scholarly journals The kinesin KIF4 mediates HBV/HDV entry through regulation of surface NTCP localization and can be targeted by RXR agonists in vitro.

2021 ◽  
Author(s):  
Sameh A. Gad ◽  
Masaya Sugiyama ◽  
Masataka Tsuge ◽  
Kosho Wakae ◽  
Kento Fukano ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Wild Type ◽  
Surface Transport ◽  
Fold Reduction ◽  
Surface Localization ◽  
Sirna Library ◽  
Hdv Infection ◽  
Protein Attachment ◽  
Kinesin Family

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP C4 cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection and suppressed both wild-type HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 regains the surface localization of NTCP in these cells. Furthermore, IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study revealed that KIF4 physically binds to NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1 mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent HBV infection in HepG2-hNTCP and primary human hepatocytes (PXB) (Bexarotene, IC 50 1.89 ± 0.98 μM). Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates that target HBV and HDV entry phase.

scholarly journals Evading the Proteasome: Absence of Lysine Residues Contributes to Pertussis Toxin Activity by Evasion of Proteasome Degradation

2007 ◽  
Vol 75 (6) ◽  
pp. 2946-2953 ◽  
Author(s):  
Zoë E. V. Worthington ◽  
Nicholas H. Carbonetti
Keyword(s):  
Pertussis Toxin ◽  
Intracellular Transport ◽  
Cellular Activity ◽  
Wild Type ◽  
Proteasome Degradation ◽  
Lysine Residues ◽  
Arginine Residues ◽  
A Subunit ◽  
Adp Ribosyltransferase

ABSTRACT Pertussis toxin (PT) is an important virulence factor produced by Bordetella pertussis. PT holotoxin comprises one enzymatically active A subunit (S1), associated with a pentamer of B subunits. PT is an ADP-ribosyltransferase that modifies several mammalian heterotrimeric G proteins. Some bacterial toxins are believed to undergo retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway involves the removal of misfolded proteins from the ER and degradation upon their return to the cytosol; this pathway may be exploited by PT and other toxins. In the cytosol, ERAD substrates are ubiquitinated at lysine residues, targeting them to the proteasome for degradation. We hypothesize that S1 avoids ubiquitination and proteasome degradation due to its lack of lysine residues. We predicted that the addition of lysine residues would reduce PT toxicity by allowing ubiquitination and degradation to occur. Variant forms of PT were engineered, replacing one, two, or three arginines with lysines in a variety of locations on S1. Several variants were identified with wild-type in vitro enzymatic activity but reduced cellular activity, consistent with our hypothesis. Significant recovery of the cellular activity of these variants was observed when CHO cells were pretreated with a proteasome inhibitor. We concluded that the replacement of arginine residues with lysine in the S1 subunit of PT renders the toxin subject to proteasomal degradation, suggesting that wild-type PT avoids proteasome degradation due to an absence of lysine residues.

scholarly journals Mutational Analysis of Superantigen Activity Responsible for the Induction of Skin Erythema by Streptococcal Pyrogenic Exotoxin C

1998 ◽  
Vol 66 (10) ◽  
pp. 5020-5026 ◽  
Author(s):  
Junichi Yamaoka ◽  
Eijiro Nakamura ◽  
Yoshifumi Takeda ◽  
Sadao Imamura ◽  
Nagahiro Minato
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mutational Analysis ◽  
Wild Type ◽  
Stimulatory Activity ◽  
Fold Reduction ◽  
Skin Erythema ◽  
Dose Dependent Fashion

ABSTRACT Streptococcal pyrogenic exotoxin C (SPEC), when injected intradermally, induces erythema in unsensitized rabbits. In the present study, we examined whether this erythema induction is due to the T-cell stimulatory activity of SPEC as a superantigen. Analysis by using single-residue mutant SPECs indicated that mutant SPECs Y15I, A16E, and Y17I, in which tyrosine 15, alanine 16, and tyrosine 17 were replaced with isoleucine, glutamic acid, and isoleucine, respectively, exhibited significantly reduced mitogenic activity for Vβ2+ human T cells in vitro, and Y15I showed as much as a 1,000-fold reduction. Y15I mutant SPEC, however, retained the ability to bind to major histocompatibility complex class II antigen and to form a homodimer, implying that residue 15 is critically important for the interaction of SPEC with T-cell antigen receptor β chains. When injected intradermally into normal rabbits, wild-type SPEC induced a characteristic erythema after 3 h in a dose-dependent fashion, which was associated with polymorphonuclear and mononuclear cell infiltration. This erythema formation was found to be severely suppressed by systemic pretreatment with cyclosporin A, suggesting the involvement of host T cells. Y15I mutant SPEC exhibited nearly 1,000-fold less erythema induction in vivo than wild-type SPEC. Altogether, the present results strongly suggest that erythema induction in rabbits by SPEC is attributable mostly to its T-cell stimulatory activity as a superantigen.

