RESUMO
Virus-like particles (VLPs) from bacteriophage MS2 provide a platform to study protein self-assembly and create engineered systems for drug delivery. Here, we aim to understand the impact of intersubunit interface mutations on the local and global structure and function of MS2-based VLPs. In previous work, our lab identified locally supercharged double mutants [T71K/G73R] that concentrate positive charge at capsid pores, enhancing uptake into mammalian cells. To study the effects of particle size on cellular internalization, we combined these double mutants with a single point mutation [S37P] that was previously reported to switch particle geometry from T = 3 to T = 1 icosahedral symmetry. These new variants retained their enhanced cellular uptake activity and could deliver small-molecule drugs with efficacy levels similar to our first-generation capsids. Surprisingly, these engineered triple mutants exhibit increased thermostability and unexpected geometry, producing T = 3 particles instead of the anticipated T = 1 assemblies. Transmission electron microscopy revealed various capsid assembly states, including wild-type (T = 3), T = 1, and rod-like particles, that could be accessed using different combinations of these point mutations. Molecular dynamics experiments recapitulated the structural rationale in silico for the single point mutation [S37P] forming a T = 1 virus-like particle and showed that this assembly state was not favored when combined with mutations that favor rod-like architectures. Through this work, we investigated how interdimer interface dynamics influence VLP size and morphology and how these properties affect particle function in applications such as drug delivery.
Assuntos
Capsídeo , Levivirus , Levivirus/genética , Levivirus/química , Levivirus/metabolismo , Capsídeo/metabolismo , Capsídeo/química , Capsídeo/ultraestrutura , Mutação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Vírion/metabolismo , Vírion/genética , Vírion/química , Mutação Puntual , Estabilidade Proteica , Humanos , Modelos MolecularesRESUMO
RNA bacteriophage MS2-derived virus-like particles (VLPs) have been widely used in biomedical research as model systems to study virus assembly, structure-function relationships, vaccine development, and drug delivery. Considering the diverse utility of these VLPs, a systemic engineering approach has been utilized to generate smaller particles with optimal serum stability and tissue penetrance. Additionally, it is crucial to demonstrate the overall stability of these mini MS2 VLPs, ensuring cargo protection until they reach their target cell/organ. However, no detailed analysis of the thermal stability and heat-induced disassembly of MS2 VLPs has yet been attempted. In this work, we investigated the thermal stability of both wild-type (WT) MS2 VLP and its "mini" variant containing S37P mutation (mini MS2 VLP). The mini MS2 VLP exhibits a higher capsid melting temperature (Tm) when compared to its WT MS2 VLP counterpart, possibly attributed to its smaller interdimer angle. Our study presents that the thermal unfolding of MS2 VLPs follows a sequential process involving particle destabilization, nucleic acid exposure/melting, and disassembly of VLP. This observation underscores the disruption of cooperative intersubunit interactions and protein-nucleic acid interactions, shedding light on the mechanism of heat-induced VLP disassembly.
Assuntos
Levivirus , Levivirus/genética , Levivirus/química , Levivirus/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Temperatura , Mutação , Temperatura Alta , Vírion/metabolismo , Vírion/química , Vírion/genética , Capsídeo/metabolismo , Capsídeo/químicaRESUMO
Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to biology. Here, we present massively parallel RNA assay combined with immunoprecipitation (MPRNA-IP) for in vivo high-throughput dissection of RNA-protein interactions and describe statistical models for identifying RNA domains and parsing the structural contributions of RNA. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, MPRNA-IP is able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest, including the long noncoding RNA NORAD, bacteriophage MS2 RNA, and human telomerase RNA, and we use it to interrogate the hitherto unknown sequence or structural RNA-binding preferences of the DNA-looping factor CTCF. By integrating systematic mutation analysis with crosslinking immunoprecipitation, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions in vivo.
