Membrane Association and Topology of Citrus Leprosis Virus C2 Movement and Capsid Proteins

Although citrus leprosis disease has been known for more than a hundred years, one of its causal agents, citrus leprosis virus C2 (CiLV-C2), is poorly characterized. This study described the association of CiLV-C2 movement protein (MP) and capsid protein (p29) with biological membranes. Our findings obtained by computer predictions, chemical treatments after membrane fractionation, and biomolecular fluorescence complementation assays revealed that p29 is peripherally associated, while the MP is integrally bound to the cell membranes. Topological analyses revealed that both the p29 and MP expose their N- and C-termini to the cell cytoplasmic compartment. The implications of these results in the intracellular movement of the virus were discussed.

Abstract 14836: Dual Dysfunction of Kir2.1 at the Sarcolemma and the Sarcoplasmic Reticulum Underlies Arrhythmogenesis in a Mouse Model of the Andersen-Tawil Syndrome Type 1

Introduction: Andersen-Tawil syndrome type 1 (ATS1) is associated with fatal cardiac arrhythmias. However, the underlying mechanisms are poorly understood. Hypothesis: Cardiac-specific expression of trafficking deficient Kir2.1 channels in mice in-vivo recapitulates the cardiac electrical phenotype of ATS1 and investigate the underlying mechanisms. Methods: We generated a new mouse model of ATS1 by a single i.v. injection of cardiac specific adeno-associated viral (AAV) transduction with Kir2.1 Δ314-315 , which recapitulated the ATS1 ECG phenotype without modifying ventricular function. The animal and cellular, structural and functional analyses were carried out by ECG, intracardiac stimulation, patch-clamping, membrane fractionation, western blot, immunolocalization and live calcium imaging. Results: AAV-Kir2.1 Δ314-315 mice were significantly more sensitive to flecainide than control, increasing the PR and QRS intervals over time. Kir2.1 Δ314-315 mice had increased vulnerability to cardiac fibrillation. Patch-clamping in ventricular cardiomyocytes from Kir2.1 Δ314-315 mice demonstrated significantly reduced I K1 and I Na , depolarized resting membrane potential and prolonged action potential. Immunolocalization in control mice revealed two bands of Kir2.1 staining, one colocalizing with Na V 1.5 and AP1 near the Z disk, the other near the H zone. Membrane fractionation and western blot experiments demonstrated two distinct levels of Kir2.1 protein expression, one at the sarcolemmal fraction together with Na V 1.5 and the other at the sarcoplasmi creticulum (SR). Kir2.1 Δ314-315 cardiomyocytes showed disruption of the Kir2.1-Nav1.5 channelosome at the sarcolemma, indicating dysfunctional trafficking o fboth channels. In addition, the SR Kir2.1 was disrupted and calcium transient dynamics were altered, resulting in frequent abnormal spontaneous calcium release events, and revealing an excitation-contraction coupling defect. Conclusions: Cardiac-specific AAV transduction with Kir2.1 Δ314-315 in mice recapitulates the ATS1 phenotype by disrupting localization and function of Kir2.1at the SR, and the Kir2.1-Na V 1.5 channelosome at the sarcolemma, revealing anovel dual mechanism of arrhythmogenesis.