RESUMEN
Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.
Asunto(s)
Baculoviridae , Capripoxvirus , Ensayo de Inmunoadsorción Enzimática , Enfermedades de las Cabras , Cabras , Proteínas del Envoltorio Viral , Capripoxvirus/genética , Capripoxvirus/aislamiento & purificación , Baculoviridae/genética , Animales , Enfermedades de las Cabras/virología , Enfermedades de las Cabras/diagnóstico , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Cabras/virología , Ensayo de Inmunoadsorción Enzimática/métodos , Infecciones por Poxviridae/diagnóstico , Infecciones por Poxviridae/veterinaria , Infecciones por Poxviridae/virología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/inmunología , Virión/genética , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Células Sf9 , Antígenos Virales/genética , Antígenos Virales/inmunología , Línea Celular , Expresión GénicaRESUMEN
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.
Asunto(s)
Cápside , Levivirus , Levivirus/genética , Levivirus/química , Levivirus/metabolismo , Cápside/metabolismo , Cápside/química , Cápside/ultraestructura , Mutación , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Virión/metabolismo , Virión/genética , Virión/química , Mutación Puntual , Estabilidad Proteica , Humanos , Modelos MolecularesRESUMEN
Plus, minus, and double-strand RNA viruses are all found in nature. We use computational models to study the relative success of these strategies. We consider translation, replication, and virion assembly inside one cell, and transmission of virions between cells. For viruses which do not incorporate a polymerase in the capsid, transmission of only plus strands is the default strategy because virions containing minus strands are not infectious. Packaging only plus strands has a significant advantage if the number of RNA strands produced per cell is larger than the number of capsids. In this case, by not packaging minus strands, the virus produces more plus-strand virions. Therefore, plus-strand viruses are selected at low multiplicity of infection. However, at high multiplicity of infection, it is preferable to package both strands because the additional minus virions produced are helpful when there are multiple infections per cell. The fact that plus-strand viruses are widespread while viruses that package both strands are not seen in nature suggests that RNA strands are indeed produced in excess over capsids, and that the multiplicity of infection is not sufficiently high to favor the production of both kinds of virions. For double-strand viruses, we show that it is advantageous to produce only plus strands from the double strand within the cell, as is observed in real viruses. The reason for the success of minus-strand viruses is more puzzling initially. For viruses that incorporate a polymerase in the virion, minus virions are infectious. However, this is not sufficient to explain the success of minus-strand viruses, because in this case, viruses that package both strands outcompete those that package only minus or only plus. Real minus-strand viruses make use of replicable strands that are coated by a nucleoprotein, and separate translatable plus strands that are uncoated. Here we show that when there are distinct replicable and translatable strands, minus-strand viruses are selected.
Asunto(s)
Virus ARN , ARN Viral , Ensamble de Virus , Replicación Viral , Virus ARN/genética , Virus ARN/fisiología , ARN Viral/genética , ARN Viral/metabolismo , Virión/genética , Evolución Molecular , Cápside/metabolismoRESUMEN
African swine fever (ASF) is an acute and severe infectious disease caused by the African Swine Fever Virus (ASFV). ASFV exhibits significant resistance and stability in the environment, which, coupled with its double-stranded DNA and large genome, predisposes it to contaminate laboratory samples. This characteristic can lead to false-positive results in swine farm settings even days after disinfection, as detectable through polymerase chain reaction (PCR) or real-time fluorescent quantitative PCR (qPCR) assays. To meet the demand for efficient clinical methods capable of discriminating between ASFV nucleic acid and ASFV virions, this study aims to ascertain the efficacy of the nuclease "BenzoNuclease" in distinguishing ASFV nucleic acid (ASFV-DNA) from ASFV virions. BenzoNuclease is a versatile nucleic acid enzyme with the capacity to degrade nearly all forms of DNA and RNA. Initially, this research established a highly sensitive general PCR detection method for ASFV. Subsequently, a positive control was constructed using the M13 bacteriophage to substitute for active ASFV, facilitating the development of an improved qPCR method. It is important to note that common disinfectants have the potential to deactivate BenzoNuclease. However, in an environment simulating actual production applications, residual disinfectants do not interfere with the enzymatic efficacy of BenzoNuclease, thus not affecting the detection capabilities of this method. Positive clinical samples from pig farms, upon testing with the improved method, revealed that three samples were positive, indicating the presence of viral particles, while the remaining samples were negative, indicating the presence of nucleic acids. This provides an additional new option for sample testing in pig farms.