Studies on DENV and ZIKV Capsid Protein Dynamics in Living Cells During Infection?

COSTA NAVARRO, GUADALUPE; DE BORBA, LUANA; GONZALEZ LOPEZ LEDESMA, MARIA MORA; GABRIEL, MANUELA; BYK, LAURA; ESTRADA, LAURA C.; PALLARÉS, HORACIO; ROSSI, ANDRÉS HUGO; GAMARNIK, ANDREA V.

Killarney

Congreso; Positive-Strand RNA Viruses (Keystone Symposia); 2019

Keystone Symposia

Zika (ZIKV) and dengue (DENV) viruses are important pathogens that belong to the flavivirus genera. These viruses contain a single stranded, positive sense, RNA genome that encodes a polyprotein that is processed into structural and non-structural proteins. Capsid is a highly basic protein that forms homodimers in solution and associates with the viral genome to form the nucleocapsid. The capsid protein releases the viral RNA during uncoating and recruits the genome during morphogenesis. Although it has been shown that the capsid protein binds a large number of host proteins and accumulates in different subcellular compartments, little is known about the mechanisms of viral genome encapsidation and uncoating.To study the intracellular dynamics of DENV and ZIKV capsid proteins in living infected cells, we generated and used genetically modified viruses labeled with mCherry or GFP along with image correlation analysis tools, such as Raster Image Correlation Spectroscopy (RICS) and two-dimensional spatial pair-correlation functions (2D-pCF). We applied RICS to determine the protein diffusion constant and concentration in different cellular compartments, including cytoplasm/ER membranes, nucleus and nucleolus. In addition, using 2D-pCF, we generated maps describing the preferential routes of capsid diffusion in the infected cell. The results obtained revealed a striking different behavior for the DENV and ZIKV capsid proteins. The highly-organized circulation of DENV capsid at the interfaces cytoplasm/nucleus and nucleus/nucleolus showed a distinct mobility compared with ZIKV capsid. Although both proteins were found to accumulate in ER membranes, lipid droplets (LDs) and nucleolus, in the case of DENV infection the capsid accumulates first in the nucleolus, while in the case of ZIKV, it was found first in LDs and around the nuclear membrane, and only at late times after infection, it was observed inside the nucleus and nucleolus. These studies provide novel and quantitative information about the concentration, location and molecular diffusion of DENV and ZIKV capsid proteins in live infected cells, revealing unique properties for each of them.