsp1a/3 during polyprotein processing have yet to be experimentally identified. To identify the junctions between viral proteins, we performed mass spectrometry and site-directed mutagenesis using epitope-tagged viral polyprotein expression plasmids. Using these strategies, we identified a consensus motif that is found throughout the polyprotein near previously proposed junctions. We found that cleavage occurs after a hydrophobic residue - X - Gln motif. Further mutagenesis of surrounding sequence identified the importance of basic residues following the motif for efficient processing. Cleavage at each junction was determined to be essential for the production of progeny virions. However, abolishing nsp1a/4-VPg cleavage allowed efficient replication, suggesting that VPg can function in an intermediate form. Overall, our results identify a conserved cleavage motif that is recognized by the nsp1a/3 protease within the viral polyprotein, and cleavage at this motif was found to be essential for the recovery of progeny virions. These findings will be instrumental in further understanding the basic functions of HAstV polyprotein processing during infection.IMPORTANCEHuman astroviruses (HAstVs) are a leading cause of non-bacterial gastroenteritis in children, elderly individuals, and immunocompromised patients. However, infection by divergent strains of HAstV is now recognized as a causative agent of severe neurological diseases, which can have fatal outcomes. Despite the global prevalence of HAstV, we currently have a limited understanding of the biology of these viruses. Translation of the viral genome leads to the production of polyproteins that are processed by viral and host proteases into functional proteins. In this study, we identified a conserved recognition sequence targeted by the viral protease for cleavage. Importantly, these findings elucidate the N- and C-termini of the nonstructural proteins within the HAstV polyprotein, offering valuable information for future studies on the function of individual viral proteins. Similar to other positive-sense RNA viruses, the necessity of proteolytic processing for the HAstV polyprotein highlights the viral protease as a promising target for antiviral development.

The maintenance of human mitochondrial DNA (mtDNA) is critical for proper cellular function as damage to mtDNA, if left unrepaired, can lead to a diverse array of pathologies. Of the pathways identified to participate in DNA repair within the mitochondria, base excision repair (BER) is the most extensively studied. Protein-protein interactions drive the step-by-step coordination required for the successful completion of this pathway and are important for crosstalk with other mitochondrial factors involved in genome maintenance. Human NEIL1 is one of seven DNA glycosylases that initiates BER in both the nuclear and mitochondrial compartments. In the current work, we scrutinized the interaction between NEIL1 and mitochondrial transcription factor A (TFAM), a protein that is essential for various aspects of mtDNA metabolism. We note, for the first time, that both the N- and C- terminal domains of NEIL1 interact with TFAM revealing a unique NEIL1 protein-binding interface. The interaction between the two proteins, as observed biochemically, appears to be transient and is most apparent at concentrations of low salt. The presence of DNA (or RNA) also positively influences the interaction between the two proteins, and molar mass estimates indicate that duplex DNA is required for complex formation at higher salt concentrations. Hydrogen deuterium exchange mass spectrometry data reveal that both proteins exchange less deuterium upon DNA binding, indicative of an interaction, and the addition of NEIL1 to the TFAM-DNA complex alters the interaction landscape. The transcriptional activity of TFAM appears to be independent of NEIL1 expression under normal cellular conditions, however, in the presence of DNA damage, we observe a significant reduction in the mRNA expression of TFAM-transcribed mitochondrial genes in the absence of NEIL1. Overall, our data indicate that the interaction between NEIL1 and TFAM can be modulated by local environment such as salt concentrations, protein availability, the presence of nucleic acids, as well as the presence of DNA damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sharma, Thompson, Arrington, Terry, Chakravarthy, Prevelige and Prakash.)

Biology shows many examples of spatially controlled assembly of cells and biomacromolecules into hierarchically organized structures, to which many of the complex biological functions are attributed. While such biological structures have inspired the design of synthetic materials, it is still a great challenge to control the spatial arrangement of individual building blocks when assembling multiple types of components into bulk materials. Here, we report self-assembly of multilayered, ordered protein arrays from mixed populations of virus-like particles (VLPs). We systematically tuned the magnitude of the surface charge of the VLPs via mutagenesis to prepare four different types of VLPs for mixing. A mixture of up to four types of VLPs selectively assembled into higher-order structures in the presence of oppositely charged dendrimers during a gradual lowering of the ionic strength of the solution. The assembly resulted in the formation of three-dimensional ordered VLP arrays with up to four distinct layers including a central core, with each layer comprising a single type of VLP. A coarse-grained computational model was developed and simulated using molecular dynamics to probe the formation of the multilayered, core-shell structure. Our findings establish a simple and versatile bottom-up strategy to synthesize multilayered, ordered materials by controlling the spatial arrangement of multiple types of nanoscale building blocks in a one-pot fabrication.

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last 2 decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that (i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication, (ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD, (iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD, (iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD, and HVD phosphorylation is mediated by CK2 kinase, and (v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication. IMPORTANCE Cellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We show that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.

