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31.
Hector C. Aguilar Zeynep Akyol Ataman Vanessa Aspericueta Angela Q. Fang Matthew Stroud Oscar A. Negrete Richard A. Kammerer Benhur Lee 《The Journal of biological chemistry》2009,284(3):1628-1635
Cellular entry of paramyxoviruses requires the coordinated action of both
the attachment (G/H/HN) and fusion (F) glycoproteins, but how receptor binding
activates G to trigger F-mediated fusion during viral entry is not known.
Here, we identify a receptor (ephrinB2)-induced allosteric activation site in
Nipah virus (NiV) G involved in triggering F-mediated fusion. We first
generated a conformational monoclonal antibody (monoclonal antibody 45
(Mab45)) whose binding to NiV-G was enhanced upon NiV-G-ephrinB2 binding.
However, Mab45 also inhibited viral entry, and its receptor binding-enhanced
(RBE) epitope was temperature-dependent, suggesting that the Mab45 RBE epitope
on G may be involved in triggering F. The Mab45 RBE epitope was mapped to the
base of the globular domain (β6S4/β1H1). Alanine scan mutants within
this region that did not exhibit this RBE epitope were also non-fusogenic
despite their ability to bind ephrinB2, oligomerize, and associate with F at
wild-type (WT) levels. Although circular dichroism revealed conformational
changes in the soluble ectodomain of WT NiV-G upon ephrinB2 addition, no such
changes were detected with soluble RBE epitope mutants or short-stalk G
mutants. Additionally, WT G, but not a RBE epitope mutant, could dissociate
from F upon ephrinB2 engagement. Finally, using a biotinylated HR2 peptide to
detect pre-hairpin intermediate formation, a cardinal feature of F-triggering,
we showed that ephrinB2 binding to WT G, but not the RBE-epitope mutants,
could trigger F. In sum, we implicate the coordinated interaction between the
base of NiV-G globular head domain and the stalk domain in mediating
receptor-induced F triggering during viral entry.The paramyxoviruses comprise a group of important human pathogens, such as
measles, mumps, human parainfluenza viruses, and the highly pathogenic Nipah
(NiV)4 and Hendra
(HeV) viruses. NiV infections have a mortality rate in humans of up to 75%,
and NiV is classified as a BSL4 pathogen because of its bio- or agro-terrorism
potential (1). The efficacy of
entry inhibitors targeted against HIV suggests that a better understanding of
Paramyxovirus entry and fusion will facilitate similarly efficacious
antiviral therapeutics.Although past studies have identified regions in either the fusion (F) or
attachment (G/H/HN) glycoproteins that are important for membrane fusion or
F-G/H/HN association
(2–10),
the region(s) in G important for receptor-activated triggering of F-mediated
fusion remains unknown. Current models of Paramyxovirus membrane
fusion posit that receptor binding to the attachment glycoprotein (G, H, or
HN) triggers a conformational cascade in the fusion protein (F). Such
F-triggering results in fusion peptide (FP) exposure, which involves formation
of a pre-hairpin intermediate and subsequent six-helix bundle formation
(11). The energy released upon
refolding into the stable six-helix bundle ground state is what drives the
fusion of the viral and host-cell membranes. These are common functional and
structural features responsible for membrane fusion for all enveloped viruses
regardless of whether the fusion protein has predominantly trimeric
α-helical coiled-coil (Class I), β (Class II), or a combination of
α and β (Class III) core structures
(12). Important human
pathogens such as the HIV, influenza, and various paramyxoviruses have Class I
fusion proteins, and their similar structural features point to similar
membrane fusion mechanisms
(11,
12). Besides sharing trimeric
coiled-coil structures, they are synthesized as precursors that are cleaved
into a metastable conformation; cleavage generates a new hydrophobic N
terminus FP that gets released and inserted into the target cell membrane upon
triggering (11,
12). Class I fusion proteins
have two heptad repeat regions, HR1 and HR2, at their N and C termini,
respectively, that fold up onto each other during six-helix bundle formation
to bring about merging of target cell and viral membranes
(12). For
Paramyxovirus F proteins, the C-terminal HR2 region is generally
thought to be pre-formed, but the N-terminal HR1 region is formed only upon
F-triggering and FP insertion
(11,
13). The formation of this
trimeric HR1 core just before six-helix bundle formation, is known as the
pre-hairpin intermediate.Despite their common features, viral fusion proteins vary in their detailed
structures, triggering factors, and number of viral surface proteins involved.