Assuntos
Proteínas de Ligação a RNA , RNA , Humanos , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Imunoprecipitação , Levivirus/genética , Levivirus/metabolismo , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , RNA/metabolismo , RNA/química , RNA/genética , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/química , RNA Viral/metabolismo , RNA Viral/química , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/química , Telomerase/metabolismo , Telomerase/genética , Modelos EstatísticosRESUMO
Self-assembly is a vital part of the life cycle of certain icosahedral RNA viruses. Furthermore, the assembly process can be harnessed to make icosahedral virus-like particles (VLPs) from coat protein and RNA in vitro. Although much previous work has explored the effects of RNA-protein interactions on the assembly products, relatively little research has explored the effects of coat-protein concentration. We mix coat protein and RNA from bacteriophage MS2, and we use a combination of gel electrophoresis, dynamic light scattering, and transmission electron microscopy to investigate the assembly products. We show that with increasing coat-protein concentration, the products transition from well-formed MS2 VLPs to "monster" particles consisting of multiple partial capsids to RNA-protein condensates consisting of large networks of RNA and partially assembled capsids. We argue that the transition from well-formed to monster particles arises because the assembly follows a nucleation-and-growth pathway in which the nucleation rate depends sensitively on the coat-protein concentration, such that at high protein concentrations, multiple nuclei can form on each RNA strand. To understand the formation of the condensates, which occurs at even higher coat-protein concentrations, we use Monte Carlo simulations with coarse-grained models of capsomers and RNA. These simulations suggest that the formation of condensates occurs by the adsorption of protein to the RNA followed by the assembly of capsids. Multiple RNA molecules can become trapped when a capsid grows from capsomers attached to two different RNA molecules or when excess protein bridges together growing capsids on different RNA molecules. Our results provide insight into an important biophysical process and could inform design rules for making VLPs for various applications.
Assuntos
Capsídeo , Levivirus , Levivirus/genética , Levivirus/metabolismo , Proteínas do Capsídeo/metabolismo , RNA Viral/genética , VírionRESUMO
Most of the defective/non-infectious enteric phages and viruses that end up in wastewater originate in human feces. Some of the causes of this high level of inactivity at the host stage are unknown. There is a significant gap between how enteric phages are environmentally transmitted and how we might design molecular tools that would only detect infectious ones. Thus, there is a need to explain the low proportion of infectious viral particles once replicated. By analyzing lysis plaque content, we were able to confirm that, under aerobic conditions, Escherichia coli produce low numbers of infectious MS2 phages (I) than the total number of phages indicated by the genome copies (G) with an I/G ratio of around 2%. Anaerobic conditions of replication and ROS inhibition increase the I/G ratio to 8 and 25%, respectively. These data cannot only be explained by variations in the total numbers of MS2 phages produced or in the metabolism of E. coli. We therefore suggest that oxidative damage impacts the molecular replication and assembly of MS2 phages.
Assuntos
Anaerobiose/fisiologia , Levivirus/metabolismo , Replicação Viral/fisiologia , Aerobiose/fisiologia , Colífagos/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/metabolismo , Fezes/virologia , Humanos , Levivirus/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , VirulênciaRESUMO
RiboNucleoProtein particles (RNPs), which are composed of RNAs and proteins, play essential roles in many biological processes. The isolation of these molecular machines is a critical step to better understand their mechanisms of action. In this chapter, we describe the MS2-MBP affinity chromatography used to purify the protein content of the RNPs formed with an RNA of interest in a nuclear extract. Substrate RNAs are furnished with a tag consisting of three stem-loops that provide specific binding sites for the phage MS2 protein. Here, we successfully applied this method to isolate RNPs formed with subfragments of the long noncoding RNA ANRIL (Antisense Noncoding RNA in the INK4 Locus).
