Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

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Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

Journal of Virology ◽

10.1128/jvi.02877-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 811-823 ◽

Cited By ~ 32

Author(s):

Yi-Ping Li ◽

Santseharay Ramirez ◽

Lotte Mikkelsen ◽

Jens Bukh

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Adaptive Mutations ◽

Culture Systems ◽

Infectious Clones ◽

Cell Culture Systems ◽

A Genome

ABSTRACTThe first discovered and sequenced hepatitis C virus (HCV) genome and the firstin vivoinfectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77Cin vivoinfectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 104.0focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 103.5and 104.4FFU/ml, respectively.IMPORTANCEHepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but notin vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developedin vitroinfectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, thein vitroinfectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

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Cell Culture Adaptation of Hepatitis C Virus and In Vivo Viability of an Adapted Variant

Journal of Virology ◽

10.1128/jvi.01362-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 13168-13179 ◽

Cited By ~ 124

Author(s):

Artur Kaul ◽

Ilka Woerz ◽

Philip Meuleman ◽

Geert Leroux-Roels ◽

Ralf Bartenschlager

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Cultured Cells ◽

Infectious Hepatitis ◽

Adaptive Mutation ◽

Viral Genomes ◽

Adaptive Mutations ◽

Spread Of Infection

ABSTRACT Production of infectious hepatitis C virus in cell culture has become possible because of the unique properties of the JFH1 isolate. However, virus titers are rather low, limiting the utility of this system. Here we describe the generation of cell culture-adapted JFH1 variants yielding higher titers of infectious particles and enhanced spread of infection in cultured cells. Sequence analysis of adapted genomes revealed a complex pattern of mutations that differed in two independent experiments. Adaptive mutations were observed both in the structural and in the nonstructural regions, with the latter having the highest impact on enhancement of virus titers. The major adaptive mutation was identified in NS5A, and it enhanced titers of three intergenotypic chimeras consisting of the structural region of a genotype 1a, 1b, or 3a isolate and the remainder of the JFH1 isolate. The mutation resides at the P3 position of the NS5A-B cleavage site and slows down processing, implying that subtle differences in replication complex formation appear to determine the efficiency of virus formation. Highly adapted JFH1 viruses carrying six mutations established a robust infection in uPA-transgenic SCID mice xenografted with human hepatocytes. However, the mutation in NS5A which enhanced virus titers in cell culture the most had reverted to wild type in nearly half of the viral genomes isolated from these animals at 15 weeks postinoculation. These results argue for some level of impaired fitness of this mutant in vivo.

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PCAF Interacts with Tax and Stimulates Tax Transactivation in a Histone Acetyltransferase-Independent Manner

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8136 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8136-8145 ◽

Cited By ~ 97

Author(s):

Hua Jiang ◽

Hanxin Lu ◽

R. Louis Schiltz ◽

Cynthia A. Pise-Masison ◽

Vasily V. Ogryzko ◽

...

Keyword(s):

Transcriptional Activation ◽

Histone Acetyltransferase ◽

Point Mutations ◽

Creb Binding Protein ◽

Independent Manner ◽

Cell Leukemia Virus Type ◽

Knock Out ◽

Human T Cell

ABSTRACT Recent studies have shown that the p300/CREB binding protein (CBP)-associated factor (PCAF) is involved in transcriptional activation. PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity. In this report, we present evidence for a HAT-independent transcription function that is activated in the presence of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. In vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP. PCAF can be recruited to the HTLV-1 Tax responsive element in the presence of Tax, and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-1 LTR over 10-fold. Point mutations at Tax amino acid 318 (TaxS318A) or 319 to 320 (Tax M47), which have decreased or no activity on the HTLV-1 promoter, are defective for PCAF binding. Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent on the PCAF HAT domain. Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF. In contrast, p300 stimulation of Tax transactivation is HAT dependent. These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral long terminal repeat.