Vesicular stomatitis virus (VSV) is an archetypical member of Mononegavirales , viruses with a genome of negative-sense single-stranded RNA (-ssRNA). Like other viruses of this order, VSV encodes a unique polymerase, a complex of viral L (large, the enzymatic component) protein and P (phosphoprotein, a cofactor component). The L protein has a modular layout consisting of a ring-shaped core trailed by three accessory domains and requires an N-terminal segment of P (P N-terminal disordered [P NTD ]) to perform polymerase activity. To date, a binding site for P on L had not been described. In this report, we show that the connector domain of the L protein, which previously had no assigned function, binds a component of P NTD We further show that this interaction is a positive regulator of viral RNA synthesis, and that the interfaces mediating it are conserved in other members of Mononegavirales Finally, we show that the connector-P interaction fits well into the existing structural information of VSV L. IMPORTANCE This study represents the first functional assignment of the connector domain of a Mononegavirales L protein. Furthermore, this study localizes P polymerase cofactor activity to specific amino acids. The functional necessity of this interaction, combined with the uniqueness of L and P proteins to the order Mononegavirales , makes disruption of the P-connector site a potential target for developing antivirals against other negative-strand RNA viruses. Furthermore, the connector domain as an acceptor site for the P protein represents a new understanding of Mononegavirales L protein biology., (Copyright © 2020 American Society for Microbiology.)

During the late phase of the HIV-1 replication cycle, the viral Gag polyproteins are targeted to the plasma membrane for assembly. The Gag-membrane interaction is mediated by binding of Gag's N-terminal myristoylated matrix (MA) domain to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). The viral envelope (Env) glycoprotein is then recruited to the assembly sites and incorporated into budding particles. Evidence suggests that Env incorporation is mediated by interactions between Gag's MA domain and the cytoplasmic tail of the gp41 subunit of Env (gp41CT). MA trimerization appears to be an obligatory step for this interaction. Insufficient production of a recombinant MA trimer and unavailability of a biologically relevant membrane system have been barriers to detailed structural and biophysical characterization of the putative MA-gp41CT-membrane interactions. Here, we engineered a stable recombinant HIV-1 MA trimer construct by fusing a foldon domain (FD) of phage T4 fibritin to the MA C terminus. Results from NMR experiments confirmed that the FD attachment does not adversely alter the MA structure. Employing hydrogen-deuterium exchange MS, we identified an MA-MA interface in the MA trimer that is implicated in Gag assembly and Env incorporation. Utilizing lipid nanodiscs as a membrane mimetic, we show that the MA trimer binds to membranes 30-fold tighter than does the MA monomer and that incorporation of PI(4,5)P 2 and phosphatidylserine enhances the binding of MA to nanodiscs. These findings advance our understanding of a fundamental mechanism in HIV-1 assembly and provide a template for investigating the interaction of MA with gp41CT., (© 2019 Murphy et al.)

Virus infections are ultimately dependent on a successful viral genome delivery to the host cell. The bacteriophage family Caudovirales evolved specialized machinery that fulfills this function: the portal proteins complex. The complexes are arranged as dodecameric rings and are a structural part of capsids incorporated at a five-fold vertex. They are involved in crucial aspects of viral replication, such as virion assembly, DNA packaging and DNA delivery. This review focuses on the organization and the mechanism through which these portal complexes achieve viral genome delivery and their similarities to other viral portal complexes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The assembly of individual molecules into hierarchical structures is a promising strategy for developing three-dimensional materials with properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular nanostructures, which are themselves hierarchically assembled from a limited number of protein subunits. Here, we demonstrate the bio-inspired modular construction of materials with two levels of hierarchy: the formation of catalytically active individual virus-like particles (VLPs) through directed self-assembly of capsid subunits with enzyme encapsulation, and the assembly of these VLP building blocks into three-dimensional arrays. The structure of the assembled arrays was successfully altered from an amorphous aggregate to an ordered structure, with a face-centered cubic lattice, by modifying the exterior surface of the VLP without changing its overall morphology, to modulate interparticle interactions. The assembly behavior and resultant lattice structure was a consequence of interparticle interaction between exterior surfaces of individual particles and thus independent of the enzyme cargos encapsulated within the VLPs. These superlattice materials, composed of two populations of enzyme-packaged VLP modules, retained the coupled catalytic activity in a two-step reaction for isobutanol synthesis. This study demonstrates a significant step toward the bottom-up fabrication of functional superlattice materials using a self-assembly process across multiple length scales and exhibits properties and function that arise from the interaction between individual building blocks.

Proteins are widely utilized as templates in biomimetic synthesis of gold nanocrystals. However, the role of proteins in mediating the pathways for gold nucleation and growth is not well understood, in part because of the lack of spatial resolution in probing the complicated biomimetic mineralization process. Self-assembled protein cages, with larger size and symmetry, can facilitate in the visualization of both biological and inorganic components. We have utilized bacteriophage P22 protein cages of ∼60 nm diameter for investigating the nucleation and growth of gold nanocrystals. By adding a gold precursor into the solution with preexisting protein cages and a reducing agent, gold nuclei/prenucleation clusters form in solution, which then locate and attach to specific binding sites on protein cages and further grow to form gold nanocrystals. By contrast, addition of the reducing agent into the solution with incubated gold precursor and protein cages leads to the formation of gold nuclei/prenucleation clusters both in solution and on the surface of protein cages that then grow into gold nanocrystals. Because of the presence of cysteine (Cys) with strong gold-binding affinity, gold nanocrystals tend to bind at specific sites of Cys, irrespective of the binding sites of gold ions. Analyzing the results obtained using these alternate routes provide important insights into the pathways of protein-mediated biomimetic nucleation of gold that challenge the importance of incubation, which is widely utilized in the biotemplated synthesis of inorganic nanocrystals.