Assuntos
Proteínas do Capsídeo/metabolismo , RNA Longo não Codificante/metabolismo , Ribonucleoproteínas/isolamento & purificação , Sítios de Ligação , Cromatografia de Afinidade , Humanos , Levivirus/metabolismo , Ribonucleoproteínas/genéticaRESUMO
Survival of respiratory viral pathogens in expelled saliva microdroplets is central to their transmission, yet the factors that determine survival in such microdroplets are not well understood. Here we combine microscopy imaging with virus viability assays to study survival of three bacteriophages suggested as good models for respiratory pathogens: the enveloped Phi6 (a surrogate for SARS-CoV-2), and the non-enveloped PhiX174 and MS2. We measured virus viability in human saliva microdroplets, SM buffer, and water following deposition on glass surfaces at various relative humidities (RH). Saliva and water microdroplets dried out rapidly, within minutes, at all tested RH levels (23%, 43%, 57%, and 78%), while SM microdroplets remained hydrated at RH ≥ 57%. Generally, the survival of all three viruses in dry saliva microdroplets was significantly greater than those in SM buffer and water under all RH (except PhiX174 in water under 57% RH survived the best among 3 media). Thus, atmosphere RH and microdroplet hydration state are not sufficient to explain virus survival, indicating that the virus-suspended medium, and association with saliva components in particular, likely play a role in virus survival. Uncovering the exact properties and components that make saliva a favorable environment for the survival of viruses, in particular enveloped ones like Phi6, is thus of great importance for reducing transmission of viral respiratory pathogens including SARS-CoV-2.
Assuntos
Bacteriófago phi X 174/metabolismo , Levivirus/metabolismo , Viabilidade Microbiana , SARS-CoV-2/metabolismo , Saliva/virologia , Bacteriófago phi 6/metabolismo , COVID-19/transmissão , Microbiologia Ambiental , Humanos , Ensaio de Placa Viral , Inativação de VírusRESUMO
The central dogma teaches us that DNA makes RNA, which in turn makes proteins, the main building blocks of the cell. But this over simplified linear transmission of information overlooks the vast majority of the genome produces RNAs that do not encode proteins and the myriad ways that RNA regulates cellular functions. Historically, one of the challenges in illuminating RNA biology has been the lack of tools for visualizing RNA in live cells. But clever approaches for exploiting RNA binding proteins, in vitro RNA evolution, and chemical biology have resulted in significant advances in RNA visualization tools in recent years. This review provides an overview of current tools for tagging RNA with fluorescent probes and tracking their dynamics, localization andfunction in live mammalian cells.
Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , RNA/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Humanos , Levivirus/genética , Levivirus/metabolismo , RNA/química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA não Traduzido/química , RNA não Traduzido/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy-infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.
Assuntos
Microscopia de Força Atômica/métodos , Nanoestruturas/química , Análise Espectral Raman/métodos , Vírion/química , Animais , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , COVID-19 , Capsídeo/química , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Análise Discriminante , Herpesvirus Humano 1/fisiologia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Análise dos Mínimos Quadrados , Levivirus/metabolismo , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , SARS-CoV-2 , Células VeroRESUMO
RNA transport and localization are evolutionarily conserved processes that allow protein translation to occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates, embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA transport to establish localized replication centers within infected cells and then to disseminate their infectious genomes between cells and throughout the plant organism with the help of their movement proteins (MP). In this chapter, we describe the transient expression of the tobacco mosaic virus movement protein (TMV-MP) and the application of the MS2 system for the in vivo labeling of the MP-encoding mRNA. The MS2 method is based on the binding of the bacteriophage coat protein (CP) to its origin of assembly (OAS) in the phage RNA. Thus, to label a specific mRNA in vivo, a tandem repetition of a 19-nucleotide-long stem-loop (SL) sequence derived from the MS2 OAS sequence (MSL) is transcriptionally fused to the RNA under investigation. The RNA is detected by the co-expression of fluorescent protein-tagged MS2 CP (MCP), which binds to each of the MSL elements. In providing a detailed protocol for the in vivo visualization of TMV-MP mRNA tagged with the MS2 system in Nicotiana benthamiana epidermal cells, we describe (1) the specific DNA constructs, (2) Agrobacterium tumefaciens-mediated transfection for their transient expression in plants, and (3) imaging conditions required to obtain high-quality mRNA imaging data.
