ABSTRACT
There is large demand for a quantitative method for rapid and ultra-sensitive detection of the influenza virus. Here, we established a digital influenza virus counting (DIViC) method that can detect a single virion without antibody. In the assay, a virion is stochastically entrapped inside a femtoliter reactor array device for the fluorogenic assay of neuraminidase, and incubated for minutes. By analyzing 600,000 reactors, the practical limit of detection reached the order of 103 (PFU)/mL, only 10-times less sensitive than RT-PCR and more than 1000-times sensitive than commercial rapid test kits (RIDTs). Interestingly, neuraminidase activity differed among virions. The coefficient of variance was 30-40%, evidently broader than that of alkaline phosphatase measured as a model enzyme for comparison, suggesting the heterogeneity in size and integrity among influenza virus particles. Sensitivity to oseltamivir also differed between virions. We also tested DIViC using clinical gargle samples that imposes less burden for sampling while with less virus titre. The comparison with RIDTs showed that DIViC was largely superior to RIDTs in the sensitivity with the clinical samples although a few false-positive signals were observed in some clinical samples that remains as a technical challenge.
Subject(s)
Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H3N2 Subtype/enzymology , Neuraminidase/chemistry , Viral Proteins/chemistry , Virion/enzymologyABSTRACT
DAZAP1 is an evolutionarily conserved RNA-binding protein expressed in many tissues in mice and humans. DAZAP1-knockout mice carrying a partial loss-of-function (hypomorphic) allele exhibited severe deficiencies in spermatogenesis and cell growth, indicating that DAZAP1 plays a pivotal role in the development of germ and somatic cells. We have identified cox6c mRNA, which encodes a subunit of complex IV of the mitochondrial respiratory chain, as a target transcript regulated by DAZAP1. We found that DAZAP1 bound to cox6c mRNA derived from either the genomic DNA or a genome-type expression vector in cells, but not to cox6c mRNA derived from an intronless expression vector. Interestingly, the presence of the last intron was sufficient for DAZAP1 binding to the mRNA, suggesting specific intron dependent DAZAP1 loading onto cox6c mRNA. Overexpression of DAZAP1 resulted in the accumulation of cox6c pre-mRNA for all introns, implying that DAZAP1 reduces pre-mRNA splicing efficiency. In addition, the reduction of mature cox6c mRNA levels led to decreases in the COX6C protein levels. Both DAZAP1 knockdown and COX6C overexpression retarded cell growth. The lines of evidence presented here reveal that DAZAP1 is a negative regulator of pre-mRNA splicing and may control energy production in mitochondria by regulating COX6C expression. The DAZAP1 functions described in this study may also account for the phenotypes observed in the DAZAP1 hypomorphic mice.
Subject(s)
Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , RNA Precursors/metabolism , RNA-Binding Proteins/metabolism , Animals , Cell Proliferation , HEK293 Cells , Humans , Introns , Mice , Protein Binding , RNA SplicingABSTRACT
Posttranslational modifications of proteins have profound effects on many aspects of their function and have received much attention due to the importance of these processes in epigenetic regulation. In this study, we report that deleted azoospermia associated protein 1 (DAZAP1)/proline-rich RNA binding protein (Prrp), a multifunctional RNA binding protein which is essential for spermatogenesis and normal cell growth, is acetylated at Lysine 150 within its RNA binding domain. The acetylation is predominantly observed in nuclear Prrp, and the nonacetylated form is in cytoplasm. Considering that Prrp is a shuttling protein, we suggest that the acetylation cycle at Prrp K150 regulates nucleocytoplasmic transport in cells.