For paramyxoviruses, receptor binding and fusion functions are carried out by
two distinct transmembrane proteins (attachment (G, H, or HN) and fusion (F)
proteins, respectively), and with few exceptions both are required for
membrane fusion. The underlying mechanism of fusion triggering by the
attachment protein may vary depending on their use of protein versus
carbohydrate receptors (14).
For example, we and others have observed an inverse correlation between
fusogenicity of the F protein and the avidity of the F/G or F/H interactions
for NiV and measles virus (2,
3,
5,
15,
16), both of which use
protein-based receptors; however, for Newcastle disease virus, a glycan-using
Paramyxovirus, there seems to be a direct correlation between
fusogenicity and the avidity of F/HN interactions
(8).For the paramyxoviruses, the early steps in the fusion cascade,
particularly how H/HN “triggers” F, are not well understood, and
the region(s) in H/HN responsible for F triggering remains unclear, although
the stalk domain of H/HN appears to be important for F triggering or for
interaction with F
(5–8).
For NiV, the G attachment glycoprotein binds either the ephrinB2 (B2) or
ephrinB3 (B3) protein receptors
(17–19),
but it is not known how receptor engagement induces G to undergo the
allosteric changes involved in triggering F. However, by homology to H or HN,
it is likely that the stalk domain in NiV-G is also involved in F-triggering
(20). Here we analyze the
early steps in the fusion cascade for NiV and identify a specific region in
NiV-G distinct from the receptor binding site that is involved in 1)
B2-induced changes that trigger FP exposure in F, 2) modulating the avidity of
F/G interactions resulting in displacement of F from G, and 3) transducing
receptor-induced membrane fusion. Our results offer testable hypotheses as to
whether this model of fusion cascade holds true for other paramyxoviruses that
use protein-based receptors. 相似文献
32.
The facultative anaerobic bacterium Lactococcus lactis has been used as a host for expression of a gene isolated from the anaerobic rumen fungus Neocallimastix sp. The coding region of the cellulase gene was obtained from the fungus with the aid of polymerase chain reaction amplification.
The gene was then transformed into pCT vector system and the constructed recombinant plasmid was introduced into two L. lactis strains (IL403 and MG1363) by electroporation. The gene encoding the fungal originated cellulase was expressed in both strains
successfully although the expression level was relatively lower in comparison with the original enzyme activity. Genetically
modified L. lactis strains were used as silage inoculants for pre-biodegradation of the plant biomass during ensiling. That treatment resulted
in a notable reduction of the acid detergent fiber (ADF) and neutral detergent fiber (NDF) contents of the plant biomass used
as silage material. Inoculation with recombinant strain IL1043 resulted in 4.8 and 9.7 % decrease in NDF and ADF contents,
respectively while the inoculation of silage with strain MG1363 decreased the ADF content by >5 %. 相似文献
33.
Frederic Vigant Jihye Lee Axel Hollmann Lukas B. Tanner Zeynep Akyol Ataman Tatyana Yun Guanghou Shui Hector C. Aguilar Dong Zhang David Meriwether Gleyder Roman-Sosa Lindsey R. Robinson Terry L. Juelich Hubert Buczkowski Sunwen Chou Miguel A. R. B. Castanho Mike C. Wolf Jennifer K. Smith Ashley Banyard Margaret Kielian Srinivasa Reddy Markus R. Wenk Matthias Selke Nuno C. Santos Alexander N. Freiberg Michael E. Jung Benhur Lee 《PLoS pathogens》2013,9(4)
LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50≤0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001''s specific inhibition of virus-cell fusion.The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen (1O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated 1O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. 1O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced 1O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides.Based on our understanding of LJ001''s mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001''s limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of 1O2 generation), and (4) 10–100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion. 相似文献
34.
