Ultrastructural and immunocytochemical evidences of core-particle formation in the methylotrophic Pichia pastoris yeast when expressing HCV structural proteins (core-E1).
Particulate antigens of the Hepatitis C virus (HCV) are reported for the first time by transmission electron microscopy in Pichia pastoris. The yeast was cloned to express the first 339 NH2-terminal amino acids of the HCV polyprotein (C-E1.339 polypeptide). The C-E1.339 polypeptide covers the putative 191 aa of the core protein (aa 1-191) and 148 aa of the E1 envelope antigen (aa 192-339). Virus-like particles (VLP) with diameters ranging from 20 nm to 30 nm were specifically observed in those cells expressing the HCV polyprotein. The VLP appeared along the membrane of the endoplasmic reticulum, but were fundamentally localized in vacuoles, either free or inside autophagic bodies. Clustered particles, chains of particles, high-density reticular structures, and crystalloid bodies were also detected, the last one being an orderly arrangement of particles with 20 nm diameters. The crystal-associated particles are well differentiated from the intracellular VLP because of their uniform size and shape. We argue that membrane components are retained in the architecture of the VLP, conferring to this particle certain heterogeneity. Both kinds of particles, the VLP formed after treatment with NP-40 and the crystal-associated particles, were core protein-positives. Whether they reflect mature HCV nucleocapsid or intermediary states in the viral nucleocapsid morphogenesis remains unknown. We conclude that, like mammalian cell lines, the P. pastoris yeast could be an appropriate host for the analysis of HCV polyprotein processing and, eventually, virus assembly.
Falcón
V
V
Electron Microscopy Department, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.
García
C
C
de la Rosa
M C
MC
Menéndez
I
I
Seoane
J
J
Grillo
J M
JM
D015870:Gene Expression; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D016253:Microscopy, Immunoelectron; D010843:Pichia / Q000648:ultrastructure; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics; D014771:Virion / Q000648:ultrastructure; D019065:Virus Assembly
C087035:E1 protein, Hepatitis C virus; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; C069130:nucleocapsid protein, Hepatitis C virus