Abstract
Viral genomes not only code the protein content, but also include silent, overlapping codes which are important to the regulation of the viral life cycle and affect its evolution. Due to the high density of these codes, their non-modular nature and the complex intracellular processes they encode, the ability of current approaches to decipher them is very limited. We describe the first computational-experimental pipeline for studying the effects of viral silent and non-silent information on its fitness. The pipeline was implemented to study the Porcine Circovirus type 2 (PCV2), the shortest known eukaryotic virus, and includes the following steps: (1) Based on the analyses of 2100 variants of PCV, suspected silent codes were inferred. (2) Five hundred variants of the PCV2 were designed to include various ‘smart’ silent mutations. (3) Using state of the art synthetic biology approaches, the genomes of these five hundred variants were generated. (4) Competition experiments between the variants were performed in Porcine kidney-15 (PK15) cell-lines. (5) The variant titers were analyzed based on novel next-generation sequencing (NGS) experiments. (6) The features related to the titer of the variants were inferred and their analyses enabled detection of various novel silent functional sequence and structural motifs. Furthermore, we demonstrate that 50 of the silent variants exhibit higher fitness than the wildtype in the analyzed conditions.
Highlights
Viral genomes code the protein content, and include silent, overlapping codes which are important to the regulation of the viral life cycle and affect its evolution
In this paper we focus on Porcine Circovirus type 2 (PCV2), which is a nonenveloped, isometric virus, 16–21 nm in diameter that contains a covalently closed, circular, single-stranded DNA genome
The general stages of the research are as follows: (1) Creation of computational models and feature selection based on big data—testing thousands of wildtype (WT) variants of viruses
Summary
Viral genomes code the protein content, and include silent, overlapping codes which are important to the regulation of the viral life cycle and affect its evolution. In particular coding regions, determine the protein products of a virus, and how their production is regulated such as the regulation of viral gene expression, the replication of the viral genetic material, and avoidance of the immune s ystem[1,2,3,4,5] This regulation is encoded by the different combinations of synonymous codons, forming an overlapping second layer of information. In this paper we focus on Porcine Circovirus type 2 (PCV2), which is a nonenveloped, isometric virus, 16–21 nm in diameter that contains a covalently closed, circular, single-stranded DNA genome. It belongs to the family Circoviridae, genus Circovirus[19,20]. All four PCV species have a similar structure: a single stranded DNA circular genome that includes two main open reading frames (ORFs) turning to opposite d irections[21]
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