104 research outputs found
Phosphorylation regulates human T-cell leukemia virus type 1 Rex function
Background: Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic complex
deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-
1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic
viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral
accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes
important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling
protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and
incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1) is a
phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore,
it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation.
Results: We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells
using a combination of affinity purification and liquid chromatography tandem mass spectrometry.
We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel
phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence
of two previously identified residues, Ser-70 and Thr-174, but found no evidence of
phosphorylation at Ser-177. The functional significance of these phosphorylation events was
evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that
phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function.
Conclusion: We have mapped completely the site-specific phosphorylation of Rex-1 identifying a
total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this
work is the first to completely map the phosphorylation sites in Rex-1 and provides important
insight into the regulation of Rex-1 function
Human T-cell leukemia virus type 2 post-transcriptional control protein p28 is required for viral infectivity and persistence in vivo
Background: Human T-cell leukemia virus (HTLV) type 1 and type 2 are related but distinct
pathogenic complex retroviruses. HTLV-1 is associated with adult T-cell leukemia and a variety of
immune-mediated disorders including the chronic neurological disease termed HTLV-1-associated
myelopathy/tropical spastic paraparesis. In contrast, HTLV-2 displays distinct biological differences
and is much less pathogenic, with only a few reported cases of leukemia and neurological disease
associated with infection. In addition to the structural and enzymatic proteins, HTLV encodes
regulatory (Tax and Rex) and accessory proteins. Tax and Rex positively regulate virus production
and are critical for efficient viral replication and pathogenesis. Using an over-expression system
approach, we recently reported that the accessory gene product of the HTLV-1 and HTLV-2 open
reading frame (ORF) II (p30 and p28, respectively) acts as a negative regulator of both Tax and Rex
by binding to and retaining their mRNA in the nucleus, leading to reduced protein expression and
virion production. Further characterization revealed that p28 was distinct from p30 in that it was
devoid of major transcriptional modulating activity, suggesting potentially divergent functions that
may be responsible for the distinct pathobiologies of HTLV-1 and HTLV-2.
Results: In this study, we investigated the functional significance of p28 in HTLV-2 infection,
proliferation, and immortaliztion of primary T-cells in culture, and viral survival in an infectious
rabbit animal model. An HTLV-2 p28 knockout virus (HTLV-2Δp28) was generated and evaluated.
Infectivity and immortalization capacity of HTLV-2Δp28 in vitro was indistinguishable from wild type
HTLV-2. In contrast, we showed that viral replication was severely attenuated in rabbits inoculated
with HTLV-2Δp28 and the mutant virus failed to establish persistent infection.
Conclusion: We provide direct evidence that p28 is dispensable for viral replication and cellular
immortalization of primary T-lymphocytes in cell culture. However, our data indicate that p28
function is critical for viral survival in vivo. Our results are consistent with the hypothesis that p28
repression of Tax and Rex-mediated viral gene expression may facilitate survival of these cells by
down-modulating overall viral gene expression
Environmental determinants of allergy and asthma in early life
Allergic disease prevalence has increased significantly in recent decades. Primary prevention efforts are being guided by study of the exposome (or collective environmental exposures beginning during the prenatal period) to identify modifiable factors that affect allergic disease risk. In this review we explore the evidence supporting a relationship between key components of the external exposome in the prenatal and early-life periods and their effect on atopy development focused on microbial, allergen, and air pollution exposures. The abundance and diversity of microbial exposures during the first months and years of life have been linked with risk of allergic sensitization and disease. Indoor environmental allergen exposure during early life can also affect disease development, depending on the allergen type, dose, and timing of exposure. Recent evidence supports the role of ambient air pollution in allergic disease inception. The lack of clarity in the literature surrounding the relationship between environment and atopy reflects the complex interplay between cumulative environmental factors and genetic susceptibility, such that no one factor dictates disease development in all subjects. Understanding the effect of the summation of environmental exposures throughout a child's development is needed to identify cost-effective interventions that reduce atopy risk in children
Forensic profiling of smokeless powders (SLPs) by gas chromatography–mass spectrometry (GC-MS): a systematic investigation into injector conditions and their effect on the characterisation of samples
Smokeless powders (SLPs) are composed of a combination of thermolabile and non-thermolabile compounds. When analysed by GC-MS, injection conditions may therefore play a fundamental role on the characterisation of forensic samples. However, no systematic investigations have ever been carried out. This casts doubt on the optimal conditions that should be adopted in advanced profiling applications (e.g. class attribution and source association), especially when a traditional split/splitless (S/SL) injector is used. Herein, a study is reported that specifically focused on the evaluation of the liner type (L type) and inlet temperature (T inj). Results showed that both could affect the exhaustiveness and repeatability of the observed chemical profiles, with L type being particularly sensitive despite typically not being clarified in published works. Perhaps as expected, degradation effects were observed for the most thermolabile compounds (e.g. nitroglycerin) at conditions maximising the heat transfer rates (L type = packed and T inj ≥ 200 °C). However, these did not seem to be as influential as, perhaps, suggested in previous studies. Indeed, the harshest injection conditions in terms of heat transfer rate (L type = packed and T inj = 260 °C) were found to lead to better performances (including better overall %RSDs and LODs) compared to the mildest ones. This suggested that implementing conditions minimising heat-induced breakdowns during injection was not necessarily a good strategy for comparison purposes. The reported findings represent a concrete step forward in the field, providing a robust body of data for the development of the next generation of SLP profiling methods. Graphical abstract: (Figure presented.).</p
Diesel Exhaust Exposure and Nasal Response to Attenuated Influenza in Normal and Allergic Volunteers
Rationale: Diesel exhaust enhances allergic inflammation, and pollutants are associated with heightened susceptibility to viral respiratory infections. The effects of combined diesel and virus exposure in humans are unknown
Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study
Enhancing antiviral host defense responses through nutritional supplementation would be an attractive strategy in the fight against influenza. Using inoculation with live attenuated influenza virus (LAIV) as an infection model, we have recently shown that ingestion of sulforaphane-containing broccoli sprout homogenates (BSH) reduces markers of viral load in the nose. To investigate the systemic effects of short-term BSH supplementation in the context of LAIV-inoculation, we examined peripheral blood immune cell populations in non-smoking subjects from this study, with a particular focus on NK cells. We carried out a randomized, double-blinded, placebo-controlled study measuring the effects of BSH (N = 13) or placebo (alfalfa sprout homogenate, ASH; N = 16) on peripheral blood mononuclear cell responses to a standard nasal vaccine dose of LAIV in healthy volunteers. Blood was drawn prior to (day-1) and post (day2, day21) LAIV inoculation and analyzed for neutrophils, monocytes, macrophages, T cells, NKT cells, and NK cells. In addition, NK cells were enriched, stimulated, and assessed for surface markers, intracellular markers, and cytotoxic potential by flow cytometry. Overall, LAIV significantly reduced NKT (day2 and day21) and T cell (day2) populations. LAIV decreased NK cell CD56 and CD158b expression, while significantly increasing CD16 expression and cytotoxic potential (on day2). BSH supplementation further increased LAIV-induced granzyme B production (day2) in NK cells compared to ASH and in the BSH group granzyme B levels appeared to be negatively associated with influenza RNA levels in nasal lavage fluid cells. We conclude that nasal influenza infection may induce complex changes in peripheral blood NK cell activation, and that BSH increases virus-induced peripheral blood NK cell granzyme B production, an effect that may be important for enhanced antiviral defense responses
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