The portal vertex in dsDNA bacteriophage serves as the site for genome encapsidation and release. In several of these viruses, efficient termination of DNA packaging has been shown to be dependent on the density of packaged DNA. The portal protein has been implicated as being part of the sensor that regulates packaging termination through DNA-dependent conformational changes during packaging. The mechanism by which DNA induces these conformational changes remains unknown. In this study, we explore how point mutants in the portal core can result in changes in genome packaging density in P22. Mutations in the portal core that subtly alter the structure or dynamics of the protein result in an increase in the amount of DNA packaged. The magnitude of the change is amino acid and location specific. Our findings suggest a mechanism wherein compression of the portal core is an essential aspect of signal transmission during packaging., (Copyright © 2017 Elsevier Inc. All rights reserved.)

During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context., Importance: Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity., (Copyright © 2016 American Society for Microbiology.)

Uracil-DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil-DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non-enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus-specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA-binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. The adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three-dimensional structure of D4. An overview of the current state of the knowledge on the structure-function relationship of D4 is provided here., (© 2016 The Protein Society.)

The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.

The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD) and that of the Fas-associated protein (FADD) interact to form the core of the death-inducing signaling complex (DISC), a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD). However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR), biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas-mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209-239 (Fas-Pep1) and 251-288 (Fas-Pep2) constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD-CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling pathway.

Bacterial viruses of the P22-like family encode a specialized tail needle essential for genome stabilization after DNA packaging and implicated in Gram-negative cell envelope penetration. The atomic structure of P22 tail needle (gp26) crystallized at acidic pH reveals a slender fiber containing an N-terminal "trimer of hairpins" tip. Although the length and composition of tail needles vary significantly in Podoviridae, unexpectedly, the amino acid sequence of the N-terminal tip is exceptionally conserved in more than 200 genomes of P22-like phages and prophages. In this paper, we used x-ray crystallography and EM to investigate the neutral pH structure of three tail needles from bacteriophage P22, HK620, and Sf6. In all cases, we found that the N-terminal tip is poorly structured, in stark contrast to the compact trimer of hairpins seen in gp26 crystallized at acidic pH. Hydrogen-deuterium exchange mass spectrometry, limited proteolysis, circular dichroism spectroscopy, and gel filtration chromatography revealed that the N-terminal tip is highly dynamic in solution and unlikely to adopt a stable trimeric conformation at physiological pH. This is supported by the cryo-EM reconstruction of P22 mature virion tail, where the density of gp26 N-terminal tip is incompatible with a trimer of hairpins. We propose the tail needle N-terminal tip exists in two conformations: a pre-ejection extended conformation, which seals the portal vertex after genome packaging, and a postejection trimer of hairpins, which forms upon its release from the virion. The conformational plasticity of the tail needle N-terminal tip is built in the amino acid sequence, explaining its extraordinary conservation in nature., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

During infection, human immunodeficiency virus type 1 (HIV-1) interacts with the cellular host factor cyclophilin A (CypA) through residues 85-93 of the N-terminal domain of HIV-1's capsid protein (CA). The role of the CA:CypA interaction is still unclear. Previous studies showed that a CypA-binding loop mutant, Δ87-97, has increased ability to assemble in vitro. We used this mutant to infer whether the CypA-binding region has an overall effect on CA stability, as measured by pressure and chemical perturbation. We built a SAXS-based envelope model for the dimer of both WT and Δ87-97. A new conformational arrangement of the dimers is described, showing the structural plasticity that CA can adopt. In protein folding studies, the deletion of the loop drastically reduces CA stability, as assayed by high hydrostatic pressure and urea. We hypothesize that the deletion promotes a rearrangement of helix 4, which may enhance the heterotypic interaction between the N- and C-terminal domains of CA dimers. In addition, we propose that the cyclophilin-binding loop may modulate capsid assembly during infection, either in the cytoplasm or near the nucleus by binding to the nuclear protein Nup385., (Copyright © 2014. Published by Elsevier B.V.)

Two- and three-dimensional assembly of nanoparticles has generated significant interest because these higher order structures could exhibit collective behaviors/properties beyond those of the individual nanoparticles. Highly specific interactions between molecules, which biology exploits to regulate molecular assemblies such as DNA hybridization, often provide inspiration for the construction of higher order materials using bottom-up approaches. In this study, higher order assembly of virus-like particles (VLPs) derived from the bacteriophage P22 is demonstrated by using a small adaptor protein, Dec, which binds to symmetry specific sites on the P22 capsid. Two types of connector proteins, which have different number of P22 binding sites and different geometries (ditopic linker with liner geometry and tetratopic linker with tetrahedral geometry) have been engineered through either a point mutation of Dec or genetic fusion with another protein, respectively. Bulk assembly and layer-by-layer deposition of P22 VLPs from solution was successfully achieved using both of the engineered multi-topic linker molecules, while Dec with only a single binding site does not mediate P22 assembly. Beyond the two types of linkers developed in this study, a wide range of different connector geometries could be envisioned using a similar engineering approach. This is a powerful strategy to construct higher order assemblies of VLP based nanomaterials., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Plasmonic photocatalytic nanostructures have been fabricated under mild conditions (room temperature aqueous solution) using genetically engineered bacteriophage P22 virus-like particles (VLP) as a nano-platform. The photodegradation of methylene blue by CdS photocatalyst confined inside VLP can be significantly enhanced by the controlled deposition of gold nanoparticles on the outer shell of VLP-CdS.