scholarly journals Epstein-Barr Virus BNRF1 Protein Allows Efficient Transfer from the Endosomal Compartment to the Nucleus of Primary B Lymphocytes

2006 ◽  
Vol 80 (19) ◽  
pp. 9435-9443 ◽  
Author(s):  
R. Feederle ◽  
B. Neuhierl ◽  
G. Baldwin ◽  
H. Bannert ◽  
B. Hub ◽  
...  
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Wild Type ◽  
Barr Virus ◽  
Fold Reduction ◽  
Viral Particles ◽  
Viral Mutant ◽  
Tumor Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a tumor virus with marked B lymphotropism. After crossing the B-cell membrane, the virus enters cytoplasmic vesicles, where decapsidation takes place to allow transfer of the viral DNA to the cell nucleus. BNRF1 has been characterized as the EBV major tegument protein, but its precise function is unknown. We have constructed a viral mutant that lacks the BNRF1 gene and report here its in vitro phenotype. A recombinant virus devoid of BNRF1 (ΔBNRF1) showed efficient DNA replication and production of mature viral particles. B cells infected with the ΔBNRF1 mutant presented viral lytic antigens as efficiently as B cells infected with wild-type or BNRF1 trans-complemented ΔBNRF1 viruses. Antigen presentation in B cells infected with either wild-type (EBV-wt) or ΔBNRF1 virus was blocked by leupeptin addition, showing that both viruses reach the endosome/lysosome compartment. These data were confirmed by direct observation of the mutant virus in endosomes of infected B cells by electron microscopy. However, we observed a 20-fold reduction in the number of B cells expressing the nuclear protein EBNA2 after infection with a ΔBNRF1 virus compared to wild-type infection. Likewise, ΔBNRF1 viruses transformed primary B cells much less efficiently than EBV-wt or BNRF1 trans-complemented viruses. We conclude from these findings that BNRF1 plays an important role in viral transport from the endosomes to the nucleus.

scholarly journals Contribution of the Collagen Adhesin Acm to Pathogenesis of Enterococcus faecium in Experimental Endocarditis

2008 ◽  
Vol 76 (9) ◽  
pp. 4120-4128 ◽  
Author(s):  
Sreedhar R. Nallapareddy ◽  
Kavindra V. Singh ◽  
Barbara E. Murray
Keyword(s):  
Enterococcus Faecium ◽  
Multidrug Resistant ◽  
Mrna Levels ◽  
Virulence Determinants ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Fold Reduction ◽  
A Cell ◽  
Peritonitis Model

ABSTRACTEnterococcus faeciumis a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstratingE. faeciumvirulence determinants. Our previous studies showed that some clinicalE. faeciumisolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenicacmdeletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Δacm::catis highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (P< 0.0001) and for valve colonization 1 and 3 hours after infection (P≤ 0.0002), making Acm the first factor shown to be important forE. faeciumpathogenesis. In contrast, no mortality differences were observed in a mouse peritonitis model. While 5 of 17 endocarditis isolates were Acm nonproducers and failed to adhere to collagen in vitro, all had an intact, highly conservedacmlocus. Highly reducedacmmRNA levels (≥50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating thatacmtranscription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction inE. faeciumcollagen adherence by affinity-purified anti-Acm antibodies fromE. faeciumendocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.

In vivo studies of the γ subunit of retinal cGMP-phophodiesterase with a substitution of tyrosine-84

2001 ◽  
Vol 353 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Stephen H. TSANG ◽  
Clyde K. YAMASHITA ◽  
Kentaro DOI ◽  
Daniel J. SALCHOW ◽  
Nicole BOUVIER ◽  
...  
Keyword(s):  
Regulatory Control ◽  
In Vivo Studies ◽  
Wild Type ◽  
Targeted Disruption ◽  
Catalytic Core ◽  
Γ Subunit ◽  
Biochemical Assays ◽  
Fold Reduction

