scholarly journals Duck enteritis virus UL21 is a late gene and encodes a protein that interacts with pUL16

2019 ◽  
Author(s):  
Linjiang Yang ◽  
Mingshu Wang ◽  
Chunhui Zeng ◽  
Yong Shi ◽  
Anchun Cheng ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Rna Binding ◽  
Gene Interaction ◽  
Duck Enteritis Virus ◽  
Late Gene ◽  
Structural Protein ◽  
Virus Transport ◽  
Enteritis Virus

Abstract Background pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. Methods In our study, recombinant pUL21 was expressed using an pET-32c (+) vector in Escherichia coli BL21 cells induced with 0.4 mM isopropyl β-D-thiogalactoside for 8 h at 30°C. The antibody used for the indirect immunofluorescence (IFA) and western blotting (WB) analysis were prepared. Pharmacological inhibition, WB and quantitative reverse transcription PCR (RT-qPCR) were performed. A coimmunoprecipitation (CO-IP) assay was conducted to test the interaction between pUL21 and pUL16. Results We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. Conclusions The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21. Keywords: Duck enteritis virus, UL21, UL16, late gene, interaction

scholarly journals Duck enteritis virus UL21 is a late gene and encodes a protein that interacts with pUL16

2019 ◽  
Author(s):  
Linjiang Yang ◽  
Mingshu Wang ◽  
Chunhui Zeng ◽  
Yong Shi ◽  
Anchun Cheng ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Rna Binding ◽  
Duck Enteritis Virus ◽  
Late Gene ◽  
Structural Protein ◽  
Virus Transport ◽  
Reverse Transcription Pcr ◽  
Enteritis Virus

Abstract Background pUL21 is a conserved protein of Alphaherpesvirinae and exhibits multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, little is known about duck enteritis virus (DEV) pUL21. Methods In our study, recombinant pUL21 was expressed using apET-32c (+) vector in Escherichia coli BL21 cells induced with 0.4 mM isopropyl β-D-thiogalactoside for 8 h at 30°C. The antibody for indirect immunofluorescence (IFA) and western blotting (WB) analysis were then prepared. Pharmacological inhibition, WB and quantitative reverse transcription PCR (RT-qPCR) were performed. A coimmunoprecipitation (CO-IP) assay was conducted to test the interaction between pUL21 and pUL16. Results We verified that DEV UL21 is a γ2 gene and encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. Conclusions The DEV UL21 gene is a late gene, and the pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interacts with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and binding partner pUL16. Our study enhances understanding of DEV pUL21.

scholarly journals Duck enteritis virus UL21 is a late gene encoding a protein that interacts with pUL16

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Linjiang Yang ◽  
Mingshu Wang ◽  
Chunhui Zeng ◽  
Yong Shi ◽  
Anchun Cheng ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Rna Binding ◽  
Duck Enteritis Virus ◽  
Late Gene ◽  
Structural Protein ◽  
Virus Transport ◽  
Hek 293 ◽  
Enteritis Virus

Abstract Background pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. Results We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293 T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. Conclusions The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21.

scholarly journals Shortening the unstructured, interdomain region of the non-structural protein NS1 of an avian H1N1 influenza virus increases its replication and pathogenicity in chickens

2014 ◽  
Vol 95 (6) ◽  
pp. 1233-1243 ◽  
Author(s):  
Sascha Trapp ◽  
Denis Soubieux ◽  
Hélène Marty ◽  
Evelyne Esnault ◽  
Thomas W. Hoffmann ◽  
...  
Keyword(s):  
Rna Binding ◽  
H1n1 Influenza ◽  
Genetically Engineered ◽  
Influenza Viruses ◽  
Epithelial Cell Line ◽  
General Effect ◽  
Structural Protein ◽  
H5n1 Influenza