Biological organisms have evolved tremendous control over the synthesis of inorganic materials in aqueous solutions at standard conditions. Such control over material properties is difficult to achieve with current synthesis strategies. Biotemplated synthesis of materials has been demonstrated to be efficient at facilitating the formation of various inorganic species. In this study, we employ a protein cage-based system to synthesize photoactive TiO2 nanoparticles less than 10 nm in diameter. We also demonstrate phase control over the material, with the ability to synthesize both anatase and rutile TiO2 using distinct biomineralization peptides within the protein cage. Finally, using analytical ultracentrifugation, we are able to resolve distinct reaction products and approximate their loading. We find that two distinct species comprise the reaction products, likely representing procapsid-like particles with early, precursor metal oxide clusters, and shells nearly full with crystalline TiO2 nanoparticles, respectively.

Bacteriophage P22 has long been considered a hallmark model for virus assembly and maturation. Repurposing of P22 and other similar virus structures for nanotechnology and nanomedicine has reinvigorated the need to further understand the protein-protein interactions that allow for the assembly, as well as the conformational shifts required for maturation. In this work, gp5, the major coat structural protein of P22, has been manipulated in order to examine the mutational effects on procapsid stability and maturation. Insertions to the P22 coat protein A-domain, while widely permissive of procapsid assembly, destabilize the interactions necessary for virus maturation and potentially allow for the tunable adjustment of procapsid stability. Future manipulation of this region of the coat protein subunit can potentially be used to alter the stability of the capsid for controllable disassembly.

Unlabelled: Purified retroviral Gag proteins can assemble in vitro to form immature virus-like particles (VLPs). By cryoelectron tomography, Rous sarcoma virus VLPs show an organized hexameric lattice consisting chiefly of the capsid (CA) domain, with periodic stalk-like densities below the lattice. We hypothesize that the structure represented by these densities is formed by amino acid residues immediately downstream of the folded CA, namely, the short spacer peptide SP, along with a dozen flanking residues. These 24 residues comprise the SP assembly (SPA) domain, and we propose that neighboring SPA units in a Gag hexamer coalesce to form a six-helix bundle. Using in vitro assembly, alanine scanning mutagenesis, and biophysical analyses, we have further characterized the structure and function of SPA. Most of the amino acid residues in SPA could not be mutated individually without abrogating assembly, with the exception of a few residues near the N and C termini, as well as three hydrophilic residues within SPA. We interpret these results to mean that the amino acids that do not tolerate mutations contribute to higher-order structures in VLPs. Hydrogen-deuterium exchange analyses of unassembled Gag compared that of assembled VLPs showed strong protection at the SPA region, consistent with a higher-order structure. Circular dichroism revealed that a 29mer SPA peptide shifts from a random coil to a helix in a concentration-dependent manner. Analytical ultracentrifugation showed concentration-dependent self-association of the peptide into a hexamer. Taken together, these results provide strong evidence for the formation of a critical six-helix bundle in Gag assembly., Importance: The structure of a retrovirus like HIV is created by several thousand molecules of the viral Gag protein, which assemble to form the known hexagonal protein lattice in the virus particle. How the Gag proteins pack together in the lattice is incompletely understood. A short segment of Gag known to be critical for proper assembly has been hypothesized to form a six-helix bundle, which may be the nucleating event that leads to lattice formation. The experiments reported here, using the avian Rous sarcoma virus as a model system, further define the nature of this segment of Gag, show that it is in a higher-order structure in the virus particle, and provide the first direct evidence that it forms a six-helix bundle in retrovirus assembly. Such knowledge may provide underpinnings for the development of antiretroviral drugs that interfere with virus assembly.

Rationale: Charge state resolution is required to determine the masses of ions in electrospray mass spectrometry, a feat which becomes increasingly difficult as the mass increases. Charge detection mass spectrometry (CDMS) circumvents this limitation by simultaneously measuring the charge and the m/z of individual ions. In this work, we have used electrospray CDMS to determine the number of scaffolding proteins associated with bacteriophage P22 procapsids., Methods: P22 procapsids containing a native cargo of scaffolding protein were assembled in E. coli and purified via differential centrifugation. Electrospray CDMS was used to measure their mass distribution., Results: The procapsid peak was centered at 23.60 MDa, which indicates that they contain an average of ~112 scaffolding proteins. The distribution is relatively narrow, less than 31 scaffolding proteins wide. In addition, a peak at 19.84 MDa with a relative abundance of ~15% is attributed to empty capsids. Despite having the same sizes in solution, the empty capsid and the procapsid have significantly different average charges., Conclusions: The detection of empty capsids is unexpected and the process that leads to them is unknown. The average charge on the empty capsids is significantly lower than expected from the charge residue model, which probably indicates that the empty capsids have contracted in the gas phase. The scaffolding protein presumably limits the contraction of the procapsids. This work shows that electrospray CDMS can provide valuable information for masses greater than 20 MDa., (Copyright © 2014 John Wiley & Sons, Ltd.)

Engineered virus-like particles (VLP) are attractive for fabricating nanostructured materials for applications in diverse areas such as catalysis, drug delivery, biomedicine, composites, etc. Basic understanding of the interaction between the inorganic guest and biomolecular host is thus important for the controlled synthesis of inorganic nanoparticles inside VLP and rational assembly of ordered VLP-based hierarchical nanostructures. We have investigated in detail the formation mechanism and growth kinetics of semiconducting nanocrystals confined inside genetically engineered bacteriophage P22 VLP using semiconducting CdS as a prototypical example. The selective nucleation and growth of CdS at the engineered sites is found to be uniform during the early stage, followed by a more stochastic growth process. Furthermore, kinetic studies reveal that the presence of an engineered biotemplate helps in significantly retarding the reaction rate. These findings provide guidance for the controlled synthesis of a wide range of other inorganic materials confined inside VLP, and are of practical importance for the rational design of VLP-based hierarchical nanostuctures.

A dodecamer of connector protein forms a conduit at a unique five-fold vertex in the capsid of many dsDNA-containing viruses providing the means for DNA entry and egress. The molecular mechanism guiding the incorporation of one connector per procapsid remains obscure; however, a recent bacteriophage ϕ29 model suggests that incorporation is coupled to nucleation between the connector and scaffolding proteins and particular amino acids may promote interactions between the two proteins. To test this model in vivo, a trans-complementation system using cloned scaffolding genes was implemented and tested for the ability to complement a ϕ29 amber-scaffolding strain. Wild type scaffolding gene induction resulted in efficient virion production, whereas synthesis of mutant scaffolding proteins displayed various phenotypes. Biochemical analyses of the resultant particles substantiate the previously identified amino acid residues in connector incorporation. Furthermore, kinetic studies of virion production using the in vivo trans-complementation system support the nucleation model., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Virus like particles, and other naturally occurring protein containers, have emerged as excellent building blocks for nanomaterials design and synthesis. Here we exploit a directed assembly and encapsulation approach to sequester multiple copies of a phosphotriesterase (PTE) enzyme within the capsid of bacteriophage P22. Phosphotriesterase, from Brevundimonas diminuta, is an intriguing enzyme as it is highly active against a wide range of harmful insecticides and nerve agents such as Soman and Sarin. However, difficulty in expressing large quantities of the active recombinant enzyme has limited efforts to scale-up its use. Additionally, as a mesophilic enzyme its low heat tolerance and susceptibility to proteolysis makes it a less than ideal candidate as a practical bioremediation tool. Through encapsulation of the PTE within the P22 capsid, we demonstrate a greatly enhanced thermal tolerance of the enzyme, maintaining 50% of its activity to 60 °C. Additionally, the P22 capsid confers protection to the enzyme from proteases, as well as stabilizing the enzyme against desiccation. Thus, our engineered P22 encapsulation system greatly enhances the stability of the mesophilic phosphotriesterase and results in a robust and active nanoparticle reactor.

The phosphoprotein (P) is virally encoded by the Rhabdoviridae and Paramyxoviridae in the order Mononegavirales. P is a self-associated oligomer and forms complexes with the large viral polymerase protein (L), the nucleocapsid protein (N), and the assembled nucleocapsid. P from different viruses has shown structural diversities even though their essential functions are the same. We systematically mapped the domains in mumps virus (MuV) P and investigated their interactions with nucleocapsid-like particles (NLPs). Similar to other P proteins, MuV P contains N-terminal, central, and C-terminal domains with flexible linkers between neighboring domains. By pulldown assays, we discovered that in addition to the previously proposed nucleocapsid binding domain (residues 343 to 391), the N-terminal region of MuV P (residues 1 to 194) could also bind NLPs. Further analysis of binding kinetics was conducted using surface plasmon resonance. This is the first observation that both the N- and C-terminal regions of a negative-strand RNA virus P are involved in binding the nucleocapsid. In addition, we defined the oligomerization domain (POD) of MuV P as residues 213 to 277 and determined its crystal structure. The tetrameric MuV POD is formed by one pair of long parallel α-helices with another pair in opposite orientation. Unlike the parallel orientation of each α-helix in the tetramer of Sendai virus POD, this represents a novel orientation of a POD where both the N- and the C-terminal domains are at either end of the tetramer. This is consistent with the observation that both the N- and the C-terminal domains are involved in binding the nucleocapsid.

Serine-rich repeat glycoproteins (SRRPs) are important bacterial adhesins conserved in streptococci and staphylococci. Fap1, a SRRP identified in Streptococcus parasanguinis, is the major constituent of bacterial fimbriae and is required for adhesion and biofilm formation. An 11-gene cluster is required for Fap1 glycosylation and secretion; however, the exact mechanism of Fap1 biogenesis remains a mystery. Two glycosylation-associated proteins within this cluster--Gap1 and Gap3--function together in Fap1 biogenesis. Here we report the role of the third glycosylation-associated protein, Gap2. A gap2 mutant exhibited the same phenotype as the gap1 and gap3 mutants in terms of Fap1 biogenesis, fimbrial assembly, and bacterial adhesion, suggesting that the three proteins interact. Indeed, all three proteins interacted with each other independently and together to form a stable protein complex. Mechanistically, Gap2 protected Gap3 from degradation by ClpP protease, and Gap2 required the presence of Gap1 for expression at the wild-type level. Gap2 augmented the function of Gap1 in stabilizing Gap3; this function was conserved in Gap homologs from Streptococcus agalactiae. Our studies demonstrate that the three Gap proteins work in concert in Fap1 biogenesis and reveal a new function of Gap2. This insight will help us elucidate the molecular mechanism of SRRP biogenesis in this bacterium and in pathogenic species.

