Project description:Purpose:MicroRNAs (miRNAs) are members of a rapidly growing class of small endogenous non-coding RNAs that play crucial roles in post-transcriptional regulator of gene expression in many biological processes. Feline Panleukopenia Virus (FPV) is a highly infectious pathogen that causes severe disease in pets, economically important animals and wildlife in worldwide. However, the molecular mechanisms underlying the pathogenicity of FPV have not been completely clear. To study the involvement of miRNAs in the FPV infection process, miRNAs expression profiles were identified via deep sequencing in the feline kidney cell line (F81) infected and uninfected with FPV. Methods:miRNA-sequencing analysis was performed on an Illumina Hiseq 2500 (LC Sciences, USA) following the vendor's recommended protocol Results:As a result, 673 known miRNAs belonging to 210 families and 278 novel miRNAs were identified. Then we found 57 significantly differential expression miRNAs by comparing the results between uninfected and FPV-infected groups. Furthermore, stem-loop qRT-PCR was applied to validate and profile the expression of the randomly selected miRNAs; the results were consistent with those by deep sequencing. Furthermore, the potential target genes were predicted. The target genes of differential expression miRNAs were analyzed by GO and KEGG pathway. Conclusions:The identification of miRNAs in feline kidney cell line before and after infection with Feline Panleukopenia Virus will provide new information and enhance our understanding of the functions of miRNAs in regulating biological processes.
Project description:<p>Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate nucleocapsid assembly by characterizing the interactions of the wild-type and truncated capsid proteins with membranes by using biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.</p>
Project description:We are here presenting a new paracrine induction of RNA granules by viruses. Infection by viruses imposes major stress on the host cell. In response to this stress, infected cells can induce several defence mechanisms, which include the activation of stress response pathways and the innate immune response. These often result in an inhibition of translation culminating in the assembly of cytoplasmic granules called stress granules (SGs). SGs assembly follows from liquid phase separation of aggregation-prone proteins such G3BP1 and TIA-1, leading to the sequestration of mRNAs. Because this threatens viral gene expression, viruses need to evade these stress response pathways to propagate. Using feline calicivirus (FCV), surrogate for norovirus, the main virus responsible for gastroenteritis outbreaks worldwide, we previously showed that FCV impairs SGs assembly by cleaving the scaffold protein G3BP1. Interestingly, we observed that uninfected bystander cells assembled G3BP1 granules, suggesting a paracrine response trigged by the infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of RNA granules. We have characterised the dynamic of the granules assembly via confocal microscopy. Moreover, we provide an understanding of paracrine granules function and specificity through their affinity purification followed by proteomics and RNAseq analysis of their proteins and mRNAs content. This helps to define rules of assembly and novel functions for paracrine granules highlighting fundamental differences with canonical stress granules.