The objective of this study was to document the frequency of occurrence of very rare fish species as well as to note the changes in diversity over time. Fish species recorded fewer than five times over the past 40 years (1969–2008) from the coasts of Izmir (central Aegean Sea) were included in the account and considered as being very rare. The records are listed chronologically. Fish species recently recorded in the area for the first time are also included. Changes in qualitative and quantitative composition of the ichthyofauna along the coasts of Izmir are also discussed. 相似文献
35.
Akyol S Ginis Z Armutcu F Ozturk G Yigitoglu MR Akyol O 《Cell biochemistry and function》2012,30(5):438-443
Protection of the patients against the side effects of chemotherapy and radiotherapy regimens has attracted increasing interest of clinicians and practitioners. Caffeic acid phenethyl ester (CAPE), which is extracted from the propolis of honeybee hives as an active component, specifically inhibits nuclear factor κB at micromolar concentrations and show ability to stop 5-lipoxygenase-catalysed oxygenation of linoleic acid and arachidonic acid. CAPE has antiinflammatory, antiproliferative, antioxidant, cytostatic, antiviral, antibacterial, antifungal and antineoplastic properties. The purpose of this review is to summarize in vivo and in vitro usage of CAPE to prevent the chemotherapy-induced and radiotherapy-induced damages and side effects in experimental animals and to develop a new approach for the potential usage of CAPE in clinical trial as a protective agent during chemotherapy and radiotherapy regimens. 相似文献
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39.
T. Çoker O. Akyol O. Özaydn S. Leblebici Z. Tosunolu 《Zeitschrift fur angewandte Ichthyologie》2008,24(1):85-87
This work focuses on reproductive biology aspects of Uranoscopus scaber from the coast of the Aegean Sea. In total, 175 U. scaber specimens were collected in Izmir Bay using trawl nets from January to December 2005. The reproduction period according to gonadosomatic index (GSI) values extents from March to September, with a peak in May (6.32 ± 1.2). Oocyte diameters taken from 59 mature gonads ranged from 62 to 1860 μm. Maximum batch fecundity (BF) was 58091 (mean: 11556 ± 1445) in large oocytes in May, and 8408 (mean: 2821 ± 945) in hydrated oocytes in September. The BF increased with total length and body weight, excluding the ovaries. Total length (TL)–BF relationships and fish weight–BF relationships were BF = 0.0034TL4.6248 (r = 0.47) and BF = 59.166W?3101 (r = 0.49), respectively, according to the oocyte size frequency method. There was a linear relationship between BF and fish size. 相似文献
40.
An enormous amount of data has been published in recent years demonstrating melatonin's defensive role against toxic free radicals. In the present study, we examined the role of melatonin as an antioxidant against the effect of continuous light exposure. Rats were divided into three groups. Control rats (group A) were kept under natural conditions whereas other group of rats (group B and C) were exposed to constant light for inhibition of melatonin secretion by the pineal gland. Group C rats also received melatonin via s.c. injection (1 mg x kg(- 1) body weight x day(- 1)). At the end of experiment, all animals were sacrificied by decapitation, serum and tissue samples were removed for determination of malondialdehyde (MDA), a product of lipid peroxidation, conjugated dienes levels and glutathione peroxidase (GSH-Px) activity levels. It was found that lipid peroxidation was increased in the rats which were exposed to constant light. Melatonin injection caused a decrease in lipid peroxidation, especially in the brain. In addition, melatonin application resulted in increased GSH-Px activity, which has an antioxidant effect. Thus, melatonin is not only a direct scavenger of toxic radicals, but also stimulates the antioxidative enzyme GSH-Px activity to detoxify hydroxyl radical produced by constant light exposure. 相似文献