RESUMO
The COVID-19 pandemic is driven by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) that emerged in 2019 and quickly spread worldwide. Genomic surveillance has become the gold standard methodology used to monitor and study this fast-spreading virus and its constantly emerging lineages. The current deluge of SARS-CoV-2 genomic data generated worldwide has put additional pressure on the urgent need for streamlined bioinformatics workflows. Here, we describe a workflow developed by our group to process and analyze large-scale SARS-CoV-2 Illumina amplicon sequencing data. This workflow automates all steps of SARS-CoV-2 reference-based genomic analysis: data processing, genome assembly, PANGO lineage assignment, mutation analysis and the screening of intrahost variants. The pipeline is capable of processing a batch of around 100 samples in less than half an hour on a personal laptop or in less than five minutes on a server with 50 threads. The workflow presented here is available through Docker or Singularity images, allowing for implementation on laptops for small-scale analyses or on high processing capacity servers or clusters. Moreover, the low requirements for memory and CPU cores and the standardized results provided by ViralFlow highlight it as a versatile tool for SARS-CoV-2 genomic analysis.
Assuntos
Automação Laboratorial/métodos , Genoma Viral , Mutação , SARS-CoV-2/classificação , SARS-CoV-2/genética , Fluxo de Trabalho , Biologia Computacional/instrumentação , Biologia Computacional/métodos , Genômica/instrumentação , Genômica/métodos , Humanos , Filogenia , Glicoproteína da Espícula de Coronavírus/genética , Montagem de Vírus/genéticaRESUMO
Designer virus-inspired proteins drive the manufacturing of more effective, safer gene-delivery systems and simpler models to study viral assembly. However, self-assembly of engineered viromimetic proteins on specific nucleic acid templates, a distinctive viral property, has proved difficult. Inspired by viral packaging signals, we harness the programmability of CRISPR-Cas12a to direct the nucleation and growth of a self-assembling synthetic polypeptide into virus-like particles (VLP) on specific DNA molecules. Positioning up to ten nuclease-dead Cas12a (dCas12a) proteins along a 48.5 kbp DNA template triggers particle growth and full DNA encapsidation at limiting polypeptide concentrations. Particle growth rate is further increased when dCas12a is dimerized with a polymerization silk-like domain. Such improved self-assembly efficiency allows for discrimination between cognate versus noncognate DNA templates by the synthetic polypeptide. CRISPR-guided VLPs will help to develop programmable bioinspired nanomaterials with applications in biotechnology as well as viromimetic scaffolds to improve our understanding of viral self-assembly.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Vírion , DNA , Nucleocapsídeo , Montagem de Vírus/genéticaRESUMO
Capsid proteins often present a positively charged arginine-rich sequence at their terminal regions, which has a fundamental role in genome packaging and particle stability for some icosahedral viruses. These sequences show little to no conservation and are structurally dynamic such that they cannot be easily detected by common sequence or structure comparisons. As a result, the occurrence and distribution of positively charged domains across the viral universe are unknown. Based on the net charge calculation of discrete protein segments, we identified proteins containing amino acid stretches with a notably high net charge (Q > + 17), which are enriched in icosahedral viruses with a distinctive bias towards arginine over lysine. We used viral particle structural data to calculate the total electrostatic charge derived from the most positively charged protein segment of capsid proteins and correlated these values with genome charges arising from the phosphates of each nucleotide. We obtained a positive correlation (r = 0.91, p-value <0001) for a group of 17 viral families, corresponding to 40% of all families with icosahedral structures described to date. These data indicated that unrelated viruses with diverse genome types adopt a common underlying mechanism for capsid assembly based on R-arms.
