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Methods in Molecular Biology (Clifton,... 2024Many negative-sense single-stranded RNA viruses within the order Mononegavirales harm humans. A common feature shared among cells infected by these viruses is the...
Many negative-sense single-stranded RNA viruses within the order Mononegavirales harm humans. A common feature shared among cells infected by these viruses is the formation of subcellular membraneless structures called biomolecular condensates, also known as inclusion bodies (IBs), that form through a process called liquid-liquid phase separation (LLPS). Like many other membraneless organelles, viral IBs enrich a specific subset of viral and host proteins involved in the formation of viral particles. Elucidation of the properties and regulation of these IBs as they mature throughout the viral replication process are important for our understanding of viral replication, which may also lead to the development of alternative antiviral treatments. The protocol outlined in this chapter aims to characterize the intrinsic properties of LLPS within the measles virus (MeV, a member of Mononegavirales) IBs by using an imaging approach that fluorescently tags an IB-associated host protein. This method uses common laboratory techniques and is generalizable to any host factors as well as other viral systems.
Topics: Humans; Inclusion Bodies, Viral; Fluorescence Recovery After Photobleaching; Measles virus; Virus Replication; Inclusion Bodies; Animals; Host-Pathogen Interactions; Phase Separation
PubMed: 38743367
DOI: 10.1007/978-1-0716-3870-5_10 -
Methods in Molecular Biology (Clifton,... 2024During the infection of a host cell by an infectious agent, a series of gene expression changes occurs as a consequence of host-pathogen interactions. Unraveling this...
During the infection of a host cell by an infectious agent, a series of gene expression changes occurs as a consequence of host-pathogen interactions. Unraveling this complex interplay is the key for understanding of microbial virulence and host response pathways, thus providing the basis for new molecular insights into the mechanisms of pathogenesis and the corresponding immune response. Dual RNA sequencing (dual RNA-seq) has been developed to simultaneously determine pathogen and host transcriptomes enabling both differential and coexpression analyses between the two partners as well as genome characterization in the case of RNA viruses. Here, we provide a detailed laboratory protocol and bioinformatics analysis guidelines for dual RNA-seq experiments focusing on - but not restricted to - measles virus (MeV) as a pathogen of interest. The application of dual RNA-seq technologies in MeV-infected patients can potentially provide valuable information on the structure of the viral RNA genome and on cellular innate immune responses and drive the discovery of new targets for antiviral therapy.
Topics: Humans; Genome, Viral; Measles; Measles virus; RNA, Viral; Host-Pathogen Interactions; Computational Biology; Sequence Analysis, RNA; RNA-Seq; Transcriptome; Gene Expression Profiling; High-Throughput Nucleotide Sequencing
PubMed: 38743366
DOI: 10.1007/978-1-0716-3870-5_9 -
Methods in Molecular Biology (Clifton,... 2024Measles is a highly infectious disease that continues to spread mainly in developing countries, often resulting in child mortality. Despite the existence of effective...
Measles is a highly infectious disease that continues to spread mainly in developing countries, often resulting in child mortality. Despite the existence of effective vaccines, no specific antivirals are available as targeted therapy to combat measles virus (MeV). The implementation of genome-wide siRNA screens can provide a powerful platform to discover host factors that mediate MeV infection and replication, which could be essential to develop novel therapeutic strategies against this disease. Here, we describe a human genome-wide siRNA screen for MeV.
Topics: Humans; RNA, Small Interfering; Measles virus; Host-Pathogen Interactions; Virus Replication; Genome, Human; RNA Interference
PubMed: 38743365
DOI: 10.1007/978-1-0716-3870-5_8 -
Methods in Molecular Biology (Clifton,... 2024The study of virus-host interactions is essential to achieve a comprehensive understanding of the viral replication process. The commonly used methods are yeast...
The study of virus-host interactions is essential to achieve a comprehensive understanding of the viral replication process. The commonly used methods are yeast two-hybrid approach and transient expression of a single tagged viral protein in host cells followed by affinity purification of interacting cellular proteins and mass spectrometry analysis (AP-MS). However, by these approaches, virus-host protein-protein interactions are detected in the absence of a real infection, not always correctly compartmentalized, and for the yeast two-hybrid approach performed in a heterologous system. Thus, some of the detected protein-protein interactions may be artificial. Here we describe a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect protein partners during the infection (AP-MS in viral context). This way, virus-host protein-protein interacting co-complexes can be purified directly from infected cells for further characterization.