Hydroxyapatite (HA) is the principal constituent of bone mineral, and synthetic HA is widely used as a biomaterial for bone repair. Previous work has shown that polyglutamate domains bind selectively to HA and that these domains can be utilized to couple bioactive peptides onto many different HA-containing materials. In the current study we have adapted this technology to engineer polyglutamate domains into cargo-loaded nanocage structures derived from the P22 bacteriophage. P22 nanocages have demonstrated significant potential as a drug delivery system due to their stability, large capacity for loading with a diversity of proteins and other types of cargo, and ability to resist degradation by proteases. Site-directed mutagenesis was used to modify the primary coding sequence of the P22 coat protein to incorporate glutamate-rich regions. Relative to wild-type P22, the polyglutamate-modified nanocages (E2-P22) exhibited increased binding to ceramic HA disks, particulate HA and allograft bone. Furthermore, E2-P22 binding was HA selective, as evidenced by negligible binding of the nanocages to non-HA materials including polystyrene, agarose, and polycaprolactone (PCL). Taken together these results establish a new mechanism for the directed coupling of nanocage drug delivery systems to a variety of HA-containing materials commonly used in diverse bone therapies., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The hierarchical organization of inorganic nanostructures has potential applications in diverse areas such as photocatalytic systems, composites, drug delivery and biomedicine. An attractive approach for this purpose is the use of biological organisms as templates since they often possess highly ordered arrays of protein molecules that can be genetically engineered for specific binding. Indeed, recent studies have shown that viruses can be used as versatile templates for the assembly of a variety of nanostructured materials because of their unique structural and chemical diversity. These highly ordered protein templates can be employed or adapted for specific binding interactions. Herein we report the directed self-assembly of independently synthesized 5 nm CdS nanocrystal quantum dots on ∼60 nm procapsid shells derived from wild-type P22 bacteriophage. The bacteriophage P22 shell is comprised of hexameric and pentameric clusters of subunits known as capsomeres. The pre-synthesized CdS QDs show the corresponding hexameric and pentameric patterns of assembly on these P22 shells, possibly by interacting with particular protein pockets.

The precise architectures of viruses and virus-like particles are proving to be highly advantageous in synthetic materials applications. Not only can these nanocontainers be harnessed as active materials, but they can be exploited for examining the effects of in vivo "cell-like" crowding and confinement on the properties of the encapsulated cargo. Here we report the first example of intermolecular communication between two proteins coencapsulated within the capsid architecture of the bacteriophage P22. Using a genetically engineered three-protein fusion between the P22 scaffold protein, and the FRET pair, GFP, and a red fluorescent protein (mCherry), we were able to direct the encapsulation of the genetic fusion when coexpressed with P22 coat protein. These self-assembled P22 capsids are densely packaged, occupying more than 24% of the available volume, and the molecular design assures a 1:1 ratio of the interacting proteins. To probe the effect of crowding and confinement on the FRET communication in this nanoenvironment, we spaced the donor-acceptor pair with variable length flexible linkers and examined the effect on FRET inside the capsid compared to the same tethered FRET pairs free in solution. The P22 system is unique in that the capsid morphology can be altered, without losing the encapsulated cargo, resulting in a doubling of the capsid volume. Thus, we have additionally examined the encapsulated fusions at two different internal concentrations. Our results indicate that FRET is sensitive to the expansion of the capsid and encapsulation enforces significant intermolecular communication, increasing FRET by 5-fold. This P22 coencapsulation system is a promising platform for studying crowding, enforced proximity, and confinement effects on communication between active proteins.

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.

The virus like particle (VLP) derived from bacteriophage P22 presents a unique platform for constructing catalytically functional nanomaterials by encapsulation of enzymes into its interior. Encapsulation has been engineered to be genetically programmed allowing "one pot" synthesis and incorporation of desired enzymes. The unique characteristic that separates P22 from other VLP systems is the ability to modulate the overall volume and porosity of the VLP structure, thus controlling substrate access to the encapsulated enzyme. The present study demonstrates incorporation of an enzyme, alcohol dehydrogenase D, with the highest internal loading for an active enzyme by any VLP described thus far. In addition, we show that not only does encapsulating AdhD inside P22 affect its kinetic parameters in comparison with the "free" enzyme, but transformation of P22 to different morphological states, which changes the internal volume of the VLP, yields changes in the overall activity of the encapsulated enzyme as well. The findings reported here clearly illustrate that P22 holds potential for synthetic approaches to create nanoreactors, by design, using the power of highly evolved enzymes for chemical transformations.

