scholarly journals Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication

2021 ◽  
Vol 12 ◽  
Author(s):  
Chengcheng Zhang ◽  
Xiuling Wang ◽  
Jiahao Sun ◽  
Mengjiao Guo ◽  
Xiaorong Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Viral Replication ◽  
Nonstructural Protein ◽  
Fever Virus ◽  
Ns5a Protein ◽  
Sequestosome 1 ◽  
Classic Swine Fever Virus ◽  
Nonstructural Protein 5A

Although classic swine fever virus (CSFV) infection has been reported to induce autophagy, the specific induced mechanism remains unrevealed. Nonstructural protein 5A (NS5A) of CSFV is a multiphosphorylated protein with multiple functions to regulate viral replication and the host cell immune responses. Herein, we demonstrated that CSFV NS5A could induce cellular autophagy and promote viral replication. In the current study, we showed that NS5A expression significantly increased the levels of autophagy-related genes (ATGs), including light chain 3 (LC3), ATG5, and Beclin 1; conversely, degradation of P62/sequestosome 1 (SQSTM1) was observed by Western blotting. The number of autophagy-like vesicles was also obviously increased in NS5A-expressing cells, as analyzed by transmission electron microscopy (TEM). Furthermore, we observed the co-localization of the NS5A and LC3 proteins by confocal immunofluorescence analysis. Direct binding of NS5A to the autophagy-related LC3 protein was confirmed by coimmunoprecipitation in vivo and by a GST pulldown assay in vitro. Through segmentation and point mutation research on the NS5A protein, we found that the N-terminal region and the phosphorylation of amino acids 81 and 92 of the NS5A protein were essential for inducing autophagy. Finally, we demonstrated that the LC3 protein had a positive effect on CSFV replication. These findings emphasize a previously unascertained interaction relationship between NS5A and LC3 in the autophagy process. Furthermore, our research revealed a new role of CSFV NS5A, particularly its N-terminal amino acids serine 81 and serine 92, as a critical regulator of CSFV-induced autophagy and have significance for extending our understanding of the CSFV-autophagy interplay.

scholarly journals Effect of Hepatitis C Virus (HCV) NS5B-Nucleolin Interaction on HCV Replication with HCV Subgenomic Replicon

2006 ◽  
Vol 80 (7) ◽  
pp. 3332-3340 ◽  
Author(s):  
Tetsuro Shimakami ◽  
Masao Honda ◽  
Takashi Kusakawa ◽  
Takayuki Murata ◽  
Kunitada Shimotohno ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Polymerase Activity ◽  
Subgenomic Replicon ◽  
Huh7 Cells ◽  
Hcv Ns5b

ABSTRACT We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

scholarly journals Interaction of Rotavirus Polymerase VP1 with Nonstructural Protein NSP5 Is Stronger than That with NSP2

2006 ◽  
Vol 81 (5) ◽  
pp. 2128-2137 ◽  
Author(s):  
F. Arnoldi ◽  
M. Campagna ◽  
C. Eichwald ◽  
U. Desselberger ◽  
O. R. Burrone
Keyword(s):  
Viral Replication ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Viral Proteins ◽  
Scaffolding Protein ◽  
Nonstructural Proteins ◽  
Viral Particles ◽  
Green Fluorescent

ABSTRACT Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1.

The 1- and 2-Naphthylalanine Analogs of Oxytocin and Vasopressin

1995 ◽  
Vol 60 (12) ◽  
pp. 2170-2177 ◽  
Author(s):  
Zdenko Procházka ◽  
Jiřina Slaninová
Keyword(s):  
Amino Acids ◽  
Solid Phase ◽  
Pressor Test

Solid phase technique on p-methylbenzhydrylamine resin was used for the synthesis of four analogs of oxytocin and four analogs of vasopressin with the non-coded amino acids L- or D- and 1- or 2-naphthylalanine and D-homoarginine. [L-1-Nal2]oxytocin, [D-1-Nal2]oxytocin, [L-2-Nal2]oxytocin, [D-2-Nal2]oxytocin, [L-1-Nal2, D-Har8]vasopressin, [D-1-Nal2, D-Har8]vasopressin, [L-2-Nal2, D-Har8]vasopressin and [D-2-Nal2, D-Har8]vasopressin were synthesized. All eight analogs were found to be uterotonic inhibitors in vitro and in vivo. Analogs with 2-naphthylalanine are stronger inhibitors, particularly in the vasopressin series than the analogs with 1-naphthylalanine. Analogs with 1-naphthylalanine have no activity in the pressor test, analogs with 2-naphthylalanine are weak pressor inhibitors.

scholarly journals Synthesis, Characterization and Evaluation of Peptide Nanostructures for Biomedical Applications

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4587
Author(s):  
Fanny d’Orlyé ◽  
Laura Trapiella-Alfonso ◽  
Camille Lescot ◽  
Marie Pinvidic ◽  
Bich-Thuy Doan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biomedical Applications ◽  
Chemical Reactivity ◽  
Physical Stability ◽  
Chemical Properties ◽  
Building Blocks ◽  
Imaging Probes ◽  
Therapeutic Molecules

