RESUMO
Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. DENV infects hundreds of millions of people per year around the world, causing a tremendous social and economic burden. DENV capsid (C) protein plays an essential role during genome encapsidation and viral particle formation. It has been previously shown that DENV C enters the nucleus in infected cells. However, whether DENV C protein exhibits nuclear export remains unclear. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. We observed that transport takes place in both directions and with similar translocation times (in the ms time scale) suggesting a bidirectional movement of both C protein import and export.Furthermore, from the pair cross-correlation functions in cytoplasmic or nuclear regions we found two populations of C molecules in each compartment with fast and slow mobilities. While in the cytoplasm the correlation times were in the 2-6 and 40-110 ms range for the fast and slow mobility populations respectively, in the cell nucleus they were 1-10 and 25-140 ms range, respectively. The fast mobility of DENV C in cytoplasmic and nuclear regions agreed with the diffusion coefficients from Brownian motion previously reported from correlation analysis. These studies provide the first evidence of DENV C shuttling from and to the nucleus in infected cells, opening new venues for antiviral interventions.
Assuntos
Proteínas do Capsídeo/ultraestrutura , Vírus da Dengue/ultraestrutura , Dengue/virologia , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , CricetinaeRESUMO
Dengue is the single most important human viral infection transmitted by insects. The function of the viral proteins andtheir interactions with the host cell is under exhaustive investigation with the aim of identifying antiviral strategies. Here,using recombinant full-length dengue virus genomes, carrying a fluorescent mCherry fused to capsid, we studied biophysicalproperties of the viral protein during one infectious cycle in living cells. Dengue virus capsid protein associates to differentcellular compartments but its function in these locations is largely unknown. We evaluated the diffusion of capsid inside the celland determined a higher effective diffusion coefficient in the cytoplasm than in the nucleus. Using advanced fluorescencecorrelation methods, including the recently developed two-dimensional pair correlation analysis, we constructed for the first timehigh resolution maps of capsid mobility in an infected cell. We observed that the motion of capsid in the nucleoplasm-nucleolusinterface was highly organized, indicating an obstacle in this interface. Although nucleoli are membraneless structures, theydisplayed liquid-liquid phase separation. Once inside nucleoli, the protein showed isotropic mobility, indicating free diffusion orimmobilized capsid inside these structures. This is the first study presenting spatial and temporal dynamics of the dengue viruscapsid protein during infection.