Assuntos
Agrobacterium tumefaciens/genética , Levivirus/metabolismo , Proteínas do Movimento Viral em Plantas/genética , Transporte de RNA/genética , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , RNA Viral/genética , Vírus do Mosaico do Tabaco/metabolismo , Transporte Biológico , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Clonagem Molecular , Expressão Gênica , Vetores Genéticos , Levivirus/genética , Proteínas Luminescentes , Microscopia de Fluorescência , Proteínas do Movimento Viral em Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plasmodesmos/metabolismo , RNA Mensageiro/genética , Nicotiana/genética , Nicotiana/metabolismo , Vírus do Mosaico do Tabaco/genéticaRESUMO
Enteric viruses, such as enterovirus, norovirus, and rotavirus, are among the leading causes of disease outbreaks due to contaminated drinking and recreational water. Viruses are difficult to remove from water through filtration based on physical size exclusion-for example by gravity-driven filters-due to their nanoscale size. To understand virus removal in drinking water treatment systems, the colloidal nanostructure of a model virus, the MS2 bacteriophage, has been investigated in relation to the effect of pH and natural organic matter in water. Dynamic light scattering, small-angle X-ray scattering, and cryogenic transmission electron microscopy demonstrated that the water pH has a major influence on the colloidal structure of the virus: The bacteriophage MS2's structure in water in the range pH = 7.0 to 9.0 was found to be spherical with core-shell-type structure with a total diameter of 27 nm and a core radius of around 8 nm. Reversible transformations from 27 nm particles at pH = 7.0 to micrometer-sized aggregates at pH = 3.0 were observed. In addition, the presence of natural organic matter that simulates the organic components present in surface water was found to enhance repulsion between virus particles, reduce the size of aggregates, and promote disaggregation upon pH increase. These findings allow a better understanding of virus interactions in water and have implications for water treatment using filtration processes and coagulation. The results will further guide the comprehensive design of advanced virus filter materials.
Assuntos
Levivirus/metabolismo , Compostos Orgânicos/metabolismo , Vírion/metabolismo , Coloides/química , Coloides/metabolismo , Hidrodinâmica , Concentração de Íons de Hidrogênio , Levivirus/química , Microscopia Eletrônica de Transmissão , Compostos Orgânicos/química , Tamanho da Partícula , Propriedades de Superfície , Vírion/química , Água/química , Água/metabolismoRESUMO
Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, in which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study, we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.
Assuntos
Proteínas do Capsídeo/ultraestrutura , Capsídeo/ultraestrutura , Levivirus/metabolismo , RNA Viral/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/virologia , Modelos MolecularesRESUMO
Unambiguous imaging of C â U edited mRNA calls for a method that distinguishes a locally high concentration of unbound probe or single nucleotide mismatched target from a locally low concentration of matched mRNA target. To address this issue, we combine FIT probes as a "chemical" detection system with the "biological" MS2 technique. Ratio measurements provide a convenient parameter to discriminate the edited from the unedited state of mRNA encoding for GlyR α2 in HEK cells.
Assuntos
Sondas de DNA/metabolismo , RNA Mensageiro/metabolismo , Benzotiazóis/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Sondas de DNA/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Levivirus/metabolismo , Microscopia de Fluorescência , Quinolinas/química , Receptores de Glicina/genéticaRESUMO
Visualization of single mRNAs in their native cellular environment provides key information to study gene expression regulation. This fundamental biological question triggered the development of the MS2-MCP (MS2-Capsid Protein) system to tag mRNAs and image their life cycle using widefield fluorescence microscopy. The last two decades have evolved toward improving the qualitative and quantitative characteristics of the MS2-MCP system. Here, we provide a protocol to use the latest versions, MS2V6 and MS2V7, to tag and visualize mRNAs in mammalian cells in culture. The motivation behind engineering MS2V6 and MS2V7 was to overcome a degradation caveat observed in S. cerevisiae with the previous MS2-MCP systems. While for yeast we recommend the use of MS2V6, we found that for live-cell imaging experiments in mammalian cells, the MS2V7 has improved reporter properties.
