Fusion of a new world Alphavirus with membrane microdomains involving partially reversible conformational changes in the viral spike proteins

This work was supported by an international grant from International Centre for Genetic Engineering and Biotechnology (ICGEB) and by Brazilian grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa de Apoio ao Desenvolvimento Cientıfíco e Tecnológico (PADCT), and Programa de Apoio a Nucleos de Excelência (PRONEX).Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Centro de Ciências da Saúde. Rio de Janeiro, RJ, Brazil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brazil / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brazil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Centro de Ciências da Saúde. Rio de Janeiro, RJ, Brazil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Centro de Ciências da Saúde. Rio de Janeiro, RJ, Brazil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Centro de Ciências da Saúde. Rio de Janeiro, RJ, Brazil.Alphaviruses are enveloped arboviruses mainly proposed to infect host cells by receptor-mediated endocytosis followed by fusion between the viral envelope and the endosomal membrane. The fusion reaction is triggered by low pH and requires the presence of both cholesterol and sphingolipids in the target membrane, suggesting the involvement of lipid rafts in the cell entry mechanism. In this study, we show for the first time the interaction of an enveloped virus with membrane microdomains isolated from living cells. Using Mayaro virus (MAYV), a New World alphavirus, we verified that virus fusion to these domains occurred to a significant extent upon acidification, although its kinetics was quite slow when compared to that of fusion with artificial liposomes demonstrated in a previous work. Surprisingly, when virus was previously exposed to acidic pH, a condition previously shown to inhibit alphavirus binding and fusion to target membranes as well as infectivity, and then reneutralized, its ability to fuse with membrane microdomains at low pH was retained. Interestingly, this observation correlated with a partial reversion of low pH-induced conformational changes in viral proteins and retention of virus infectivity upon reneutralization. Our results suggest that MAYV entry into host cells could alternatively involve internalization via lipid rafts and that the conformational changes triggered by low pH in the viral spike proteins during the entry process are partially reversible

The interaction of dengue virus capsid protein with negatively charged interfaces drives the in vitro assembly of nucleocapsid-like particles.

Dengue virus (DENV) causes a major arthropod-borne viral disease, with 2.5 billion people living in risk areas. DENV consists in a 50 nm-diameter enveloped particle in which the surface proteins are arranged with icosahedral symmetry, while information about nucleocapsid (NC) structural organization is lacking. DENV NC is composed of the viral genome, a positive-sense single-stranded RNA, packaged by the capsid (C) protein. Here, we established the conditions for a reproducible in vitro assembly of DENV nucleocapsid-like particles (NCLPs) using recombinant DENVC. We analyzed NCLP formation in the absence or presence of oligonucleotides in solution using small angle X-ray scattering, Rayleigh light scattering as well as fluorescence anisotropy, and characterized particle structural properties using atomic force and transmission electron microscopy imaging. The experiments in solution comparing 2-, 5- and 25-mer oligonucleotides established that 2-mer is too small and 5-mer is sufficient for the formation of NCLPs. The assembly process was concentration-dependent and showed a saturation profile, with a stoichiometry of 1:1 (DENVC:oligonucleotide) molar ratio, suggesting an equilibrium involving DENVC dimer and an organized structure compatible with NCLPs. Imaging methods proved that the decrease in concentration to sub-nanomolar concentrations of DENVC allows the formation of regular spherical NCLPs after protein deposition on mica or carbon surfaces, in the presence as well as in the absence of oligonucleotides, in this latter case being surface driven. Altogether, the results suggest that in vitro assembly of DENV NCLPs depends on DENVC charge neutralization, which must be a very coordinated process to avoid unspecific aggregation. Our hypothesis is that a specific highly positive spot in DENVC α4-α4' is the main DENVC-RNA binding site, which is required to be firstly neutralized to allow NC formation

Dengue virus nonstructural 3 protein interacts directly with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and reduces its glycolytic activity

Submitted by Sandra Infurna ([email protected]) on 2019-05-02T15:36:16Z No. of bitstreams: 1 Marciano_Paes_etal_IOC_2019.pdf: 2757474 bytes, checksum: f484f68c0e57cb9176253927e0d15291 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-05-02T15:49:17Z (GMT) No. of bitstreams: 1 Marciano_Paes_etal_IOC_2019.pdf: 2757474 bytes, checksum: f484f68c0e57cb9176253927e0d15291 (MD5)Made available in DSpace on 2019-05-02T15:49:17Z (GMT). No. of bitstreams: 1 Marciano_Paes_etal_IOC_2019.pdf: 2757474 bytes, checksum: f484f68c0e57cb9176253927e0d15291 (MD5) Previous issue date: 2019Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Faculdade de Farmácia. Departamento de Biotecnologia Farmacêutica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biodiversidade e Sustentabilidade. Macaé, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisa Médica. Rio de Janeiro, RJ. Brasil.Universidade do Estado do Rio de Janeiro. Laboratório de Ultraestrutura e Biologia Tecidual. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Física Biológica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Física Biológica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Genômica Estrutural. Rio de Janeiro, RJ, Brasil.Dengue is an important mosquito-borne disease and a global public health problem. The disease is caused by dengue virus (DENV), which is a member of the Flaviviridae family and contains a positive single-stranded RNA genome that encodes a single precursor polyprotein that is further cleaved into structural and non-structural proteins. Among these proteins, the non-structural 3 (NS3) protein is very important because it forms a non-covalent complex with the NS2B cofactor, thereby forming the functional viral protease. NS3 also contains a C-terminal ATPase/helicase domain that is essential for RNA replication. Here, we identified 47 NS3-interacting partners using the yeast two-hybrid system. Among those partners, we highlight several proteins involved in host energy metabolism, such as apolipoprotein H, aldolase B, cytochrome C oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH directly binds full-length NS3 and its isolated helicase and protease domains. Moreover, we observed an intense colocalization between the GAPDH and NS3 proteins in DENV2-infected Huh7.5.1 cells, in NS3-transfected BHK-21 cells and in hepatic tissue from a fatal dengue case. Taken together, these results suggest that the human GAPDH-DENV NS3 interaction is involved in hepatic metabolic alterations, which may contribute to the appearance of steatosis in dengue-infected patients. The interaction between GAPDH and full-length NS3 or its helicase domain in vitro as well as in NS3-transfected cells resulted in decreased GAPDH glycolytic activity. Reduced GAPDH glycolytic activity may lead to the accumulation of metabolic intermediates, shifting metabolism to alternative, non-glycolytic pathways. This report is the first to identify the interaction of the DENV2 NS3 protein with the GAPDH protein and to demonstrate that this interaction may play an important role in the molecular mechanism that triggers hepatic alterations