Uncoupling cis-Acting RNA elements from coding sequences revealed a requirement of the N-terminal region of dengue virus capsid protein in virus particle formation

Little is known about the mechanism of flavivirus genome encapsidation. Here, functional elements of the dengue virus (DENV) capsid (C) protein were investigated. Study of the N-terminal region of DENV C has been limited by the presence of overlapping cis-acting RNA elements within the protein-coding region. To dissociate these two functions, we used a recombinant DENV RNA with a duplication of essential RNA structures outside the C coding sequence. By the use of this system, the highly conserved amino acids FNML, which are encoded in the RNA cyclization sequence 5'CS, were found to be dispensable for C function. In contrast, deletion of the N-terminal 18 amino acids of C impaired DENV particle formation. Two clusters of basic residues (R5-K6-K7-R9 and K17-R18-R20-R22) were identified as important. A systematic mutational analysis indicated that a high density of positive charges, rather than particular residues at specific positions, was necessary. Furthermore, a differential requirement of N-terminal sequences of C for viral particle assembly was observed in mosquito and human cells. While no viral particles were observed in human cells with a virus lacking the first 18 residues of C, DENV propagation was detected in mosquito cells, although to a level about 50-fold less than that observed for a wild-type (WT) virus. We conclude that basic residues at the N terminus of C are necessary for efficient particle formation in mosquito cells but that they are crucial for propagation in human cells. This is the first report demonstrating that the N terminus of C plays a role in DENV particle formation. In addition, our results suggest that this function of C is differentially modulated in different host cellsFil: Samsa, Marcelo Mario Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Mondotte, Juan Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Caramelo, Julio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentin

Dengue Virus Uses a Non-Canonical Function of the Host GBF1-Arf-COPI System for Capsid Protein Accumulation on Lipid Droplets

Dengue viruses cause the most important human viral disease transmittedby mosquitoes. In recent years, a great deal has been learnedabout molecular details of dengue virus genome replication; however,little is known about genome encapsidation and the functions of theviral capsid protein. During infection, dengue virus capsid progressivelyaccumulates around lipid droplets (LDs) by an unknown mechanism.Here, we examined the process by which the viral capsid is transportedfrom the endoplasmic reticulum (ER) membrane, where the protein issynthesized, to LDs. Using different methods of intervention, we foundthat the GBF1-Arf1/Arf4-COPI pathway is necessary for capsid transportto LDs, while the process is independent of both COPII componentsand Golgi integrity. The transport was sensitive to Brefeldin A, while adrug resistant form of GBF1 was sufficient to restore capsid subcellulardistribution in infected cells. The mechanism by which LDs gain or loseproteins is still an open question. Our results support a model in whichthe virus uses a non-canonical function of the COPI system for capsidaccumulation on LDs, providing new ideas for antiviral strategies.Fil: Iglesias, Nestor Gabriel. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mondotte, Juan Alberto. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Byk, Laura Andrea. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de Maio, Federico Andres. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Samsa, Marcelo Mario Alejandro. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alvarez, Cecilia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Bioquímica Clínica E Inmunología; ArgentinaFil: Gamarnik, Andrea Vanesa. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin