In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells

Ignacio Sallaberry,Andrea V Gamarnik,Guadalupe S Costa Navarro,Natalia Philipp,Manuela V Gabriel,Enrico Gratton,Alexis Luszczak,Laura C Estrada

doi:10.1038/s41598-021-03854-z

Abstract

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.

Highlights

Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others
To examine whether the fusion C-mCherry protein behaved as the WT C protein, we performed viral RNA transfections with the WT and the recombinant viral genomes
We reveal molecular dynamics and protein translocation by applying the pair correlation function method to the diffusion of dengue virus C-protein in infected cells

Summary

Introduction

Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. 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. 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. The pair Correlation Function (pCF) approach is one of the correlation-based techniques that has grown in the last years to study single molecule mobility. The pCF approach has proven to be a powerful tool to study molecular shuttling across the N E9, mobility within intracellular structures or obstacles such fibers or filaments (chromatin, actin, or microtubules)[10], microdomains (as the endoplasmic reticulum, ER), organelles, lipid droplets, among others

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