Membrane association of greasy grouper nervous necrosis virus protein A and characterization of its mitochondrial localization targeting signal.

Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. The betanodavirus greasy grouper (Epinephelus tauvina) nervous necrosis virus (GGNNV) is a positive-RNA virus with one of the smallest genomes among RNA viruses replicating in fish cells. To understand the localization of GGNNV replication complexes, we generated polyclonal antisera against protein A, the GGNNV RNA-dependent RNA polymerase. Protein A was detected at 5 h postinfection in infected sea bass cells. Biochemical fractionation experiments revealed that GGNNV protein A sedimented with intracellular membranes upon treatment with an alkaline pH and a high salt concentration, indicating that GGNNV protein A is tightly associated with intracellular membranes in infected cells. Confocal immunofluorescence microscopy and bromo-UTP incorporation studies identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. In addition, protein A fused with green fluorescent protein (GFP) was detected in the mitochondria in transfected cells and was demonstrated to be tightly associated with intracellular membranes by biochemical fractionation analysis and membrane flotation assays, indicating that protein A alone was sufficient for mitochondrial localization in the absence of RNA replication, nonstructural protein B, or capsid proteins. Three sequence analysis programs showed two regions of hydrophobic amino acid residues, amino acids 153 to 173 and 229 to 249, to be transmembrane domains (TMD) that might contain a membrane association domain. Membrane fraction analysis showed that the major domain is N-terminal amino acids 215 to 255, containing the predicted TMD from amino acids 229 to 249. Using GFP as the reporter by systematically introducing deletions of these two regions in the constructs, we further confirmed that the N-terminal amino acids 215 to 255 of protein A function as a mitochondrial targeting signal.

Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein alpha as an apoptosis inducer.

Betanodaviruses, members of the Nodaviridae family, are the causative agents of viral nervous necrosis in fish and infection by which cause high mortality in larvae and juveniles in a wide range of marine fish species in Asia, Europe, Australia, Martinique, and Tahit. Greasy grouper (Epinephelus tauvina) nervous necrosis viruses (GGNNV) were investigated for their apoptotic activity in culture cells. GGNNV infection of sea bass (SB) cells appeared to induce a typical cytopathic effect (CPE), i.e., cytoplasmic vacuolation, thinning, rounding up, detachment of infected cells from the cultured dish, and eventually cell lysis and death. The infected SB cells underwent DNA fragmentation and stained positive in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay, suggesting that GGNNV infection induced apoptosis in SB cells. In addition, GGNNV-infected SB cells showed an increased activity of caspase-8-like proteases (IETDase) and caspase-3-like proteases (IETDase), whereas inhibitor of caspase-8 and caspase-3 reduced GGNNV-induced apoptosis. This suggests that GGNNV may promote apoptosis via the extrinsic pathway in SB cells. Protein alpha, the precursor of GGNNV capsid proteins, was transiently expressed in SB and Cos-7 cells. The DNA fragmentation and TUNEL positive signal were apparent in SB and Cos-7 cells expressing protein alpha, suggesting that protein alpha may serve as an apoptotic inducer in these cells. Moreover, expression of protein alpha resulted in the activation of caspase-3-like proteases in both cells, which could be inhibited by a caspase-3-like protease specific inhibitor DEVD-CHO peptide. These results suggest that fish caspases are important elements in GGNNV-meditated apoptosis.

Identification of nucleolus localization signal of betanodavirus GGNNV protein alpha.

Betanodavirus greasy grouper (Epinephelus tauvina) nervous necrosis viruses (GGNNV) protein alpha, a virus capsid protein, was detected in both nucleolus and cytoplasm of infected cells of Asian sea bass (SB) and transfected cells of SB and Cos-7 with pcDNA3.1/RNA2. To study its subcellular localization, ORF of protein alpha with 338 aa was fused with enhanced green fluorescent protein (EGFP) gene and was detected in transfected cells in the absence of other viral proteins. In both SB and Cos-7 cells, protein alpha was found to localize EGFP to the nucleolus and cytoplasm. Deletion mutants of protein alpha indicated that N-terminal 43 amino acid residues were required to import EGFP-alpha protein into the nucleolus. Further deletions within the 43 amino acid backbone, EGFP/33aa(1-33) and EGFP/30aa(14-43), localized to the nucleolus, suggesting that the 20 amino acids from 14 to 33 of protein alpha were the domain of nucleolus localization. To further determine the nucleolus targeting sequence, deletion mutations within the 20 amino acids of protein alpha were constructed. It was found that the deletion of (23)RRR(25), (29)RRR(31), or (23)RRRANNRRR(31) prevented the accumulation of EGFP fusion proteins into the nucleolus, demonstrating that (23)RRRANNRRR(31) contain the signal required for nucleolar localization. A similar distribution pattern of localization of protein alpha and its deletion mutants in SB and Cos-7 cells suggested that N-terminal residues of protein alpha (23)RRRANNRRR(31) constitute a nucleolus localization signal that functions in both fish and mammalian cells.

Cloning and Expression of Human CD20 Gene on NIH-3T3 Cell Membrane.

CD20 is a specific antigen expressed on normal and neoplastic B cells exclusively. Recent researches showed that in B cell leukemia, CD20 was over-expressed. Therefore monoclonal antibody (McAb) to CD20 may be of clinical value in diagnosis and treatment of some leukemias and lymphomas. In this study, the full length gene of CD20 cDNA were cloned from total RNA of Raji cells, inserted into an eukaryotic expression vector pcDNA3.1, forming a recombinant plasmid pcDNA3.1/CD20. NIH-3T3 cells were transfected with pcDNA3.1/CD20 and selected with G418 for the transfected cells. Alkaline phosphatase against alkaline phosphatase assay(APAAP) experiments showed that the selected cells could express the human CD20 onto its surface. Balb/c mice were immunized with CD20( ) NIH-3T3 cells once three weeks for 3 shuts. Indirect immunofluorescence experiments were done with the Raji cells and the serum of the immunized mice, and the results showed that the spleen of the immunized mice could be used to prepare the McAb to CD20.