The Human Respiratory Syncytial Virus Nonstructural Protein 1 Regulates Type I and Type II Interferon Pathways

Abstract

Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon-γ signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets.

Oxygen-dependent hydroxylation by Factor Inhibiting HIF (FIH) regulates the TRPV3 ion channel

“…Purified protein was then separated by SDS-PAGE and subjected to in-gel tryptic digestion as described previously (Hastie et al, 2012;Peet et al, 2004). In-gel digests were subjected to capillary high performance liquid chromatography (HPLC) and either arrayed onto matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF)/TOF targets for MALDI-TOF/ TOF-MS and -MS/MS (Dave et al, 2011) or sprayed directly into the ion-source of a High-Performance LTQ-Orbitrap Elite mass spectrometer essentially as described previously (Joubert et al, 2014).…”

A Comprehensive Proteomic View of Responses of A549 Type II Alveolar Epithelial Cells to Human Respiratory Syncytial Virus Infection

“…For instance, the use of fractionation of cells into cytoplasmic and nuclear fractions (23,(25)(26)(27)) is likely to have perturbed the ability to reliably quantify protein abundance changes at a global cellular level. Some of these studies may have been limited because of the lack of penetrating coverage of the proteomes using twodimensional (2D)-gel-based protocols (23)(24)(25)29), with (23,25) and without (24,29) subcellular fractionation steps. In some instances, proteome coverage may have been limited from an analytical technology standpoint (23)(24)(25)(26)(27)(28)(29).…”

“…Although this may be beneficial to the virus, a better understanding of how hRSV impairs host antiviral responses may also be the basis for design of effective vaccines and/or therapeutic strategies. Accordingly, a variety of studies have been conducted to define host cell responses to hRSV infection at both the transcriptional (18 -22) and proteomic (23)(24)(25)(26)(27)(28)(29) levels. This has included studies with forms of hRSV lacking genes that encode proteins known to impair host innate antiviral responses, such as the nonstructural protein1 (NS1) (24).…”

Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway

“…Nanoflow ultra-highpressure liquid chromatography (nUHPLC)-tandem MS (MS/MS) analysis was performed using a Waters (Milford, MA) NanoAcquity system (maximum pressure, 10,000 lb/in 2 ) interfaced with a linear ion trap (LTQ)-Orbitrap-Velos Pro hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The tryptic digests were acidified, loaded on the trap, and separated using nUHPLC as described previously (53,54). Peptides were separated by a set of three linear gradients: up to 27% solvent B over 45 min, up to 60% solvent B over 4 min, and up to 95% solvent B over 5 min.…”

“…In-gel trypsin digestion of excised SDS-PAGE gel bands and mass spectrometry analysis of the extracted peptides were performed as described previously (53). Nanoflow ultra-highpressure liquid chromatography (nUHPLC)-tandem MS (MS/MS) analysis was performed using a Waters (Milford, MA) NanoAcquity system (maximum pressure, 10,000 lb/in 2 ) interfaced with a linear ion trap (LTQ)-Orbitrap-Velos Pro hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).…”