Protein cage nanoparticles (PCNs) are attractive platforms for developing functional nanomaterials using biomimetic approaches for functionalization and cargo encapsulation. Many strategies have been employed to direct the loading of molecular cargos inside a wide range of PCN architectures. Here we demonstrate the exploitation of a metal-ligand coordination bond with respect to the direct packing of guest molecules on the interior interface of a virus-like PCN derived from Salmonella typhimurium bacteriophage P22. The incorporation of these guest species was assessed using mass spectrometry, multiangle laser light scattering, and analytical ultracentrifugation. In addition to small-molecule encapsulation, this approach was also effective for the directed synthesis of a large macromolecular coordination polymer packed inside of the P22 capsid and initiated on the interior surface. A wide range of metals and ligands with different thermodynamic affinities and kinetic stabilities are potentially available for this approach, highlighting the potential for metal-ligand coordination chemistry to direct the site-specific incorporation of cargo molecules for a variety of applications., (© 2011 American Chemical Society)

For a machine to function, it must first be assembled. The morphogenesis of the simplest icosahedral virus would require only 60 copies of a single capsid protein to coalesce. If the capsid protein's structure could be entirely dedicated to this endeavor, the morphogenetic mechanism would be relatively uncomplicated. However, capsid proteins have had to evolve other functions, such as receptor recognition, immune system evasion, and the incorporation of other structure proteins, which can detract from efficient assembly. Moreover, evolution has mandated that viruses obtain additional proteins that allow them to adapt to their hosts or to more effectively compete in their respective niches. Consequently, genomes have increased in size, which has required capsids to do likewise. This, in turn, has lead to more complex icosahedral geometries. These challenges have driven the evolution of scaffolding proteins, which mediate, catalyze, and promote proper virus assembly. The mechanisms by which these proteins perform their functions are discussed in this review.

The human immunodeficiency virus (HIV) is an enveloped virus constituted by two monomeric RNA molecules that encode for 15 proteins. Among these are the structural proteins that are translated as the gag polyprotein. In order to become infectious, HIV must undergo a maturation process mediated by the proteolytic cleavage of gag to give rise to the isolated structural protein matrix, capsid (CA), nucleocapsid as well as p6 and spacer peptides 1 and 2. Upon maturation, the 13 N-terminal residues from CA fold into a β-hairpin, which is stabilized mainly by a salt bridge between Pro1 and Asp51. Previous reports have shown that non-formation of the salt bridge, which potentially disrupts proper β-hairpin arrangement, generates noninfectious virus or aberrant cores. To date, however, there is no consensus on the role of the β-hairpin. In order to shed light in this subject, we have generated mutations in the hairpin region to examine what features would be crucial for the β-hairpin's role in retroviral mature core formation. These features include the importance of the proline at the N-terminus, the amino acid sequence, and the physical structure of the β-hairpin itself. The presented experiments provide biochemical evidence that β-hairpin formation plays an important role in regard to CA protein conformation required to support proper mature core arrangement. Hydrogen/deuterium exchange and in vitro assembly reactions illustrated the importance of the β-hairpin structure, its dynamics, and its influence on the orientation of helix 1 for the assembly of the mature CA lattice., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The pressing need to develop antivirals active against resistant strains of HIV-1 has led to efforts to target steps in the virus life cycle other than reverse transcription and Gag proteolysis. Among those steps are entry, integration, and assembly and/or maturation. Advances in understanding the structural biology of both the immature and the mature forms of the HIV capsid have made it possible to design or discover small molecules and peptides that interfere with both assembly and maturation. Here, we review the current state of the art in assembly and maturation inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Biomimetic chemistry offers new approaches to supramolecular materials synthesis and assembly. We have demonstrated that an assembled viral protein cage, comprising an organic core-shell structure, can be used as a template for the size constrained synthesis of Fe(2)O(3). Particle nucleation is directed by the inner scaffold protein layer, while the size constraints are determined by the outer capsid layer., (This journal is © The Royal Society of Chemistry 2011)

DNA viruses such as bacteriophages and herpesviruses deliver their genome into and out of the capsid through large proteinaceous assemblies, known as portal proteins. Here, we report two snapshots of the dodecameric portal protein of bacteriophage P22. The 3.25-Å-resolution structure of the portal-protein core bound to 12 copies of gene product 4 (gp4) reveals a ~1.1-MDa assembly formed by 24 proteins. Unexpectedly, a lower-resolution structure of the full-length portal protein unveils the unique topology of the C-terminal domain, which forms a ~200-Å-long α-helical barrel. This domain inserts deeply into the virion and is highly conserved in the Podoviridae family. We propose that the barrel domain facilitates genome spooling onto the interior surface of the capsid during genome packaging and, in analogy to a rifle barrel, increases the accuracy of genome ejection into the host cell.

Ordered ZnS and CdS nanocrystal assemblies have been synthesized by a facile bioinspired approach consisting of an initial self-assembly of engineered proteins into spherical biotemplates and a subsequent protein-directed nucleation and growth of ZnS and CdS nanocrystals symmetrically distributed over the self-assembled biotemplates.