Assuntos
Proteínas do Capsídeo/metabolismo , Genoma Viral , Ácidos Nucleicos/metabolismo , Proteínas Virais/metabolismo , Vírus/genética , Vírus/metabolismo , Sequência de Aminoácidos , Nucleotídeos/metabolismo , Fosfatos/metabolismo , Domínios Proteicos , Eletricidade Estática , Vírion/metabolismo , Montagem de Vírus/genéticaRESUMO
The viral capsid is a macromolecular complex formed by a defined number of self-assembled proteins, which, in many cases, are biopolymers with an identical amino acid sequence. Specific protein-protein interactions (PPI) drive the capsid self-assembly process, leading to several distinct protein interfaces. Following the PPI hot spot hypothesis, we present a conservation-based methodology to identify those interface residues hypothesized to be crucial elements on the self-assembly and thermodynamic stability of the capsid. We validate the predictions through a rigorous physical framework which integrates molecular dynamics simulations and free energy calculations by Umbrella sampling and the potential of mean force using an all-atom molecular representation of the capsid proteins of an icosahedral virus in an explicit solvent. Our results show that a single mutation in any of the structure-conserved hot spots significantly perturbs the quaternary protein-protein interaction, decreasing the absolute value of the binding free energy, without altering the protein's secondary nor tertiary structure. Our conservation-based hot spot prediction methodology can lead to strategies to rationally modulate the capsid's thermodynamic properties.
Assuntos
Proteínas do Capsídeo/genética , Capsídeo/fisiologia , Montagem de Vírus/genética , Montagem de Vírus/fisiologia , Sequência de Aminoácidos , Simulação de Dinâmica Molecular , Mutação/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Conformação Proteica , Mapas de Interação de Proteínas/genética , Mapas de Interação de Proteínas/fisiologia , TermodinâmicaRESUMO
Virus-like particles (VLPs) are being used for therapeutic developments such as vaccines and drug nanocarriers. Among these, plant virus capsids are gaining interest for the formation of VLPs because they can be safely handled and are noncytotoxic. A paradigm in virology, however, is that plant viruses cannot transfect and deliver directly their genetic material or other cargos into mammalian cells. In this work, we prepared VLPs with the CCMV capsid and the mRNA-EGFP as a cargo and reporter gene. We show, for the first time, that these plant virus-based VLPs are capable of directly transfecting different eukaryotic cell lines, without the aid of any transfecting adjuvant, and delivering their nucleic acid for translation as observed by the presence of fluorescent protein. Our results show that the CCMV capsid is a good noncytotoxic container for genome delivery into mammalian cells.
Assuntos
Bromovirus/genética , Técnicas de Transferência de Genes , Vírus de Plantas/genética , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Células Eucarióticas/virologia , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Transfecção/métodos , Montagem de Vírus/genéticaRESUMO
The expression of several structural proteins from a wide variety of viruses in heterologous cell culture systems results in the formation of virus-like particles (VLPs). These VLPs structurally resemble the wild-type virus particles and have been used to study viral assembly process and as antigens for diagnosis and/or vaccine development. Tomato blistering mosaic virus (ToBMV) is a tymovirus that has a 6.3-kb positive-sense ssRNA genome. We have employed the baculovirus expression vector system (BEVS) for the production of tymovirus-like particles (tVLPs) in insect cells. Two recombinant baculoviruses containing the ToBMV wild-type coat protein (CP) gene or a modified short amino-terminal deletion (Δ2-24CP) variant were constructed and used to infect insect cells. Both recombinant viruses were able to express ToBMV CP and Δ2-24CP from infected insect cells that self-assembled into tVLPs. Therefore, the N-terminal residues (2-24) of the native ToBMV CP were shown not to be essential for self-assembly of tVLPs. We also constructed a third recombinant baculovirus containing a small sequence coding for the major epitope of the chikungunya virus (CHIKV) envelope protein 2 (E2) replacing the native CP N-terminal 2-24 amino acids. This recombinant virus also produced tVLPs. In summary, ToBMV VLPs can be produced in a baculovirus/insect cell heterologous expression system, and the N-terminal residues 2-24 of the CP are not essential for this assembly, allowing its potential use as a protein carrier that facilitates antigen purification and might be used for diagnosis.
Assuntos
Baculoviridae/genética , Proteínas do Capsídeo/biossíntese , Tymovirus/crescimento & desenvolvimento , Tymovirus/genética , Proteínas do Envelope Viral/biossíntese , Montagem de Vírus/genética , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Vírus Chikungunya/genética , Expressão Gênica/genética , Solanum lycopersicum/virologia , Mariposas/citologia , Proteínas do Envelope Viral/genéticaRESUMO
The packaging of genomic RNA in positive-sense single-stranded RNA viruses is a key part of the viral infectious cycle, yet this step is not fully understood. Unlike double-stranded DNA and RNA viruses, this process is coupled with nucleocapsid assembly. The specificity of RNA packaging depends on multiple factors: (i) one or more packaging signals, (ii) RNA replication, (iii) translation, (iv) viral factories, and (v) the physical properties of the RNA. The relative contribution of each of these factors to packaging specificity is different for every virus. In vitro and in vivo data show that there are different packaging mechanisms that control selective packaging of the genomic RNA during nucleocapsid assembly. The goals of this article are to explain some of the key experiments that support the contribution of these factors to packaging selectivity and to draw a general scenario that could help us move towards a better understanding of this step of the viral infectious cycle.
