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

MANUELA GABRIEL; LAURA BYK; LAURA ESTRADA; HORACIO M. PALLARES; ANDRES ROSSI; ANDREA V. GAMARNIK; GUADALUPE COSTA NAVARRO; LUANA DE BORBA; MARÍA MORA GONZALEZ LOPES LEDESMA

Killarney

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

Keystone Symposia

Zika (ZIKV) and dengue (DENV) viruses are importantpathogens that belong to the flavivirus genera. These viruses contain a singlestranded, positive sense, RNA genome that encodes a polyprotein that isprocessed into structural and non-structural proteins. Capsid is a highly basicprotein that forms homodimers in solution and associates with the viral genometo form the nucleocapsid. The capsid protein releases the viral RNA duringuncoating and recruits the genome during morphogenesis. Although it has beenshown that the capsid protein binds a large number of host proteins andaccumulates in different subcellular compartments, little is known about themechanisms of viral genome encapsidation and uncoating.To study the intracellular dynamics of DENV and ZIKVcapsid proteins in living infected cells, we generated and used geneticallymodified viruses labeled with mCherry or GFP along with image correlation analysistools, such as Raster Image Correlation Spectroscopy (RICS) and two-dimensionalspatial pair-correlation functions (2D-pCF). We applied RICS to determine theprotein diffusion constant and concentrationin different cellular compartments, includingcytoplasm/ER membranes, nucleus and nucleolus. In addition, using 2D-pCF,we generated maps describing the preferential routes of capsid diffusion in theinfected cell. The results obtained revealed a striking different behavior forthe DENV and ZIKV capsid proteins. The highly-organized circulation of DENV capsidat the interfaces cytoplasm/nucleus and nucleus/nucleolus showed a distinctmobility compared with ZIKV capsid. Although both proteins were found to accumulate in ERmembranes, lipid droplets (LDs) and nucleolus, in the case of DENV infectionthe capsid accumulates first in the nucleolus, while in the case of ZIKV, itwas found first in LDs and around the nuclear membrane, and only at late timesafter infection, it was observed inside the nucleus and nucleolus. Thesestudies provide novel and quantitative information about the concentration, locationand molecular diffusion of DENV and ZIKV capsid proteins in live infected cells,revealing unique properties for eachof them.