Intracellular accumulation of hepatitis C virus proteins in a human hepatoma cell line.
OBJECTIVE
The establishment of HCV replicon systems strongly improved the research on the replication processes but poorly advanced our knowledge on the subcellular localization of the structural glycoproteins, mainly due to their low expression. We sought to verify whether reinforcing E1E2 expression in the context of both HCV genomic and subgenomic replicon from either homologous or heterologous strains leads to formation of supramolecular structures including structural and nonstructural proteins.


METHODS
Robust expression of HCV glycoproteins was achieved by stable expression of E1E2p7 from genotype 1a and 1b.


RESULTS
In these cells, E1 and E2 triggered the formation of dot-like structures in which they co-localized with core and the nonstructural proteins NS3 and NS5A. Confocal microscopy analyses suggested that accumulation of HCV proteins occurs in an ER-derived subcompartment. Moreover, by labeling de novo-synthesized HCV RNA, we showed that these structures constitute a site of viral RNA synthesis.


CONCLUSIONS
Expression in trans of HCV glycoproteins in the context of replicative viral genome or subgenome generates accumulation of structural and nonstructural viral proteins in peculiar cytoplasmic structures. The simultaneous presence of viral RNA, structural and nonstructural protein suggests that these complexes represent not only sites of HCV replication but also potential places of viral pre-budding.
Brazzoli
Michela
M
Novartis Vaccines and Diagnostics, via Fiorentina 1, 53100 Siena, Italy.
Crotta
Stefania
S
Bianchi
Alessia
A
Bagnoli
Fabio
F
Monaghan
Paul
P
Wileman
Thomas
T
Abrignani
Sergio
S
Merola
Marcello
M
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000821:virology; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000821:virology; D015870:Gene Expression; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008113:Liver Neoplasms / Q000821:virology; D046912:Multiprotein Complexes; D012093:Replicon; D013347:Subcellular Fractions / Q000821:virology; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D004279:DNA, Viral; D046912:Multiprotein Complexes; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C087035:E1 protein, Hepatitis C virus; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C091015:glycoprotein E2, Hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase