A 6K-deletion variant of salmonid alphavirus is non-viable but can be rescued through RNA recombination

• Published in: PloS one Vol. 9; no. 7; p. e100184
• Main Authors: Guo, Tz-Chun, Johansson, Daniel X, Haugland, Øyvind, Liljeström, Peter, Evensen, Øystein
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.07.2014; Public Library of Science (PLoS)
• Subjects: Alphavirus; Alphavirus - drug effects; Alphavirus - genetics; Alphavirus - physiology; Amino Acid Sequence; Analysis; Animals; Antibodies - immunology; Aquaculture; Biology and Life Sciences; Budding; Cell Line; Cell surface; Clonal deletion; Cytoplasm; Cytoplasm - metabolism; DNA, Complementary - genetics; Fish; Fishes; Gene Deletion; Genetics; Genomes; Genomics; Health aspects; Hydrophobicity; Infectivity; Interferon-alpha - pharmacology; Medicin och hälsovetenskap; Medicine and Health Sciences; Membrane proteins; Mice; Microbial Viability - genetics; Molecular Sequence Data; Molecular Weight; Nucleotide sequence; Oncorhynchus kisutch - virology; Oncorhynchus mykiss; Pancreas; Proteins; Recombination; Recombination, Genetic; Research and Analysis Methods; Ribonucleic acid; RNA; RNA, Viral - genetics; Salmo salar; Salmon; Salmonids; Transfection; Trout; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - immunology; Viral Proteins - metabolism; Virion - drug effects; Virion - physiology; Virus replication; Viruses; Scotland; Sweden; United Kingdom > UK; Norway; Ireland
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0100184

Pancreas disease (PD) of Atlantic salmon is an emerging disease caused by Salmonid alphavirus (SAV) which mainly affects salmonid aquaculture in Western Europe. Although genome structure of SAV has been characterized and each individual viral protein has been identified, the role of 6K protein in viral replication and infectivity remains undefined. The 6K protein of alphaviruses is a small and hydrophobic protein which is involved in membrane permeabilization, protein processing and virus budding. Because these common features are shared across many viral species, they have been named viroporins. In the present study, we applied reverse genetics to generate SAV3 6K-deleted (Δ6K) variant and investigate the role of 6K protein. Our findings show that the 6K-deletion variant of salmonid alphavirus is non-viable. Despite viral proteins of Δ6K variant are detected in the cytoplasm by immunostaining, they are not found on the cell surface. Further, analysis of viral proteins produced in Δ6K cDNA clone transfected cells using radioimmunoprecipitation (RIPA) and western blot showed a protein band of larger size than E2 of wild-type SAV3. When Δ6K cDNA was co-transfected with SAV3 helper cDNA encoding the whole structural genes including 6K, the infectivity was rescued. The development of CPE after co-transfection and resolved genome sequence of rescued virus confirmed full-length viral genome being generated through RNA recombination. The discovery of the important role of the 6K protein in virus production provides a new possibility for the development of antiviral intervention which is highly needed to control SAV infection in salmonids.