ABSTRACT
BACKGROUND: Nelson Bay orthoreovirus (NBV) was first isolated over 40 years ago from a fruit bat in Australia. Normally, NBV does not cause human diseases, but recently several NBV strains have been associated with human respiratory tract infections, thus attracting clinical attention. Autophagy, an evolutionarily conserved process in eukaryotic cells, degrades intracellular substrates, participates in multiple physiological processes, and maintains cellular homeostasis. In addition, autophagy is intimately involved in viral infection. METHODS: A new strain of NBV, isolated from a patient with a respiratory tract infection who returned to Japan from Bali, Indonesia, in 2007, was used in this study. NBV was rescued using a reverse genetics system involving cotransfection of BHK cells with 11 plasmids (pT7-L1 MB, pT7-L2 MB, pT7-L3 MB, pT7-M1 MB, pT7-M2 MB, pT7-M3 MB, pT7-S1 MB, pT7-S2 MB, pT7-S3 MB, pT7-S4 MB, and pcDNA3.1-T7), yielding NBV-MB. Recovered viruses were confirmed by immunofluorescence. The effect of NBV-MB on autophagy was evaluated by measuring the LC3-I/II proteins by immunoblot analysis after infection of BHK cells. Furthermore, after treatment with rapamycin (RAPA), 3-methyladenine (3-MA), chloroquine (CQ), or plasmid (GFP-LC3) transfection, the changes in expression of the LC3 gene and the amount of LC3-I/II protein were examined. In addition, variations in viral titer were assayed after treatment of BHK cells with drugs or after transfection with plasmids pCAGM3 and pCAGS3, which encode virus nonstructural proteins µNS and σNS, respectively. RESULTS: NBV-MB infection induced autophagy in host cells; however, the level of induction was dependent on viral replication. Induction of autophagy increased viral replication. By contrast, inhibiting autophagy suppressed NBV replication, albeit not significantly. The NBV-MB nonstructural protein µNS was involved in the induction of autophagy with viral infection. CONCLUSIONS: NBV-MB infection triggered autophagy. Also, the NBV nonstructural protein µNS may contribute to augmentation of autophagy upon viral infection.
Subject(s)
Autophagy , Host Microbial Interactions , Orthoreovirus/physiology , Virus Replication , Cell Line , HEK293 Cells , Humans , Reoviridae Infections/virology , Reverse Genetics , Viral Load , Viral Proteins/geneticsABSTRACT
Human astrovirus non-structural protein nsP1a/4, located at the C-terminal end of nsP1a, is thought to be involved in regulating RNA replication. Here, we show that host protein CD63 interacts with the nsP1a protein. Further research showed that the large loop (LEL) domain of CD63 also interacts with nsP1a/4. Confocal microscopy showed that nsP1a/4 protein and CD63 co-localized in the cytoplasm of co-transfected cells. Co-localization of nsP1a/4 and CD63 was also observed in HAstV-1-infected cells. Overexpression of CD63 promoted replication of HAstV-1, whereas knockdown of CD63 reduced production of HAstV-1 viral progeny. These results suggest that CD63 plays a critical role in HAstV-1 replication, and provide an avenue to further understanding the interactions between host and virus proteins during replication and pathogenesis of HAstV.
Subject(s)
Astroviridae/genetics , RNA, Viral/genetics , Tetraspanin 30/genetics , Viral Nonstructural Proteins/genetics , Virus Replication/genetics , Caco-2 Cells , Cell Line , Cell Line, Tumor , HEK293 Cells , HumansABSTRACT
Lactose intolerance in northern Europeans is strongly associated with a single-nucleotide polymorphism (SNP) located 14 kb upstream of the human lactase gene: -13,910 C/T. We examined whether SNPs in the 5' flanking region of the pig lactase gene are similar to those in the human gene and whether these polymorphisms play a functional role in regulating pig lactase gene expression. The 5' flanking region of the lactase gene from several different breeds of pigs was cloned and analyzed for gene regulatory activity of a luciferase reporter gene. One SNP was found in the enhancer region (-797 G/A) and two were found in the promoter region (-308G/C and -301 A/G). The promoter C-308,G-301(Pro-CG) strongly promotes the expression of the lactase gene, but the promoter G-308,A-301(Pro-GA) does not. The enhancer A-797(Enh-A) genotype for Pro-GA can significantly enhance promoter activity, but has an inhibitory effect on Pro-CG. The Enhancer G-797(Enh-G) has a significant inhibitory effect on both promoters. In conclusion, the order of effectiveness on the pig lactase gene is Enh-A+Pro-GA>Enh-A/G+Pro-CG>Enh-G+Pro-GA.