Assuntos
Proteínas do Capsídeo/metabolismo , Hibridização in Situ Fluorescente , Levivirus/metabolismo , Microscopia de Fluorescência , Imagem Molecular/métodos , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , Animais , Aptâmeros de Nucleotídeos/genética , Proteínas do Capsídeo/genética , Linhagem Celular , Regulação Fúngica da Expressão Gênica , Humanos , Levivirus/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Estabilidade de RNA , RNA Fúngico/genética , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Fatores de TempoRESUMO
The self-assembly of viral capsids is an essential step to the formation of infectious viruses. Elucidating the kinetic mechanisms of how a capsid or virus-like particle assembles could advance our knowledge about the viral lifecycle, as well as the general principles in self-assembly of biomaterials. However, current understanding of capsid assembly remains incomplete for many viruses due to the fact that the transient intermediates along the assembling pathways are experimentally difficult to be detected. In this paper, we constructed a new multiscale computational framework to simulate the self-assembly of virus-like particles. We applied our method to the coat proteins of bacteriophage MS2 as a specific model system. This virus-like particle of bacteriophage MS2 has a unique feature that its 90 sequence-identical dimers can be classified into two structurally various groups: one is the symmetric CC dimer, and the other is the asymmetric AB dimer. The homotypic interactions between AB dimers result in a 5-fold symmetric contact, while the heterotypic interactions between AB and CC dimers result in 6-fold symmetric contact. We found that the assembly can be described as a physical process of phase transition that is regulated by various factors such as concentration and specific stoichiometry between AB and CC dimers. Our simulations also demonstrate that heterotypic and homotypic interfaces play distinctive roles in modulating the assembling kinetics. The interaction between AB and CC dimers is much more dynamic than that between two AB dimers. We therefore suggest that the alternate growth of viral capsid through the heterotypic dimer interactions dominates the assembling pathways. This is, to the best of our knowledge, the first multiscale model to simulate the assembling process of coat proteins in bacteriophage MS2. The generality of this approach opens the door to its further applications in assembly of other viral capsids, virus-like particles, and novel drug delivery systems.
Assuntos
Proteínas do Capsídeo/metabolismo , Levivirus/metabolismo , Modelos Moleculares , Proteínas do Capsídeo/química , Cinética , Ligação Proteica , Conformação ProteicaRESUMO
The binding of MS2 bacteriophage coat protein (MCP) to MS2 binding site (MBS) RNA stem-loop sequences has been widely used to label mRNA for live-cell imaging at single-molecule resolution. However, concerns have been raised recently from studies with budding yeast showing aberrant mRNA metabolism following the MS2-GFP labeling. To investigate the degradation pattern of MS2-GFP-labeled mRNA in mammalian cells and tissues, we used Northern blot analysis of ß-actin mRNA extracted from the Actb-MBS knock-in and MBS×MCP hybrid mouse models. In the immortalized mouse embryonic cell lines and various organ tissues derived from the mouse models, we found no noticeable accumulation of decay products of ß-actin mRNA compared with the wild-type mice. Our results suggest that accumulation of MBS RNA decay fragments does not always happen depending on the mRNA species and the model organisms used.
Assuntos
Actinas/química , Actinas/genética , Proteínas do Capsídeo/metabolismo , Levivirus/genética , Actinas/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Embrião de Mamíferos/citologia , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Levivirus/metabolismo , Camundongos , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Organismos Livres de Patógenos EspecíficosRESUMO
Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes-a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.
