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Production and characterization of novel ssRNA bacteriophage virus-like particles from metagenomic sequencing data.

ABSTRACT: BACKGROUND:Protein shells assembled from viral coat proteins are an attractive platform for development of new vaccines and other tools such as targeted bioimaging and drug delivery agents. Virus-like particles (VLPs) derived from the single-stranded RNA (ssRNA) bacteriophage coat proteins (CPs) have been important and successful contenders in the area due to their simplicity and robustness. However, only a few different VLP types are available that put certain limitations on continued developments and expanded adaptation of ssRNA phage VLP technology. Metagenomic studies have been a rich source for discovering novel viral sequences, and in recent years have unraveled numerous ssRNA phage genomes significantly different from those known before. Here, we describe the use of ssRNA CP sequences found in metagenomic data to experimentally produce and characterize novel VLPs. RESULTS:Approximately 150 ssRNA phage CP sequences were sourced from metagenomic sequence data and grouped into 14 different clusters based on CP sequence similarity analysis. 110 CP-encoding sequences were obtained by gene synthesis and expressed in bacteria which in 80 cases resulted in VLP assembly. Production and purification of the VLPs was straightforward and compatible with established protocols, with the only exception that a considerable proportion of the CPs had to be produced at a lower temperature to ensure VLP assembly. The VLP morphology was similar to that of the previously studied phages, although a few deviations such as elongated or smaller particles were noted in certain cases. In addition, stabilizing inter-subunit disulfide bonds were detected in six VLPs and several possible candidate RNA structures in the phage genomes were identified that might bind to the coat protein and ensure specific RNA packaging. CONCLUSIONS:Compared to the few types of ssRNA phage VLPs that were used before, several dozens of new particles representing ten distinct similarity groups are now available with a notable potential for biotechnological applications. It is believed that the novel VLPs described in this paper will provide the groundwork for future development of new vaccines and other applications based on ssRNA bacteriophage VLPs.

SUBMITTER: Lieknina I

PROVIDER: S-EPMC6513524 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Production and characterization of novel ssRNA bacteriophage virus-like particles from metagenomic sequencing data.

Liekniņa Ilva I Kalniņš Gints G Akopjana Ināra I Bogans Jānis J Šišovs Mihails M Jansons Juris J Rūmnieks Jānis J Tārs Kaspars K

Journal of nanobiotechnology 20190513 1

<h4>Background</h4>Protein shells assembled from viral coat proteins are an attractive platform for development of new vaccines and other tools such as targeted bioimaging and drug delivery agents. Virus-like particles (VLPs) derived from the single-stranded RNA (ssRNA) bacteriophage coat proteins (CPs) have been important and successful contenders in the area due to their simplicity and robustness. However, only a few different VLP types are available that put certain limitations on continued d ...[more]

PMID: 31084612

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Project description:BackgroundThe prediction of bacteriophage sequences in metagenomic datasets has become a topic of considerable interest, leading to the development of many novel bioinformatic tools. A comparative analysis of ten state-of-the-art phage identification tools was performed to inform their usage in microbiome research.MethodsArtificial contigs generated from complete RefSeq genomes representing phages, plasmids, and chromosomes, and a previously sequenced mock community containing four phage species, were used to evaluate the precision, recall, and F1 scores of the tools. We also generated a dataset of randomly shuffled sequences to quantify false-positive calls. In addition, a set of previously simulated viromes was used to assess diversity bias in each tool's output.ResultsVIBRANT and VirSorter2 achieved the highest F1 scores (0.93) in the RefSeq artificial contigs dataset, with several other tools also performing well. Kraken2 had the highest F1 score (0.86) in the mock community benchmark by a large margin (0.3 higher than DeepVirFinder in second place), mainly due to its high precision (0.96). Generally, k-mer-based tools performed better than reference similarity tools and gene-based methods. Several tools, most notably PPR-Meta, called a high number of false positives in the randomly shuffled sequences. When analysing the diversity of the genomes that each tool predicted from a virome set, most tools produced a viral genome set that had similar alpha- and beta-diversity patterns to the original population, with Seeker being a notable exception.ConclusionsThis study provides key metrics used to assess performance of phage detection tools, offers a framework for further comparison of additional viral discovery tools, and discusses optimal strategies for using these tools. We highlight that the choice of tool for identification of phages in metagenomic datasets, as well as their parameters, can bias the results and provide pointers for different use case scenarios. We have also made our benchmarking dataset available for download in order to facilitate future comparisons of phage identification tools. Video Abstract.

Project description:Viruses are a significant component of the intestinal microbiota in mammals. In recent years, advances in sequencing technologies and data analysis techniques have enabled detailed metagenomic studies investigating intestinal viromes (collections of bacteriophage and eukaryotic viral nucleic acids) and their potential contributions to the ecology of the microbiota. An important component of virome studies is the isolation and purification of virus-like particles (VLPs) from intestinal contents or feces. Several methods have been applied to isolate VLPs from intestinal samples, yet to our knowledge, the efficiency and reproducibility between methods have not been explored. A rigorous evaluation of methods for VLP purification is critical as many studies begin to move from descriptive analyses of virus diversity to studies striving to quantitatively compare viral abundances across many samples. Therefore, reproducible VLP purification methods which allow for high sample throughput are needed. Here we compared and evaluated four methods for VLP purification using artificial intestinal microbiota samples of known bacterial and viral composition.We compared the following four methods of VLP purification from fecal samples: (i) filtration?+?DNase, (ii) dithiothreitol treatment?+?filtration?+?DNase, (iii) filtration?+?DNase?+?PEG precipitation and (iv) filtration?+?DNase?+?CsCl density gradient centrifugation. Three of the four tested methods worked well for VLP purification. We observed several differences between methods related to the removal efficiency of bacterial and host DNAs and biases against specific phages. In particular the CsCl density gradient centrifugation method, which is frequently used for VLP purification, was most efficient in removing host derived DNA, but also showed strong discrimination against specific phages and showed a lower reproducibility of quantitative results.Based on our data we recommend the use of methods (i) or (ii) for large scale studies when quantitative comparison of viral abundances across samples is required. The CsCl density gradient centrifugation method, while being excellently suited to achieve highly purified samples, in our opinion, should be used with caution when performing quantitative studies.

Dataset Information

Production and characterization of novel ssRNA bacteriophage virus-like particles from metagenomic sequencing data.

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Production and characterization of novel ssRNA bacteriophage virus-like particles from metagenomic sequencing data.

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