Quantitative Proteomic Analyses of Influenza Virus-Infected Cultured Human Lung Cells

Abstract

Because they are obligate intracellular parasites, all viruses are exclusively and intimately dependent upon host cells for replication. Viruses, in turn, induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays, which measure the cellular "transcriptome." Until recently, it has not been possible to extend comparable types of studies to globally examine all the host cellular proteins, which are the actual effector molecules. We have used stable isotope labeling by amino acids in cell culture (SILAC), combined with high-throughput two-dimensional (2-D) high-performance liquid chromatography (HPLC)/mass spectrometry, to determine quantitative differences in host proteins after infection of human lung A549 cells with human influenza virus A/PR/8/34 (H1N1) for 24 h. Of the 4,689 identified and measured cytosolic protein pairs, 127 were significantly upregulated at >95% confidence, 153 were significantly downregulated at >95% confidence, and a total of 87 proteins were upregulated or downregulated more than 5-fold at >99% confidence. Gene ontology and pathway analyses indicated differentially regulated proteins and included those involved in host cell immunity and antigen presentation, cell adhesion, metabolism, protein function, signal transduction, and transcription pathways.

Differential proteomic analysis of respiratory samples from patients suffering from influenza

“…Till date, however, only one scientific report [19] has been documented in literature studying nasal secretions from naturally influenza infected patients. Proteins identified in this study are different from those described in influenza-infected cell lines and primary macrophages [7,22]. Ten proteins were found to be differentially upregulated in the infected children including PLUNC, cystatin S, cystatin SA, S100A9, lipocalin 1 fragments (n52), truncated lactotransferrin, two immunoglobulin (Ig) kappa fragments and one immunoglobulin (Ig) lambda fragment.…”

Influenza A Virus-induced expression of ISG20 inhibits viral replication by interacting with nucleoprotein

“…Gene ontology enrichment and pathway analysis demonstrate that the regulated genes are involved in various biological processes, including immune responses, cell adhesion, cell metabolism, antigen presentation, and signal transduction. Notably, among these biological processes, most genes focus on immune response regulation [9,13]. The ISG20 gene was identified on the basis of induced expression level by both type I and type II IFNs in several cell lines and encodes an IFN-stimulated gene product of 20 kDa [14].…”

Lipidomics identifies a requirement for peroxisomal function during influenza virus replication

“…Infl uenza viruses hijack host cell machineries for efficient replication and acquire a host-derived lipid envelope during budding. Recent systems-scale studies have primarily addressed the individual roles of genes ( 1-5 ) and proteins (6)(7)(8) in this process, yet have failed to illustrate how they function together to generate macromolecular precursors Supernatants and cell lysates were subjected to plaque assay and Western blot at 18 hpi. Cell viability after drug treatment was assessed using Vybrant MTT cell proliferation assay kit (V13154) (Invitrogen®, Life Technologies Co., San Diego, CA) according to the manufacturer's protocol.…”