Following budding, HIV-1 virions undergo a maturation process where the Gag polyprotein in the immature virus is cleaved by the viral protease and rearranges to form the mature infectious virion. Despite the wealth of structures of isolated capsid domains and an in vitro-assembled mature lattice, models of the immature lattice do not provide an unambiguous model of capsid-molecule orientation and no structural information is available for the capsid maturation pathway. Here we have applied hydrogen/deuterium exchange mass spectrometry to immature, mature, and mutant Gag particles (CA5) blocked at the final Gag cleavage event to examine the molecular basis of capsid assembly and maturation. Capsid packing arrangements were very similar for all virions, whereas immature and CA5 virions contained an additional intermolecular interaction at the hexameric, 3-fold axis. Additionally, the N-terminal β-hairpin was observed to form as a result of capsid-SP1 cleavage rather than driving maturation as previously postulated., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Human immunodeficiency virus type 1 (HIV-1) encodes a polypeptide called Gag that is capable of forming virus-like particles (VLPs) in vitro in the absence of other cellular or viral constituents. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. A combination of in vivo, in vitro, and structural studies have shown that Gag targeting and assembly on the PM are mediated by specific interactions between the myristoylated matrix [myr(+)MA] domain of Gag and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P2 binding and is enhanced by factors that promote protein self-association. In the studies reported here, we demonstrate that myr exposure in MA is modulated by pH. Our data show that deprotonation of the His89 imidazole ring in myr(+)MA destabilizes the salt bridge formed between His89(Hδ2) and Glu12(COO-), leading to tight sequestration of the myr group and a shift in the equilibrium from trimer to monomer. Furthermore, we show that oligomerization of a Gag-like construct containing matrix-capsid is also pH-dependent. Disruption of the His−Glu salt bridge by single-amino acid substitutions greatly altered the myr-sequestered−myr-exposed equilibrium. In vivo intracellular localization data revealed that the H89G mutation retargets Gag to intracellular compartments and severely inhibits virus production. Our findings reveal that the MA domain acts as a “pH sensor” in vitro, suggesting that the effect of pH on HIV-1 Gag targeting and binding to the PM warrants investigation.

Cryo-electron microscopy (Cryo-EM) and cryo-electron tomography (Cryo-ET) provide structural insights into complex biological processes. The podoviridae are dsDNA containing phage with short, non-contractile tails which nevertheless translocate their DNA into the cytoplasm of their host cells. Liu et al. [1] used a combination of cryo-EM and cryo-ET to study the structural changes accompanying infection of P. marinus by the phage P-SSP7 and thereby provide unique molecular insight into the process by which the DNA transits from phage to host during infection.

Viral capsids are dynamic macromolecular machines which self-assemble and undergo concerted conformational changes during their life cycle. We have taken advantage of the inherent structural flexibility of viral capsids and generated two morphologically different types of viral nanoplatforms from the bacteriophage P22 capsids. Their interior surfaces were genetically manipulated for site-specific attachment of a biotin linker. The extent of internal modifications in each capsid form was characterized by high-resolution mass spectrometry and the analyses revealed that the reactivity of the genetically introduced residues located on the internal surface changes according to the structural transformation of the capsid. Internally modified capsids having 10 nm diameter pores at the 12 icosahedral vertices, so-called wiffle-balls (WB), exhibited the capability to entrap the large tetrameric protein complex streptavidin via the biotin linker anchored onto the interior surface of the WB.

The molecular mechanism of scaffolding protein-mediated incorporation of one and only one DNA packaging motor/connector dodecamer at a unique vertex during lambdoid phage assembly has remained elusive because of the lack of structural information on how the connector and scaffolding proteins interact. We assembled and characterized a phi29 connector-scaffolding complex, which can be incorporated into procapsids during in vitro assembly. Native mass spectrometry revealed that the connector binds at most 12 scaffolding molecules, likely organized as six dimers. A data-driven docking model, using input from chemical cross-linking and mutagenesis data, suggested an interaction between the scaffolding protein and the exterior of the wide domain of the connector dodecamer. The connector binding region of the scaffolding protein lies upstream of the capsid binding region located at the C terminus. This arrangement allows the C terminus of scaffolding protein within the complex to both recruit capsid subunits and mediate the incorporation of the single connector vertex.

The incorporation of the DNA packaging connector complex during lambdoid phage assembly in vivo is strictly controlled-one and only one of the twelve identical icosahedral vertices is differentiated by the inclusion of a portal or connector dodecamer. Proposed control mechanisms include obligate nucleation from a connector containing complex, addition of the connector as the final step during assembly, and a connector-mediated increase in the growth rate. The inability to recapitulate connector incorporation in vitro has made it difficult to obtain direct biochemical evidence in support of one model over another. Here we report the development an in vitro assembly system for the well characterized dsDNA phage Phi29 which results in the co-assembly of connector with capsid and scaffolding proteins to form procapsid-like particles (PLPs). Immuno-electron microscopy demonstrates the specific incorporation of connector vertex in PLPs. The connector protein increases both the yield and the rate of capsid assembly suggesting that the incorporation of the connector in Phi29 likely promotes nucleation of assembly.

The nucleocapsid protein (NP) of mumps virus (MuV), a paramyxovirus, was coexpressed with the phosphoprotein (P) in Escherichia coli. The NP and P proteins form a soluble complex containing RNA. Under a transmission electron microscope, the NP-RNA complex appears as a nucleocapsidlike ring that has a diameter of approximately 20 nm with 13 subunits. There is a piece of single-stranded RNA with a length of 78 nucleotides in the NP-RNA ring. Shorter RNA pieces are also visible. The MuV NP protein may provide weaker protection of the RNA than the NP protein of some other negative-strand RNA viruses, reflecting the degree of NP protein association.