scholarly journals Activities of Thrombin and Factor Xa Are Essential for Replication of Hepatitis E Virus and Are Possibly Implicated in ORF1 Polyprotein Processing

2018 ◽  
Vol 92 (6) ◽  
Author(s):  
Gayatri D. Kanade ◽  
Kunal D. Pingale ◽  
Yogesh A. Karpe
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Liver Cell ◽  
Serine Proteases ◽  
Hepatitis E Virus ◽  
Factor Xa ◽  
Hepatitis E ◽  
Cleavage Sites ◽  
Polyprotein Processing ◽  
Liver Cell Line

ABSTRACTHepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a nonstructural polyprotein of 1,693 amino acids. It is not clear whether the ORF1 polyprotein (pORF1) is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave various results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bears conserved cleavage sites of thrombin and factor Xa. Using a reverse genetics approach, we demonstrated that an HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstratedin vitroprocessing when we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of a liver cell line with a serine protease inhibitor as well as small interfering RNA (siRNA) knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both of these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in a liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV.IMPORTANCEHepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. A high mortality rate of about 30% is observed in HEV-infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to the variability of study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine proteases, thrombin and factor Xa, that are conserved among HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study sheds light on the presence and roles of clotting factors with respect to virus replication in the cells.

scholarly journals Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces

2019 ◽  
Vol 24 (3) ◽  
pp. 503-508
Author(s):  
Carolina Quintero-Gil ◽  
Santiago Mirazo ◽  
Jaime Parra-Suescún ◽  
Albeiro López-Herrera ◽  
Victoria Mainardi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Post Infection ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Viral Rna ◽  
Human Feces ◽  
Genotype 3 ◽  
Virus Genotype

Hepatitis E virus (HEV) is considered one of the leading causes of acute viral hepatitis worldwide, and about 20 million infections and approximately 57 000 deaths occurred every year. However, little is known about the replicative virus cycle due to the absence of a consensus cell culture model. A549 cell line is considered susceptible to HEV genotype 3, however, both viral strain and cell culture conditions could affect the viral isolation in vitro. The objective of this work was to isolate in vitro an HEV-3 strain obtained from human feces. To this, a genotype 3 HEV strain previously identified by genetic characterization was inoculated in A549 monolayers, and incubated for two hours at 37 °C. Five days post-infection, cells were passaged (subcultured) for the first time, and serial passages were done on average every four days during 41 days. HEV replication was evaluated through RT-qPCR in each passage, and reinfection of the cell line with the viral progeny derived from A549 infected monolayers was assessed through immunofluorescence and RT-qPCR. Viral RNA was detected in each passage from infected monolayers, and the highest amount was found after 26 days (2 x 106 copies/μL). In reinfection assay, capsid antigen was detected perinuclearly and forming foci, and 1x104 copies/μL of viral RNA was detected after 96 hours post infection. This shows that HEV recovered from the cell lysate monolayers was infectious. This viral isolate offers a critical tool to study the unknown aspect of HEV infection.

scholarly journals Evidence for AMPK-dependent regulation of exocytosis of lipoproteins in a model liver cell line

2008 ◽  
Vol 314 (10) ◽  
pp. 2100-2109 ◽  
Author(s):  
Livia Puljak ◽  
Vinay Parameswara ◽  
Svjetlana Dolovcak ◽  
Shar L. Waldrop ◽  
Daniel Emmett ◽  
...  
Keyword(s):  
Cell Line ◽  
Liver Cell ◽  
Liver Cell Line

scholarly journals Establishment of an Immortalized Porcine Liver Cell Line JSNK-1 with Retroviral Transduction of SV40T

