Project description:This experiment was undertaken to document changes in gene expression in the skin of tick-resistant Brahman (Bos indicus) and tick-susceptible Holstein-Friesian (Bos taurus) cattle prior to, and following, infestation with the cattle tick Rhipicephalus (Boophilus) microplus Experiment Overall Design: RNA was extracted from skin samples of tick-naïve cattle (animals with no previous R.microplus exposure) and tick-infested cattle after a period of successive, heavy infestations with R. microplus. Skin samples taken from tick-infested animals were taken at sites where tick larvae (approximately 24 h old) were attached to the skin sample. Skin samples were of 8 mm diameter and full skin thickness (approximately 10 mm). RNA samples from 12 animals (3 tick-naive Holstein-Friesian, 3 tick-naive Brahman, 3 tick-infested Holstein-Friesian and 3 tick-infested Brahman) were processed and hybridised to individual slides.
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>