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , ADN Viral , Virión , Virus de la Fiebre Porcina Africana/genética , Animales , Porcinos , Fiebre Porcina Africana/virología , ADN Viral/genética , Virión/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
The BK polyomavirus (BKPyV) is a small DNA non-enveloped virus whose infection is asymptomatic in most of the world's adult population. However, in cases of immunosuppression, the reactivation of the virus can cause various complications, and in particular, nephropathies in kidney transplant recipients or hemorrhagic cystitis in bone marrow transplant recipients. Recently, it was demonstrated that BKPyV virions can use extracellular vesicles to collectively traffic in and out of cells, thus exiting producing cells without cell lysis and entering target cells by diversified entry routes. By a comparison to other naked viruses, we investigated the possibility that BKPyV virions recruit the Endosomal-Sorting Complexes Required for Transport (ESCRT) machinery through late domains in order to hijack extracellular vesicles. We identified a single potential late domain in the BKPyV structural proteins, a YPX3L motif in the VP1 protein, and used pseudovirions to study the effect of point mutations found in a BKPyV clinical isolate or known to ablate the interaction of such a domain with the ESCRT machinery. Our results suggest that this domain is not involved in BKPyV association with extracellular vesicles but is crucial for capsomere interaction and thus viral particle assembly.
Asunto(s)
Secuencias de Aminoácidos , Virus BK , Proteínas de la Cápside , Vesículas Extracelulares , Virión , Ensamble de Virus , Virus BK/genética , Virus BK/fisiología , Virus BK/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virología , Humanos , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/química , Virión/metabolismo , Virión/genética , Infecciones por Polyomavirus/virología , Infecciones por Polyomavirus/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células HEK293RESUMEN
Phasmaviridae is a family for negative-sense RNA viruses with genomes of about 9.7-15.8 kb. These viruses are maintained in and/or transmitted by insects. Phasmavirids produce enveloped virions containing three single-stranded RNA segments that encode a nucleoprotein (N), a glycoprotein precursor (GPC), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Phasmaviridae, which is available at ictv.global/report/phasmaviridae.
Asunto(s)
Genoma Viral , ARN Viral , Animales , ARN Viral/genética , Virus ARN de Sentido Negativo/genética , Virus ARN de Sentido Negativo/clasificación , Virión/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Insectos/virología , Filogenia , Replicación ViralRESUMEN
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.
Asunto(s)
Levivirus , Levivirus/genética , Levivirus/química , Levivirus/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Temperatura , Mutación , Calor , Virión/metabolismo , Virión/química , Virión/genética , Cápside/metabolismo , Cápside/químicaRESUMEN
Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the surface of the AAV or AAV VLPs has been used to redirect the AAV to different target tissues and for vaccine development. Usually, the VLPs self-assemble intracellularly, and an extraction step must be performed before purification. Here, we describe the method we have used to extract AAV VLPs from insect cells successfully with peptide insertions on their surface.
Asunto(s)
Dependovirus , Péptidos , Dependovirus/genética , Animales , Péptidos/química , Péptidos/genética , Vectores Genéticos/genética , Virión/genética , Baculoviridae/genética , Células Sf9 , Humanos , Línea Celular , Proteínas de la Cápside/genética , Proteínas de la Cápside/aislamiento & purificaciónRESUMEN
Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).
Asunto(s)
Bromovirus , Bromovirus/genética , Animales , Baculoviridae/genética , Vectores Genéticos/genética , Cromatografía por Intercambio Iónico/métodos , Virión/aislamiento & purificación , Virión/genética , Virión/metabolismoRESUMEN
Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.