2010 ◽  
Vol 19 (6-7) ◽  
pp. 849-856 ◽  
Author(s):  
Javed M. Shahid ◽  
Masaya Iwamuro ◽  
Hiromi Sasamoto ◽  
Yasuhiro Kubota ◽  
Masayuki Seita ◽  
...  
Keyword(s):  
Cell Line ◽  
Liver Cell ◽  
Porcine Liver ◽  
Retroviral Transduction ◽  
Liver Cell Line

scholarly journals Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line

2016 ◽  
Vol 17 (4) ◽  
pp. 534 ◽  
Author(s):  
Binhai Ren ◽  
Chang Tao ◽  
Margaret Swan ◽  
Nichole Joachim ◽  
Rosetta Martiniello-Wilks ◽  
...  
Keyword(s):  
Cell Line ◽  
Human Insulin ◽  
Rat Liver ◽  
Liver Cell ◽  
Liver Cell Line

Multiplication stimulating activity (MSA) from the BRL 3A rat liver cell line: Relation to human somatomedins and insulin

1981 ◽  
Vol 15 (3) ◽  
pp. 253-286 ◽  
Author(s):  
Matthew M. Rechler ◽  
S. Peter Nissley ◽  
George L. King ◽  
Alan C. Moses ◽  
Ellen E. Van Obberghen-Schilling ◽  
...  
Keyword(s):  
Cell Line ◽  
Rat Liver ◽  
Liver Cell ◽  
Liver Cell Line

Evidence of prooxidant and antioxidant action of melatonin on human liver cell line HepG2

Life Sciences ◽  
2000 ◽  
Vol 68 (4) ◽  
pp. 387-399 ◽  
Author(s):  
Raouf A. Osseni ◽  
Patrice Rat ◽  
André Bogdan ◽  
Jean-Michel Warnet ◽  
Yvan Touitou
Keyword(s):  
Cell Line ◽  
Liver Cell ◽  
Human Liver ◽  
Antioxidant Action ◽  
Liver Cell Line ◽  
Human Liver Cell

scholarly journals Cell Culture Isolation and Whole Genome Characterization of Hepatitis E Virus Strains from Wild Boars in Germany

2021 ◽  
Vol 9 (11) ◽  
pp. 2302
Author(s):  
Katja Schilling-Loeffler ◽  
Oliver Viera-Segura ◽  
Victor Max Corman ◽  
Julia Schneider ◽  
Ashish K. Gadicherla ◽  
...  
Keyword(s):  
Cell Culture ◽  
Wild Boar ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Enriched Library ◽  
Genome Copy ◽  
Genotype 3 ◽  
Wild Boars ◽  
Copy Numbers ◽  
Cell Culture Isolation

Infection with hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. The HEV genotype 3 can be zoonotically transmitted from animals to humans, with wild boars representing an important reservoir species. Cell culture isolation of HEV is generally difficult and mainly described for human isolates so far. Here, five sera and five liver samples from HEV-RNA-positive wild boar samples were inoculated onto PLC/PRF/5 cells, incubated for 3 months and thereafter passaged for additional 6 weeks. As demonstrated by RT-qPCR, immunofluorescence and immune electron microscopy, virus was successfully isolated from two liver samples, which originally contained high HEV genome copy numbers. Both isolates showed slower growth than the culture-adapted HEV strain 47832c. In contrast to this strain, the isolated strains had no insertions in their hypervariable genome region. Next generation sequencing using an HEV sequence-enriched library enabled full genome sequencing. Strain Wb108/17 belongs to subtype 3f and strain Wb257/17 to a tentative novel subtype recently described in Italian wild boars. The results indicate that HEV can be successfully isolated in cell culture from wild boar samples containing high HEV genome copy numbers. The isolates may be used further to study the zoonotic potential of wild boar-derived HEV subtypes.

scholarly journals The Capsid (ORF2) Protein of Hepatitis E Virus in Feces Is C-Terminally Truncated

Pathogens ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 24
Author(s):  
Takashi Nishiyama ◽  
Koji Umezawa ◽  
Kentaro Yamada ◽  
Masaharu Takahashi ◽  
Satoshi Kunita ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Structure Prediction ◽  
Hepatitis E Virus ◽  
Culture Media ◽  
Molecular Size ◽  
Hepatitis E ◽  
Terminal Region ◽  
Translation Product ◽  
Orf2 Protein

The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.

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