Currently circulating H5N1 influenza viruses have undergone a complex evolution since the appearance of their progenitor A/Goose/Guangdong/1/96 in 1996. After the eradication of the H5N1 viruses that emerged in Hong Kong in 1997 (HK/97 viruses), new genotypes of H5N1 viruses emerged in the same region in 2000 that were more pathogenic for both chickens and mice than HK/97 viruses. These, as well as virtually all highly pathogenic H5N1 viruses since 2000, harbour a deletion of aa 80–84 in the unstructured region of the non-structural (NS) protein NS1 linking its RNA-binding domain to its effector domain. NS segments harbouring this mutation have since been found in non-H5N1 viruses and we asked whether this 5 aa deletion could have a general effect not limited to the NS1 of H5N1 viruses. We genetically engineered this deletion in the NS segment of a duck-origin avian H1N1 virus, and compared the in vivo and in vitro properties of the WT and NSdel8084 viruses. In experimentally infected chickens, the NSdel8084 virus showed both an increased replication potential and an increased pathogenicity. This in vivo phenotype was correlated with a higher replicative efficiency in vitro, both in embryonated eggs and in a chicken lung epithelial cell line. Our data demonstrated that the increased replicative potential conferred by this small deletion was a general feature not restricted to NS1 from H5N1 viruses and suggested that viruses acquiring this mutation may be selected positively in the future.

scholarly journals The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mrna Processing ◽  
Key Factors ◽  
Pol Ii ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

scholarly journals Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zizhen Si ◽  
Lei Yu ◽  
Haoyu Jing ◽  
Lun Wu ◽  
Xidi Wang
Keyword(s):  
Colorectal Cancer ◽  
Rna Binding ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Cancer Proliferation ◽  
Mouse Xenograft ◽  
Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

scholarly journals RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽  
2021 ◽  
Author(s):  
Qiuxia Yan ◽  
Peng Zeng ◽  
Xiuqin Zhou ◽  
Xiaoying Zhao ◽  
Runqiang Chen ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Aerobic Glycolysis ◽  
Histological Grade ◽  
Migration And Invasion ◽  
Binding Motif ◽  
Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

scholarly journals Andrographis paniculata (Burm. F.) Wall. Ex Nees, Andrographolide, and Andrographolide Analogues as SARS-CoV-2 Antivirals? A Rapid Review

2021 ◽  
Vol 16 (5) ◽  
pp. 1934578X2110166
Author(s):  
Xin Yi Lim ◽  
Janice Sue Wen Chan ◽  
Terence Yew Chin Tan ◽  
Bee Ping Teh ◽  
Mohd Ridzuan Mohd Abd Razak ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
In Silico ◽  
Rna Binding ◽  
Symptomatic Relief ◽  
Andrographis Paniculata ◽  
Spike Protein ◽  
Rapid Review ◽  
In Silico Studies

Drug repurposing is commonly employed in the search for potential therapeutic agents. Andrographis paniculata, a medicinal plant commonly used for symptomatic relief of the common cold, and its phytoconstituent andrographolide, have been repeatedly identified as potential antivirals against SARS-CoV-2. In light of new evidence emerging since the onset of the COVID-19 pandemic, this rapid review was conducted to identify and evaluate the current SARS-CoV-2 antiviral evidence for A. paniculata, andrographolide, and andrographolide analogs. A systematic search and screen strategy of electronic databases and gray literature was undertaken to identify relevant primary articles. One target-based in vitro study reported the 3CLpro inhibitory activity of andrographolide as being no better than disulfiram. Another Vero cell-based study reported potential SARS-CoV-2 inhibitory activity for both andrographolide and A. paniculata extract. Eleven in silico studies predicted the binding of andrographolide and its analogs to several key antiviral targets of SARS-CoV-2 including the spike protein-ACE-2 receptor complex, spike protein, ACE-2 receptor, RdRp, 3CLpro, PLpro, and N-protein RNA-binding domain. In conclusion, in silico and in vitro studies collectively suggest multi-pathway targeting SARS-CoV-2 antiviral properties of andrographolide and its analogs, but in vivo data are needed to support these predictions.

scholarly journals Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

2001 ◽  
Vol 183 (4) ◽  
pp. 1423-1433 ◽  
Author(s):  
Susan R. Heimer ◽  
Harry L. T. Mobley
Keyword(s):  
Proteus Mirabilis ◽  
Accessory Proteins ◽  
Structural Protein ◽  
Yeast Two Hybrid ◽  
Nickel Ions ◽  
Catalytically Active ◽  
Tract Infections ◽  
Two Hybrid

ABSTRACT Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

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