Asunto(s)
Baculoviridae , Vectores Genéticos , Ensayo de Placa Viral , Baculoviridae/genética , Células Sf9 , Ensayo de Placa Viral/métodos , Animales , Vectores Genéticos/genética , Transgenes , Virión/genética , Dependovirus/genética , Spodoptera/virologíaRESUMEN
Rotavirus causes severe diarrhea in infants. Although live attenuated rotavirus vaccines are available, vaccine-derived infections have been reported, which warrants development of next-generation rotavirus vaccines. A single-round infectious virus is a promising vaccine platform; however, this platform has not been studied extensively in the context of rotavirus. Here, we aimed to develop a single-round infectious rotavirus by impairing the function of the viral intermediate capsid protein VP6. Recombinant rotaviruses harboring mutations in VP6 were rescued using a reverse genetics system. Mutations were targeted at VP6 residues involved in virion assembly. Although the VP6-mutated rotavirus expressed viral proteins, it did not produce progeny virions in wild-type cells; however, the virus did produce progeny virions in VP6-expressing cells. This indicates that the VP6-mutated rotavirus is a single-round infectious rotavirus. Insertion of a foreign gene, and replacement of the VP7 gene segment with that of human rotavirus clinical isolates, was successful. No infectious virions were detected in mice infected with the single-round infectious rotavirus. Immunizing mice with the single-round infectious rotavirus induced neutralizing antibody titers as high as those induced by wild-type rotavirus. Taken together, the data suggest that this single-round infectious rotavirus has potential as a safe and effective rotavirus vaccine. This system is also applicable for generation of safe and orally administrable viral vectors.IMPORTANCERotavirus, a leading cause of acute gastroenteritis in infants, causes an annual estimated 128,500 infant deaths worldwide. Although live attenuated rotavirus vaccines are available, they are replicable and may cause vaccine-derived infections. Thus, development of safe and effective rotavirus vaccine is important. In this study, we report the development of a single-round infectious rotavirus that can replicate only in cells expressing viral VP6 protein. We demonstrated that (1) the single-round infectious rotavirus did not replicate in wild-type cells or in mice; (2) insertion of foreign genes and replacement of the outer capsid gene were possible; and (3) it was as immunogenic as the wild-type virus. Thus, the mutated virus shows promise as a next-generation rotavirus vaccine. The system is also applicable to orally administrable viral vectors, facilitating development of vaccines against other enteric pathogens.
Asunto(s)
Antígenos Virales , Proteínas de la Cápside , Mutación , Infecciones por Rotavirus , Vacunas contra Rotavirus , Rotavirus , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Rotavirus/genética , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Ratones , Infecciones por Rotavirus/virología , Infecciones por Rotavirus/prevención & control , Vacunas contra Rotavirus/genética , Vacunas contra Rotavirus/inmunología , Vacunas contra Rotavirus/administración & dosificación , Humanos , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Ratones Endogámicos BALB C , Línea Celular , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Virión/genética , FemeninoRESUMEN
Plant viruses such as brome mosaic virus and cowpea chlorotic mottle virus are effectively purified through PEG precipitation and sucrose cushion ultracentrifugation. Increasing ionic strength and an alkaline pH cause the viruses to swell and disassemble into coat protein subunits. The coat proteins can be reassembled into stable virus-like particles (VLPs) that carry anionic molecules at low ionic strength and through two-step dialysis from neutral pH to acidic buffer. VLPs have been extensively studied due to their ability to protect and deliver cargo, particularly RNA, while avoiding degradation under physiological conditions. Furthermore, chemical functionalization of the surface of VLPs allows for the targeted drug delivery. VLPs derived from plants have demonstrated great potential in nanomedicine by offering a versatile platform for drug delivery, imaging, and therapeutic applications.
Asunto(s)
Virus de Plantas , Virus de Plantas/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Virión/química , Virión/genética , Bromovirus/química , Bromovirus/genética , ARN/química , Concentración de Iones de Hidrógeno , ARN Viral/genéticaRESUMEN
Genetic screens have transformed our ability to interrogate cellular factor requirements for viral infections1,2, but most current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information that is often based on survival of infected cells2-4. Here, by engineering human cytomegalovirus to express single guide RNA libraries directly from the viral genome, we developed virus-encoded CRISPR-based direct readout screening (VECOS), a sensitive, versatile, viral-centric approach that enables profiling of different stages of viral infection in a pooled format. Using this approach, we identified hundreds of host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on virus-host interactions. These high-resolution measurements reveal that perturbations altering late stages in the life cycle of human cytomegalovirus (HCMV) mostly regulate viral particle quality rather than quantity, establishing correct virion assembly as a critical stage that is heavily reliant on virus-host interactions. Overall, VECOS facilitates systematic high-resolution dissection of the role of human proteins during the infection cycle, providing a roadmap for in-depth study of host-herpesvirus interactions.