The inhibitory rod cGMP phosphodiesterase γ subunit (PDEγ) is a major component of the photoresponse and is required to support rod integrity. Pdegtm1/Pdegtm1 mice (which lack PDEγ owing to a targeted disruption of the Pdeg gene) suffer from a very rapid and severe photoreceptor degeneration. The Y84G (Tyr84 → Gly) allele of PDEγ has previously been shown in experiments carried out in vitro to reduce the regulatory control of the PDE catalytic core (PDEαβ) exerted by the wild-type γ subunit. To determine the effects of this mutation on in vivo function, the murine opsin promoter was used to direct expression to the photoreceptors of +/Pdegtm1 mice of a mutant Y84G and a wild-type PDEγ control transgene. The transgenic mice were crossed with Pdegtm1/Pdegtm1 mice to generate animals able to synthesize only the transgenic PDEγ. Our results showed that wild-type PDEγ and Y84G transgenes could complement the Pdegtm1/Pdegtm1 mutant for photoreceptor survival. The mutation caused a significant biochemical defect in PDE activation by transducin. However, the Y84G mutation did not fully eliminate the control of PDEγ on the PDE catalytic core in vivo; the expression of the mutant subunit was associated with only a 10-fold reduction in the amplitude of the a-wave and a 1.5-fold decrease in the b-wave of the corneal electroretinogram. Unexpectedly, the mutation caused a much ‘milder’ phenotype in vivo than was predicted from the biochemical assays in vitro.

scholarly journals A dominant mutation in mec-7/β-tubulin affects axon development and regeneration in Caenorhabditis elegans neurons

2013 ◽  
Vol 24 (3) ◽  
pp. 285-296 ◽  
Author(s):  
Leonie Kirszenblat ◽  
Brent Neumann ◽  
Sean Coakley ◽  
Massimo A. Hilliard
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Shape ◽  
Intracellular Transport ◽  
Wild Type ◽  
Axonal Regrowth ◽  
Axon Development ◽  
Developing Neurons ◽  
Β Tubulin

Microtubules have been known for decades to be basic elements of the cytoskeleton. They form long, dynamic, rope-like structures within the cell that are essential for mitosis, maintenance of cell shape, and intracellular transport. More recently, in vitro studies have implicated microtubules as signaling molecules that, through changes in their stability, have the potential to trigger growth of axons and dendrites in developing neurons. In this study, we show that specific mutations in the Caenorhabditis elegans mec-7/β-tubulin gene cause ectopic axon formation in mechanosensory neurons in vivo. In mec-7 mutants, the ALM mechanosensory neuron forms a long ectopic neurite that extends posteriorly, a phenotype that can be mimicked in wild-type worms with a microtubule-stabilizing drug (paclitaxel), and suppressed by mutations in unc-33/CRMP2 and the kinesin-related gene, vab-8. Our results also reveal that these ectopic neurites contain RAB-3, a marker for presynaptic loci, suggesting that they have axon-like properties. Interestingly, in contrast with the excessive axonal growth observed during development, mec-7 mutants are inhibited in axonal regrowth and remodeling following axonal injury. Together our results suggest that MEC-7/β-tubulin integrity is necessary for the correct number of neurites a neuron generates in vivo and for the capacity of an axon to regenerate.

COVID-19: Opinion in Prevention and dietary based management hypothesis

Coronaviruses ◽  
2020 ◽  
Vol 01 ◽  
Author(s):  
Ashraf Talaat Youssef
Keyword(s):  
Fatty Acids ◽  
Saturated Fatty Acids ◽  
Lung Damage ◽  
Gastric Aspirate ◽  
Enveloped Viruses ◽  
Multiple Organs ◽  
Fold Reduction ◽  
Antiviral Activities

The pandemic of COVID-19 had started in Wuhan city china in late 2019 with a subsequent worldwide spread. The viral infection can seriousely affect multiple organs mainly lungs, kidneys, heart, liver and brain and may lead to respiratory, renal, cardiac or hepatic failure.Vascular thrombosis of unexplained mechanism that may lead to widespread blood clots in multiple organs and cytokine storms that result of overstimulation of the immune system subsequent of lung damage may lead to sudden decompensation due to hypotension and more damage to liver, kidney, brain or lungs.Until now no drug had proved efficient in getting rid of the problem and controlling the pandemic mainly depends on preventive measures.Many preventive measures can be considered to prevent the worldwide spread of viral transmission. Polyunsaturated long chain fatty acids (PUFAs) and the medium chain saturated fatty acids (MCSFAs) and their corresponding monoglycerides had high antiviral activities against the enveloped viruses which reach to more than 10,000 -fold reduction in the viral titres in vitro and in vivo after testing of its gastric aspirate, and can contribute to the systemic immunity against the enveloped viruses.

The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

1999 ◽  
Vol 73 (12) ◽  
pp. 10551-10555 ◽  
Author(s):  
Armin Ensser ◽  
André Pfinder ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein
Keyword(s):  
Cell Culture ◽  
Herpesvirus Saimiri ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Human T Cell ◽  
Human T Cells ◽  
Small Nuclear Rnas ◽  
T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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