Topics: Measles virus; Humans; Host-Pathogen Interactions; Reverse Genetics; Viral Proteins; Two-Hybrid System Techniques; Virus Replication; Mass Spectrometry; Protein Interaction Mapping; Measles; Animals; Protein Binding
PubMed: 38743364
DOI: 10.1007/978-1-0716-3870-5_7 -
Methods in Molecular Biology (Clifton,... 2024Mononegaviruses are promising tools as oncolytic and transgene vectors for gene therapy and regenerative medicine. However, when mononegaviruses are used for therapeutic...
Mononegaviruses are promising tools as oncolytic and transgene vectors for gene therapy and regenerative medicine. However, when mononegaviruses are used for therapeutic applications, the viral activity must be strictly controlled due to concerns about toxicity and severe side effects. With this technology, mononegavirus vectors can be grown where they are intended and can be easily removed when they are no longer needed. In particular, a photoswitch protein called Magnet (consisting of two magnet domains) is incorporated into the hinge region between the connector and methyltransferase domains of the mononegavirus polymerase protein (L protein) to disrupt the L protein functions. Blue light (470 ± 20 nm) irradiation causes the dimerization of the two magnet domains, and the L protein is restored to activity, allowing viral gene expression and virus replication. Since the magnet domains' dimerization is reversible, viral gene expression and replication cease when blue light irradiation is stopped.
Topics: Virus Replication; Gene Expression Regulation, Viral; Humans; Viral Proteins; Light; Animals; Genetic Vectors
PubMed: 38743361
DOI: 10.1007/978-1-0716-3870-5_4 -
Methods in Molecular Biology (Clifton,... 2024Morbilliviruses such as measles virus (MeV) are responsible for major morbidity and mortality worldwide, despite the availability of an effective vaccine and global...
Morbilliviruses such as measles virus (MeV) are responsible for major morbidity and mortality worldwide, despite the availability of an effective vaccine and global vaccination campaigns. MeV belongs to the mononegavirus order of viral pathogens that store their genetic information in non-segmented negative polarity RNA genomes. Genome replication and viral gene expression are carried out by a virus-encoded RNA-dependent RNA polymerase (RdRP) complex that has no immediate host cell analog. To better understand the organization and regulation of the viral RdRP and mechanistically characterize antiviral candidates, biochemical RdRP assays have been developed that employ purified recombinant polymerase complexes and synthetic RNA templates to monitor the initiation of RNA synthesis and RNA elongation in vitro. In this article, we will discuss strategies for the efficient expression and preparation of mononegavirus polymerase complexes, provide detailed protocols for the execution and optimization of RdRP assays, evaluate alternative options for the choice of template and detection system, and describe the application of the assay for the characterization of inhibitor candidates. Although MeV RdRP assays are the focus of this article, the general strategies and experimental approaches are readily transferable to related viruses in the mononegavirus order.
Topics: Measles virus; RNA-Dependent RNA Polymerase; Virus Replication; RNA, Viral; Mononegavirales; Animals; Viral Proteins; Humans
PubMed: 38743360
DOI: 10.1007/978-1-0716-3870-5_3 -
Methods in Molecular Biology (Clifton,... 2024Protein-fragment complementation assays (PCAs) are powerful tools to investigate protein-protein interactions in a cellular context. These are especially useful to study...
Protein-fragment complementation assays (PCAs) are powerful tools to investigate protein-protein interactions in a cellular context. These are especially useful to study unstable proteins and weak interactions that may not resist protein isolation or purification. The PCA based on the reconstitution of the Gaussia princeps luciferase (split-luc) is a sensitive approach allowing the mapping of protein-protein interactions and the semiquantitative measurement of binding affinity. Here, we describe the split-luc protocol we used to map the viral interactome of measles virus polymerase complex.
Topics: Protein Interaction Mapping; Measles virus; Protein Binding; Humans; Luciferases; Viral Proteins; RNA-Dependent RNA Polymerase
PubMed: 38743359
DOI: 10.1007/978-1-0716-3870-5_2 -
Methods in Molecular Biology (Clifton,... 2024We have adopted a real-time assay based on a dual-split reporter to assess cell-cell fusion mediated by the measles virus (MeV) membrane fusion machinery. This reporter...
