The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach.
Dengue is an important mosquito borne viral disease in the world. Dengue virus (DENV) encodes a polyprotein, which is cleaved in ten proteins, including the non-structural protein 1 (NS1). In this work, we analyzed the effect of NS1 expression in one hepatic cell line, HepG2, through a shotgun proteomic approach. Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) and pMAXGFP (GFP, a protein unrelated to dengue). Expression of NS1 was detected by immunofluorescence, western blot and flow cytometry. We identified 14,138 peptides that mapped to 4,756 proteins in all analyzed conditions. We found 41 and 81 differentially abundant proteins when compared to cells transfected with plasmids pcDNA3 and pMAXGFP, respectively. Besides, 107 proteins were detected only in the presence of NS1. We identified clusters of proteins involved mainly in mRNA process and viral RNA replication. Down regulation expression of one protein (MARCKS), identified by the proteomic analysis, was also confirmed by real time PCR in HepG2 cells infected with DENV2. Identification of proteins modulated by the presence of NS1 may improve our understanding of its role in virus infection and pathogenesis, contributing to development of new therapies and vaccines.



Dengue is an important viral disease, with epidemics in tropical and subtropical regions of the world. The disease is complex, with different manifestations, in which the liver is normally affected. The NS1 is found in infected cells associated with plasma membrane and secreted into the circulation as a soluble multimer. This protein is essential for virus viability, although its function is not elucidated. Some reports indicate that the NS1 can be used as a protective antigen for the development of a dengue vaccine, while others suggest its involvement in viral pathogenesis. In this work, we report an in-depth comprehensive proteomic profiling resulting from the presence of NS1 in HepG2 cells. These results can contribute to a better understanding of the NS1 role during infection.
Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
Kíssila
K
Rabelo
Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
Monique R O
MRO
Trugilho
Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
Simone M
SM
Costa
Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
Bernardo A S
BAS
Pereira
Laboratory of Molecular Biology and Endemic Diseases, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
Otacílio C
OC
Moreira
Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
André T S
ATS
Ferreira
Laboratory for Proteomics and Protein Engineering, Carlos Chagas Institute, Fiocruz, Paraná, Brazil.
Paulo C
PC
Carvalho
Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
Jonas
J
Perales
Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil. Electronic address: ada@ioc.fiocruz.br.
Ada M B
AMB
Alves
D016000:Cluster Analysis; D003716:Dengue Virus / Q000737:chemistry / Q000502:physiology; D056945:Hep G2 Cells / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D008099:Liver / Q000821:virology; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000032:analysis / Q000502:physiology
C079340:NS1 protein, Dengue virus type 2; D012333:RNA, Messenger; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins
Dengue; HepG2; NS1; Proteomics