Project description:Current mass spectrometry (MS) methods and new instrumentation now allow for more accurate identification of proteins in low abundance than previous protein fractionation and identification methods. It was of interest if this method could serve to define the virus proteome of a membrane-containing virus. To evaluate the efficacy of mass spec to determine the proteome of medically important viruses, Sindbis virus (SINV), the prototypical alphavirus was chosen for evaluation. This model system was chosen specifically because the alphaviruses contain members which are human pathogens, this virus is well defined biochemically and structurally, and grows to high titers in both vertebrate and non-vertebrate host cells. The SINV proteome was investigated using this method to determine if host proteins are specifically packaged into infectious virions. It was also of interest if the SINV proteome, when grown in multiple host cells representing vertebrate and mosquito hosts, incorporated specific host proteins from all hosts. Observation of recurrent or distinctive proteins in the virus proteome aided in the determination of proteins incorporated into the virion as opposed to those bound to the particle exterior. Mass spectrometry analysis identified the total protein content of purified virions within limits of detection. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress, identifying at least one host factor integrally involved in alphavirus replication. Key to the success of this analysis is the method of virus purification which must deliver measurably infectious virus free of high levels of contaminants. For SINV and other members of the alphavirus family, this is accomplished by isopycnic centrifugation through potassium tartrate, followed by a high salt wash.
Project description:Nipah virus (NiV) is a highly pathogenic, negative strand RNA paramyxovirus that has recently emerged from flying foxes to cause serious human disease. To study the poorly-understood role of nonstructural NiV proteins in NiV pathogenesis, we generated recombinant viruse lacking the expression of accesory NiV C protein (NiV∆C).
Project description:Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins' conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Results and discussion: Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.
Project description:Noroviruses cause immense sporadic gastroenteritis outbreaks worldwide. Upcoming genotypes, which are divided based on VP1 sequence, further enhance this public thread regularly. Self-assembling properties of the human norovirus major capsid protein VP1 are crucial for using virus-like particles (VLPs) for vaccine development. However, there is no vaccine available yet. Here, VLPs from different variants produced in insect cells are characterized in detail using a set of biophysical and structural tools.
Project description:The project aims at studying in vitro the TOPOVIBL∆C25 protein. This protein is essential to the formation of DNA double strand breaks, which initiates homologous recombination in meiosis in the mice. We set up a purification procedure of the recombinant TOPOVIBL∆C25 protein expressed in insect cells, which was validated using MS/MS approaches.
Project description:Virus-like particles (VLPs) are hollow nanoparticles composed of recombinant viral surface proteins without a virus genome. In the present study, we investigated the production of influenza VLPs using recombinant insect cells. DNA fragments encoding influenza A virus hemagglutinin (HA) and matrix protein 1 (M1) were cloned with the Drosophila BiP signal sequence in plasmid vectors containing a blasticidin and a neomycin resistance gene, respectively. After Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with a pair of constructed plasmid vectors, stably transformed cells were established via incubation with blasticidin and G418. Western blot analyses showed that recombinant High Five cells secreted HA and M1 proteins into the culture supernatant. Immunoprecipitation of the culture supernatant with an anti-HA antibody and transmission electron microscopy suggested that secreted HA and M1 proteins were in a particulate structure with a morphology similar to that of an influenza virus. Hemagglutination assay indicated that expressed HA molecules retained hemagglutination activity. In a shake-flask culture, recombinant cells achieved a high HA yield (≈ 10 μg/ml) comparable to the yields obtained using the baculovirus-insect cell system. Recombinant insect cells may serve as excellent platforms for the efficient production of influenza VLPs for use as safe and effective vaccines and diagnostic antigens.