There is a challenging need for the development of new alternative nanostructures that can allow the coupling and/or encapsulation of therapeutic/diagnostic molecules while reducing their toxicity and improving their circulation and in-vivo targeting. Among the new materials using natural building blocks, peptides have attracted significant interest because of their simple structure, relative chemical and physical stability, diversity of sequences and forms, their easy functionalization with (bio)molecules and the possibility of synthesizing them in large quantities. A number of them have the ability to self-assemble into nanotubes, -spheres, -vesicles or -rods under mild conditions, which opens up new applications in biology and nanomedicine due to their intrinsic biocompatibility and biodegradability as well as their surface chemical reactivity via amino- and carboxyl groups. In order to obtain nanostructures suitable for biomedical applications, the structure, size, shape and surface chemistry of these nanoplatforms must be optimized. These properties depend directly on the nature and sequence of the amino acids that constitute them. It is therefore essential to control the order in which the amino acids are introduced during the synthesis of short peptide chains and to evaluate their in-vitro and in-vivo physico-chemical properties before testing them for biomedical applications. This review therefore focuses on the synthesis, functionalization and characterization of peptide sequences that can self-assemble to form nanostructures. The synthesis in batch or with new continuous flow and microflow techniques will be described and compared in terms of amino acids sequence, purification processes, functionalization or encapsulation of targeting ligands, imaging probes as well as therapeutic molecules. Their chemical and biological characterization will be presented to evaluate their purity, toxicity, biocompatibility and biodistribution, and some therapeutic properties in vitro and in vivo. Finally, their main applications in the biomedical field will be presented so as to highlight their importance and advantages over classical nanostructures.

Turnover of total proteins and ornithine aminotransferase during liver regeneration in rats

1980 ◽  
Vol 238 (1) ◽  
pp. E46-E52
Author(s):  
S. L. Augustine ◽  
R. W. Swick
Keyword(s):  
Amino Acids ◽  
Liver Regeneration ◽  
Protein Degradation ◽  
Partial Hepatectomy ◽  
Free Amino ◽  
Liver Protein ◽  
Ornithine Aminotransferase ◽  
Fractional Rate

The recovery of approximately 40% of the total liver protein during the first day after partial hepatectomy was shown to be due to the near cessation of protein breakdown rather than to an increase in protein synthesis. The decrease in degradation of total protein was less if rats were adrenalectomized or protein-depleted prior to partial hepatectomy. The effect of these treatments originally suggested that changes in free amino acid levels in liver might be related to the rate of protein degradation. However, no correlation was found between levels of total free amino acids and rates of breakdown. Measurements of individual amino acids during liver regeneration suggested that levels of free methionine and phenylalanine, amino acids that have been found to lower rates of protein degradation in vitro, are not correlated with rates of breakdown in vivo. The difference between the fractional rate of ornithine aminotransferase degradation (0.68/day and 0.28/day in sham-hepatectomized and partially hepatectomized rats, respectively) was sufficient to account for the higher level of this protein 3 days after surgery in the latter group.

scholarly journals Synergistic In Vitro Antiretroviral Activity of a Humanized Monoclonal Anti-CD4 Antibody (TNX-355) and Enfuvirtide (T-20)

2006 ◽  
Vol 50 (6) ◽  
pp. 2231-2233 ◽  
Author(s):  
Xing-Quan Zhang ◽  
Meredith Sorensen ◽  
Michael Fung ◽  
Robert T. Schooley
Keyword(s):  
Viral Replication ◽  
Viral Entry ◽  
Antiretroviral Agents ◽  
Entry Inhibitors ◽  
High Level ◽  
Antiretroviral Activity

ABSTRACT Recently, antiretroviral agents directed at several steps involved in viral entry have been shown to reduce viral replication in vitro and in vivo. We have demonstrated a high level of in vitro synergistic antiretroviral activity for two entry inhibitors that are directed at sequential steps in the entry process.

scholarly journals Influenza virus hemagglutinin-specific cytotoxic T cell response induced by polypeptide produced in Escherichia coli.

1985 ◽  
Vol 162 (2) ◽  
pp. 663-674 ◽  
Author(s):  
A Yamada ◽  
M R Ziese ◽  
J F Young ◽  
Y K Yamada ◽  
F A Ennis
Keyword(s):  
Influenza Virus ◽  
Recombinant Dna ◽  
Cytotoxic T Lymphocyte ◽  
Nonstructural Protein ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
Protein Immunization ◽  
Recombinant Dna Techniques

We have tested the abilities of various polypeptides of A/PR/8/34 (H1N1) virus, constructed by recombinant DNA techniques, to induce influenza virus-specific secondary cytotoxic T lymphocyte (CTL) responses. A hybrid protein (c13 protein), consisting of the first 81 amino acids of viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutinin (HA), induced H-2-restricted, influenza virus subtype-specific secondary CTL in vitro, although other peptides did not. Using a recombinant virus, the viral determinant responsible for recognition was mapped to the HA2 portion of c13 protein. Immunization of mice with c13 protein induced the generation of memory CTL in vivo. The CTL precursor frequencies of A/PR/8/34 virus- and c13 protein-immune mice were estimated as one in 8,047 and 50,312, respectively. These results indicate that c13 protein primed recipient mice, even though the level of precursor frequency was below that observed in virus-immune mice.

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