Assuntos
Aminoácidos/química , Proteínas do Capsídeo/química , Capsídeo/química , Levivirus/química , Mutação , Vírion/química , Aminoácidos/metabolismo , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Levivirus/metabolismo , Levivirus/ultraestrutura , Modelos Moleculares , Mutagênese Sítio-Dirigida , Engenharia de Proteínas/métodos , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteólise , Eletricidade Estática , Vírion/metabolismo , Vírion/ultraestruturaRESUMO
MS2 phage-like particles (MS2 PLP) are artificially constructed pseudo-viral particles derived from bacteriophage MS2. They are able to carry a specific single stranded RNA (ssRNA) sequence of choice inside their capsid, thus protecting it against the effects of ubiquitous nucleases. Such particles are able to mimic ssRNA viruses and, thus, may serve as the process control for molecular detection and quantification of such agents in several kinds of matrices, vaccines and vaccine candidates, drug delivery systems, and systems for the display of immunologically active peptides or nanomachines. Currently, there are several different in vivo plasmid-driven packaging systems for production of MS2 PLP. In order to combine all the advantages of the available systems and to upgrade and simplify the production and purification of MS2 PLP, a one-plasmid double-expression His-tag system was designed. The described system utilizes a unique fusion insertional mutation enabling purification of particles using His-tag affinity. Using this new production system, highly pure MS2 PLP can be quickly produced and purified by a fast performance liquid chromatography (FPLC) approach. The system can be easily adapted to produce other MS2 PLP with different properties.
Assuntos
Proteínas do Capsídeo , Levivirus , Plasmídeos , Proteínas Recombinantes de Fusão , Vírion , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Levivirus/química , Levivirus/genética , Levivirus/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Vírion/química , Vírion/genética , Vírion/isolamento & purificação , Vírion/metabolismoRESUMO
For bacteriophage infections, the cell walls of bacteria, consisting of a single highly polymeric molecule of peptidoglycan (PG), pose a major problem for the release of progeny virions. Phage lysis proteins that overcome this barrier can point the way to new antibacterial strategies 1 , especially small lytic single-stranded DNA (the microviruses) and RNA phages (the leviviruses) that effect host lysis using a single non-enzymatic protein 2 . Previously, the A2 protein of levivirus Qß and the E protein of the microvirus ÏX174 were shown to be 'protein antibiotics' that inhibit the MurA and MraY steps of the PG synthesis pathway 2-4 . Here, we investigated the mechanism of action of an unrelated lysis protein, LysM, of the Escherichia coli levivirus M 5 . We show that LysM inhibits the translocation of the final lipid-linked PG precursor called lipid II across the cytoplasmic membrane by interfering with the activity of MurJ. The finding that LysM inhibits a distinct step in the PG synthesis pathway from the A2 and E proteins indicates that small phages, particularly the single-stranded RNA (ssRNA) leviviruses, have a previously unappreciated capacity for evolving novel inhibitors of PG biogenesis despite their limited coding potential.
Assuntos
Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/virologia , Levivirus/metabolismo , Peptidoglicano/biossíntese , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Proteínas Virais/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Bacteriólise/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Conformação Proteica , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo , Proteínas Virais/genética , VírionRESUMO
Small single-stranded nucleic acid phages effect lysis by expressing a single protein, the amurin, lacking muralytic enzymatic activity. Three amurins have been shown to act like 'protein antibiotics' by inhibiting cell-wall biosynthesis. However, the L lysis protein of the canonical ssRNA phage MS2, a 75 aa polypeptide, causes lysis by an unknown mechanism without affecting net peptidoglycan synthesis. To identify residues important for lytic function, randomly mutagenized alleles of L were generated, cloned into an inducible plasmid and the transformants were selected on agar containing the inducer. From a total of 396 clones, 67 were unique single base-pair changes that rendered L non-functional, of which 44 were missense mutants and 23 were nonsense mutants. Most of the non-functional missense alleles that accumulated in levels comparable to the wild-type allele are localized in the C-terminal half of L, clustered in and around an LS dipeptide sequence. The LS motif was used to align L genes from ssRNA phages lacking any sequence similarity to MS2 or to each other. This alignment revealed a conserved domain structure, in terms of charge, hydrophobic character and predicted helical content. None of the missense mutants affected membrane-association of L. Several of the L mutations in the central domains were highly conservative and recessive, suggesting a defect in a heterotypic protein-protein interaction, rather than in direct disruption of the bilayer structure, as had been previously proposed for L.