Asunto(s)
Sistemas CRISPR-Cas , Infecciones por Citomegalovirus , Citomegalovirus , Interacciones Huésped-Patógeno , ARN Guía de Sistemas CRISPR-Cas , Replicación Viral , Humanos , Línea Celular , Sistemas CRISPR-Cas/genética , Citomegalovirus/genética , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Genoma Viral/genética , Interacciones Huésped-Patógeno/genética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Virión/genética , Virión/metabolismo , Ensamble de Virus/genética , Liberación del Virus/genética , Replicación Viral/genéticaRESUMEN
The HIV-1 nucleocapsid protein (NC) is a multifunctional viral protein necessary for HIV-1 replication. Recent studies have demonstrated that reverse transcription (RT) completes in the intact viral capsid, and the timing of RT and uncoating are correlated. How the small viral core stably contains the ~10 kbp double stranded (ds) DNA product of RT, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates dsDNA in a non-specific electrostatic binding mode that triggers uniform DNA self-attraction, condensing dsDNA into a tight globule against extending forces up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to characterize the role of NC's basic residues in dsDNA condensation. Basic residue mutations of NC lead to defective interaction with the dsDNA substrate, with the constant force plateau condensation observed with wild-type (WT) NC missing or diminished. These results suggest that NC's high positive charge is essential to its dsDNA condensing activity, and electrostatic interactions involving NC's basic residues are responsible in large part for the conformation, size, and stability of the dsDNA-protein complex inside the viral core. We observe DNA re-solubilization and charge reversal in the presence of excess NC, consistent with the electrostatic nature of NC-induced DNA condensation. Previous studies of HIV-1 replication in the presence of the same cationic residue mutations in NC showed significant defects in both single- and multiple-round viral infectivity. Although NC participates in many stages of viral replication, our results are consistent with the hypothesis that cationic residue mutations inhibit genomic DNA condensation, resulting in increased premature capsid uncoating and contributing to viral replication defects.
Asunto(s)
ADN Viral , VIH-1 , Transcripción Reversa , VIH-1/genética , VIH-1/fisiología , VIH-1/química , VIH-1/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Humanos , Cationes/metabolismo , Replicación Viral , Microscopía de Fuerza Atómica , Virión/metabolismo , Virión/genética , Virión/química , MutaciónRESUMEN
HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm. At the plasma membrane, the membrane competes with gRNA for Gag binding, resulting in a transition to the extended form of Gag found in immature particles with MA bound to membrane lipids and NC to gRNA. The Gag compact form was previously evidenced in vitro. Here, we demonstrated the compact form of Gag in cells by confocal microscopy, using a bimolecular fluorescence complementation approach with a split-GFP bipartite system. Using wild-type Gag and Gag mutants, we showed that the compact form is highly dependent on the binding of MA and NC domains to RNA, as well as on interactions between MA and CA domains. In contrast, Gag multimerization appears to be less critical for the accumulation of the compact form. Finally, mutations altering the formation of Gag compact form led to a strong reduction in viral particle production and infectivity, revealing its key role in the production of infectious viral particles.
Asunto(s)
VIH-1 , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , VIH-1/metabolismo , VIH-1/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Ensamble de Virus/genética , Humanos , Virión/metabolismo , Virión/genética , Unión Proteica , ARN Viral/metabolismo , ARN Viral/genética , Membrana Celular/metabolismo , Membrana Celular/virologíaRESUMEN
Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.
Asunto(s)
Citidina , Virus de la Hepatitis B , ARN Viral , Transcripción Reversa , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , Virus de la Hepatitis B/fisiología , ARN Viral/genética , ARN Viral/metabolismo , Citidina/análogos & derivados , Citidina/metabolismo , Citidina/genética , Humanos , Transcripción Reversa/genética , Metilación , Replicación Viral/genética , Epigénesis Genética , Virión/metabolismo , Virión/genética , TranscriptomaRESUMEN
Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.
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Dependovirus , Vectores Genéticos , Transfección , Humanos , Dependovirus/genética , Dependovirus/aislamiento & purificación , Células HEK293 , Vectores Genéticos/genética , Vectores Genéticos/aislamiento & purificación , Transfección/métodos , Plásmidos/genética , Plásmidos/aislamiento & purificación , Polietileneimina/química , Reactores Biológicos , Cromatografía por Intercambio Iónico/métodos , Virión/genética , Virión/aislamiento & purificaciónRESUMEN
Nipah virus (NiV), a highly pathogenic Henipavirus in humans, has been responsible for annual outbreaks in recent years. Experiments involving live NiV are highly restricted to biosafety level 4 (BSL-4) laboratories, which impedes NiV research. In this study, we developed transcription and replication-competent NiV-like particles (trVLP-NiV) lacking N, P, and L genes. This trVLP-NiV exhibited the ability to infect and continuously passage in cells ectopically expressing N, P, and L proteins while maintaining stable genetic characteristics. Moreover, the trVLP-NiV displayed a favourable safety profile in hamsters. Using the system, we found the NiV nucleoprotein residues interacting with viral RNA backbone affected viral replication in opposite patterns. This engineered system was sensitive to well-established antiviral drugs, innate host antiviral factors, and neutralizing antibodies. We then established a high-throughput screening platform utilizing the trVLP-NiV, leading to the identification of tunicamycin as a potential anti-NiV compound. Evidence showed that tunicamycin inhibited NiV replication by decreasing the infectivity of progeny virions. In conclusion, this trVLP-NiV system provided a convenient and versatile molecular tool for investigating NiV molecular biology and conducting antiviral drug screening under BSL-2 conditions. Its application will contribute to the development of medical countermeasures against NiV infections.
Asunto(s)
Infecciones por Henipavirus , Virus Nipah , Replicación Viral , Virus Nipah/fisiología , Virus Nipah/genética , Virus Nipah/efectos de los fármacos , Animales , Cricetinae , Humanos , Infecciones por Henipavirus/virología , Transcripción Genética , Virión/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Antivirales/farmacología , Células Vero , Chlorocebus aethiops , Línea Celular , ARN Viral/genéticaRESUMEN
Antigen-presenting cells (APCs) play an important role in virus infection control by bridging innate and adaptive immune responses. Macrophages and dendritic cells (DCs) possess various surface receptors to recognize/internalize antigens, and antibody binding can enhance pathogen-opsonizing uptake by these APCs via interaction of antibody fragment crystallizable (Fc) domains with Fc receptors, evoking profound pathogen control in certain settings. Here, we examined phagocytosis-enhancing potential of Fc domains directly oriented on a retroviral virion/virus-like particle (VLP) surface. We generated an expression vector coding a murine Fc fragment fused to the transmembrane region (TM) of a retroviral envelope protein, deriving expression of the Fc-TM fusion protein on the transfected cell surface and production of virions incorporating the chimeric Fc upon co-transfection. Incubation of Fc-displaying simian immunodeficiency virus (SIV) with murine J774 macrophages and bone marrow-derived DCs derived Fc receptor-dependent enhanced uptake, being visualized by imaging cytometry. Alternative preparation of a murine leukemia virus (MLV) backbone-based Fc-displaying VLP loading an influenza virus hemagglutinin (HA) antigen resulted in enhanced HA internalization by macrophages, stating antigen compatibility of the design. Results show that the Fc-TM fusion molecule can be displayed on certain viruses/VLPs and may be utilized as a molecular adjuvant to facilitate APC antigen uptake.
Asunto(s)
Células Presentadoras de Antígenos , Células Dendríticas , Fragmentos Fc de Inmunoglobulinas , Virión , Animales , Ratones , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/inmunología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Virión/metabolismo , Virión/genética , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Macrófagos/metabolismo , Macrófagos/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Línea Celular , Virus de la Leucemia Murina/genética , Fagocitosis , HumanosRESUMEN
Baculoviruses enter insect midgut epithelial cells via a set of occlusion-derived virion (ODV) envelope proteins called per os infectivity factors (PIFs). P74 of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which was the first identified PIF, is cleaved by an endogenous proteinase embedded within the occlusion body during per os infection, but the target site(s) and function of the cleavage have not yet been ascertained. Here, based on bioinformatics analyses, we report that cleavage was predicted at an arginine and lysine-rich region in the middle of P74. A series of recombinant viruses with site-directed mutants in this region of P74 were generated. R325 or R334 was identified as primary cleavage site. In addition, we showed that P74 is also cleaved by brush border membrane vesicles (BBMV) of the host insect at R325 or R334, instead of R195, R196, and R199, as previously reported. Simultaneous mutations in R195, R196, and R199 lead to instability of P74 during ODV release. Bioassays showed that mutations at both R325 and R334 significantly affected oral infectivity. Taken together, our data show that both R325 and R334 of AcMNPV P74 are the primary cleavage site for both occlusion body endogenous proteinase and BBMV proteinase during ODV release and are critical for oral infection. IMPORTANCE: Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a per os infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus per os infection.