13 research outputs found
Identification of putative interactions between swine and human influenza A virus nucleoprotein and human host proteins
Abstract
Background
Influenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. IAVs are enveloped, negative single-stranded RNA viruses whose genome encodes at least ten proteins. The IAV nucleoprotein (NP) is a structural protein that associates with the viral RNA and is essential for virus replication. Understanding how IAVs interact with host proteins is essential for elucidating all of the required processes for viral replication, restrictions in species host range, and potential targets for antiviral therapies.
Methods
In this study, the NP from a swine IAV was cloned into a yeast two-hybrid âbaitâ vector for expression of a yeast Gal4 binding domain (BD)-NP fusion protein. This âbaitâ was used to screen a Y2H human HeLa cell âpreyâ library which consisted of human proteins fused to the Gal4 proteinâs activation domain (AD). The interaction of âbaitâ and âpreyâ proteins resulted in activation of reporter genes.
Results
Seventeen positive bait-prey interactions were isolated in yeast. All of the âpreyâ isolated also interact in yeast with a NP âbaitâ cloned from a human IAV strain. Isolation and sequence analysis of the cDNAs encoding the human prey proteins revealed ten different human proteins. These host proteins are involved in various host cell processes and structures, including purine biosynthesis (PAICS), metabolism (ACOT13), proteasome (PA28B), DNA-binding (MSANTD3), cytoskeleton (CKAP5), potassium channel formation (KCTD9), zinc transporter function (SLC30A9), Na+/K+ ATPase function (ATP1B1), and RNA splicing (TRA2B).
Conclusions
Ten human proteins were identified as interacting with IAV NP in a Y2H screen. Some of these human proteins were reported in previous screens aimed at elucidating host proteins relevant to specific viral life cycle processes such as replication. This study extends previous findings by suggesting a mechanism by which these host proteins associate with the IAV, i.e., physical interaction with NP. Furthermore, this study revealed novel host protein-NP interactions in yeast.http://deepblue.lib.umich.edu/bitstream/2027.42/110223/1/12985_2014_Article_228.pd
Identification of putative interactions between swine and human influenza A virus nucleoprotein and human host proteins
Impact of infection on proteome-wide glycosylation revealed by distinct signatures for bacterial and viral pathogens
Mechanisms of infection and pathogenesis have predominantly been studied based on differential gene or protein expression. Less is known about posttranslational modifications, which are essential for protein functional diversity. We applied an innovative glycoproteomics method to study the systemic proteome-wide glycosylation in response to infection. The protein site-specific glycosylation was characterized in plasma derived from well-defined controls and patients. We found 3862 unique features, of which we identified 463 distinct intact glycopeptides, that could be mapped to more than 30 different proteins. Statistical analyses were used to derive a glycopeptide signature that enabled significant differentiation between patients with a bacterial or viral infection. Furthermore, supported by a machine learning algorithm, we demonstrated the ability to identify the causative pathogens based on the distinctive host blood plasma glycopeptide signatures. These results illustrate that glycoproteomics holds enormous potential as an innovative approach to improve the interpretation of relevant biological changes in response to infection
Genomic investigations of unexplained acute hepatitis in children
Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children
Glucose-mediated inhibition of calcium-activated potassium channels limits α-cell calcium influx and glucagon secretion
Pancreatic alpha-cells exhibit oscillations in cytosolic Ca2+ (Ca-c(2+)), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate alpha-cell Ca-c(2+) oscillations have not been elucidated. As beta-cell Ca-c(2-) oscillations are regulated in part by Ca2+-activated K+(K-SLOW) currents, this work investigated the role of K-SLOW, in alpha-cell Ca(2+ )handling and GCG secretion. alpha-Cells displayed K-SLOW, currents that were dependent on Ca2+ influx through L- and P/Q-type voltage-dependent Ca2+ channels (VDCCs) as well as Ca2+ released from endoplasmic reticulum stores. a-Cell K s i m ., was decreased by small-conductance Ca'activated K+ (SK) channel inhibitors apamin and UCL 1684. largeconductance Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+ (BK) channel inhibitor TRAM 34. Moreover, partial inhibition of alpha-cell K-SLOW, with apamin depolarized membrane potential (V-m) (3.8 +/- 0.7 mV) and reduced action potential (AP) amplitude (10.4 +/- 1.9 mV). Although apamin transiently increased Ca2+ influx into a-cells at low glucose (42.9 +/- 10.6%), sustained SK (38.5 +/- 10.4%) or BK channel inhibition (31.0 +/- 11.7%) decreased alpha-cell Ca2+ influx. Total alpha-cell Ca-c(2+) was similarly reduced (28.3 +/- 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced alpha-cell Ca-c(2+) following prolonged K-SLOW, inhibition. apamin decreased GCG secretion from mouse (20.4 +/- 4.2%) and human (27.7 +/- 13.1%) islets at low glucose. These data demonstrate that K-SLOW, activation provides a hyperpolarizing influence on alpha-cell V-m that sustains Ca2+ entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when alpha-cell Ca-c(2+) is elevated during secretagogue stimulation, K-SLOW activation helps to preserve GCG secretion