We have adopted a real-time assay based on a dual-split reporter to assess cell-cell fusion mediated by the measles virus (MeV) membrane fusion machinery. This reporter system is comprised of two expression vectors, each encoding a segment of Renilla luciferase fused to a segment of GFP. To regain function, the two segments need to associate, which is dependent on cell-cell fusion between effector cells expressing the MeV fusion machinery and target cells expressing the corresponding MeV receptor. By measuring reconstituted luciferase activity, we can follow the kinetics of cell-cell fusion and quantify the extent of fusion. This assay lends itself to the study of the MeV fusion machinery comprised of the attachment and fusion glycoproteins, the matrix protein, and the MeV receptors. Moreover, entry inhibitors targeting attachment or fusion can be readily screened using this assay. Finally, this assay can be easily adopted to study the entry of other members of the Paramyxoviridae, as we have demonstrated for the henipaviruses.
Topics: Measles virus; Virus Internalization; Humans; Animals; Cell Fusion; Genes, Reporter; Green Fluorescent Proteins; Chlorocebus aethiops; Cell Line; Vero Cells; Luciferases, Renilla
PubMed: 38743358
DOI: 10.1007/978-1-0716-3870-5_1 -
Microbial Genomics May 2024Paramyxoviruses are a group of single-stranded, negative-sense RNA viruses, some of which are responsible for acute human disease, including parainfluenza virus, measles...
Paramyxoviruses are a group of single-stranded, negative-sense RNA viruses, some of which are responsible for acute human disease, including parainfluenza virus, measles virus, Nipah virus and Hendra virus. In recent years, a large number of novel paramyxoviruses, particularly members of the genus , have been discovered in wild mammals, suggesting that the diversity of paramyxoviruses may be underestimated. Here we used hemi-nested reverse transcription PCR to obtain 190 paramyxovirus sequences from 969 small mammals in Hubei Province, Central China. These newly identified paramyxoviruses were classified into four clades: genera , , and , with most of them belonging to the genus . Using Illumina sequencing and Sanger sequencing, we successfully recovered six near-full-length genomes with different genomic organizations, revealing the more complex genome content of paramyxoviruses. Co-divergence analysis of jeilongviruses and their known hosts indicates that host-switching occurred more frequently in the evolutionary histories of the genus . Together, our findings demonstrate the high prevalence of paramyxoviruses in small mammals, especially jeilongviruses, and highlight the diversity of paramyxoviruses and their genome content, as well as the evolution of jeilongviruses.
Topics: Paramyxovirinae; Paramyxoviridae Infections; Mammals; China; Phylogeny; Genome, Viral; Host Specificity
PubMed: 38700925
DOI: 10.1099/mgen.0.001229 -
Viruses Apr 2024As SARS-CoV-2 continues to evolve and COVID-19 cases rapidly increase among children and adults, there is an urgent need for a safe and effective vaccine that can elicit...
As SARS-CoV-2 continues to evolve and COVID-19 cases rapidly increase among children and adults, there is an urgent need for a safe and effective vaccine that can elicit systemic and mucosal humoral immunity to limit the emergence of new variants. Using the Chinese Hu191 measles virus (MeV-hu191) vaccine strain as a backbone, we developed MeV chimeras stably expressing the prefusion forms of either membrane-anchored, full-length spike (rMeV-preFS), or its soluble secreted spike trimers with the help of the SP-D trimerization tag (rMeV-S+SPD) of SARS-CoV-2 Omicron BA.2. The two vaccine candidates were administrated in golden Syrian hamsters through the intranasal or subcutaneous routes to determine the optimal immunization route for challenge. The intranasal delivery of rMeV-S+SPD induced a more robust mucosal IgA antibody response than the subcutaneous route. The mucosal IgA antibody induced by rMeV-preFS through the intranasal routine was slightly higher than the subcutaneous route, but there was no significant difference. The rMeV-preFS vaccine stimulated higher mucosal IgA than the rMeV-S+SPD vaccine through intranasal or subcutaneous administration. In hamsters, intranasal administration of the rMeV-preFS vaccine elicited high levels of NAbs, protecting against the SARS-CoV-2 Omicron BA.2 variant challenge by reducing virus loads and diminishing pathological changes in vaccinated animals. Encouragingly, sera collected from the rMeV-preFS group consistently showed robust and significantly high neutralizing titers against the latest variant XBB.1.16. These data suggest that rMeV-preFS is a highly promising COVID-19 candidate vaccine that has great potential to be developed into bivalent vaccines (MeV/SARS-CoV-2).
Topics: Animals; Spike Glycoprotein, Coronavirus; SARS-CoV-2; Antibodies, Viral; Immunity, Mucosal; Antibodies, Neutralizing; Immunity, Humoral; COVID-19; COVID-19 Vaccines; Measles virus; Cricetinae; Immunoglobulin A; Humans; Administration, Intranasal; Mesocricetus; Female
PubMed: 38675901
DOI: 10.3390/v16040559