Assuntos
Genoma Viral , Nucleocapsídeo/genética , Vírus de RNA/genética , RNA Viral/genética , Montagem de Vírus/genética , Proteínas do Capsídeo/genética , Vírion/genéticaRESUMO
INTRODUCTION: Pseudo-infectious yellow fever viral particles (YFV-PIVs) have been used to study vaccines and viral packaging. Here, we report the development of a packaging cell line, which expresses the YFV prM/E proteins. METHODS: HEK293 cells were transfected with YFV prM/E and C (84 nt) genes to generate HEK293-YFV-PrM/E-opt. The cells were evaluated for their ability to express the heterologous proteins and to package the replicon repYFV-17D-LucIRES, generating YFV-PIVs. RESULTS: The expression of prM/E proteins was confirmed, and the cell line trans-packaged the replicon for recovery of a reporter for the YFV-PIVs. CONCLUSIONS: HEK293-YFV-prM/E-opt trans-packaging capacity demonstrates its possible biotechnology application.
Assuntos
Vacinas de Partículas Semelhantes a Vírus/imunologia , Montagem de Vírus/imunologia , Replicação Viral/imunologia , Vírus da Febre Amarela/imunologia , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Vacinas de Partículas Semelhantes a Vírus/genética , Montagem de Vírus/genética , Replicação Viral/genética , Vírus da Febre Amarela/genéticaRESUMO
Abstract INTRODUCTION: Pseudo-infectious yellow fever viral particles (YFV-PIVs) have been used to study vaccines and viral packaging. Here, we report the development of a packaging cell line, which expresses the YFV prM/E proteins. METHODS: HEK293 cells were transfected with YFV prM/E and C (84 nt) genes to generate HEK293-YFV-PrM/E-opt. The cells were evaluated for their ability to express the heterologous proteins and to package the replicon repYFV-17D-LucIRES, generating YFV-PIVs. RESULTS: The expression of prM/E proteins was confirmed, and the cell line trans-packaged the replicon for recovery of a reporter for the YFV-PIVs. CONCLUSIONS: HEK293-YFV-prM/E-opt trans-packaging capacity demonstrates its possible biotechnology application.
Assuntos
Humanos , Replicação Viral/imunologia , Vírus da Febre Amarela/imunologia , Montagem de Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Replicação Viral/genética , Vírus da Febre Amarela/genética , Montagem de Vírus/genética , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde , Células HEK293 , Vacinas de Partículas Semelhantes a Vírus/genética , Citometria de FluxoRESUMO
Chronic infection with HCV is a leading cause of cirrhosis, hepatocellular carcinoma and liver failure. One of the least understood steps in the HCV life cycle is the morphogenesis of new viral particles. HCV infection alters the lipid metabolism and generates a variety of microenvironments in the cell cytoplasm that protect viral proteins and RNA promoting viral replication and assembly. Lipid droplets (LDs) have been proposed to link viral RNA synthesis and virion assembly by physically associating these viral processes. HCV assembly, envelopment, and maturation have been shown to take place at specialized detergent-resistant membranes in the ER, rich in cholesterol and sphingolipids, supporting the synthesis of luminal LDs-containing ApoE. HCV assembly involves a regulated allocation of viral and host factors to viral assembly sites. Then, virus budding takes place through encapsidation of the HCV genome and viral envelopment in the ER. Interaction of ApoE with envelope proteins supports the viral particle acquisition of lipids and maturation. HCV secretion has been suggested to entail the ion channel activity of viral p7, several components of the classical trafficking and autophagy pathways, ESCRT, and exosome-mediated export of viral RNA. Here, we review the most recent advances in virus morphogenesis and the interplay between viral and host factors required for the formation of HCV virions.