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Human Astrovirus MLB Replication In Vitro: Persistence in Extraintestinal Cell Lines

Journal of Virology ◽

10.1128/jvi.00557-19 ◽

2019 ◽

Vol 93 (13) ◽

Cited By ~ 3

Author(s):

Diem-Lan Vu ◽

Albert Bosch ◽

Rosa M. Pintó ◽

Enric Ribes ◽

Susana Guix

Keyword(s):

Cell Culture ◽

Cell Line ◽

Cell Lines ◽

Culture System ◽

Unknown Etiology ◽

Cell Culture System ◽

Persistent Infections ◽

A Cell ◽

Replicative Cycle

ABSTRACT MLB astroviruses were identified 10 years ago in feces from children with gastroenteritis of unknown etiology and have been unexpectedly detected in severe cases of meningitis/encephalitis, febrile illness of unknown etiology, and respiratory syndromes. The aim of this study was to establish a cell culture system supporting MLB astrovirus replication. We used two clinical strains to infect several cell lines, an MLB1 strain from a gastroenteritis case, and an MLB2 strain associated with a neurologic infection. Efforts to propagate the viruses in the Caco-2 cell line were unsuccessful. In contrast, we identified two human nonintestinal cell lines, Huh-7 and A549, permissive for both genotypes. After serial passages in the Huh-7.5 cell line, the adapted strains were able to establish persistent infections in the Huh-7.5, Huh-7AI, and A549 cell lines, with high viral loads (up to 10 log10 genome copies/ml) detected by quantitative reverse transcription-PCR (RT-qPCR) in the culture supernatant. Immunofluorescence assays demonstrated infection in about 10% of cells in persistently infected cultures. Electron microscopy revealed particles of 32 to 33 nm in diameter after negative staining of cell supernatants and capsid arrays in ultrathin sections with a particularly high production in Huh-7.5 cells. Interferon (IFN) expression by infected cells and effect of exogenous IFN varied depending on the type of infection and the cell line. The availability of a cell culture system to propagate MLB astroviruses represents a key step to better understand their replicative cycle, as well as a source of viruses to conduct a wide variety of basic virologic studies. IMPORTANCE MLB astroviruses are emerging viruses infecting humans. More studies are required to determine their exact epidemiology, but several reports have already identified them as the cause of unexpected clinical diseases, including severe neurologic diseases. Our study provides the first description of a cell culture system for the propagation of MLB astroviruses, enabling the study of their replicative cycle. Moreover, we demonstrated the unknown capacity of MLB astrovirus to establish persistent infections in cell culture. Whether these persistent infections are also established in vivo remains unknown, but the clinical consequences would be of high interest if persistence was confirmed in vivo. Finally, our analysis of IFN expression provides some trails to understand the mechanism by which MLB astroviruses can cause persistent infections in the assayed cultures.

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The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5643-5649.1989 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5643-5649

Author(s):

H Ma ◽

L M Bloom ◽

C T Walsh ◽

D Botstein

Keyword(s):

Saccharomyces Cerevisiae ◽

Glucose Repression ◽

Point Mutations ◽

Inverse Correlation ◽

Invertase Activity ◽

Yeast Cells ◽

Hexokinase Ii ◽

Mutant Proteins ◽

Hexokinase Activity

Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.

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Ionizing Irradiation Protection and Mitigation by Carbamazepine Is p53 and Autophagy Independent,

Blood ◽

10.1182/blood.v118.21.3400.3400 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3400-3400

Author(s):

Hyun Kim ◽

Mark E. Bernard ◽

Amy Farkas ◽

Frank Houghton ◽

Donna Shields ◽

...

Keyword(s):

Cell Lines ◽

Hind Limb ◽

Conflicts Of Interest ◽

Survival Curve ◽

Tumor Growth Inhibition ◽

Independent Manner ◽

Mitochondrial Membrane Depolarization ◽

Significant Difference

Abstract Abstract 3400 Introduction: Carbamazepine (CBZ), an established sodium channel blocker, used in treatment of epilepsy and trigeminal neuralgia, induces autophagy. CBZ has recently been demonstrated to be an ionizing radiation mitigator and protector (Kim,H.et. al IJRB-in press). CBZ protected C57BL/6NHsd female mice from irradiation if given 24 hr before total body irradiation (TBI) and mitigated if given 12 hours after irradiation. Materials and Methods: To elucidate the mechanism of CBZ action, autophagy incompetent (ATG5−/−) and competent (ATG5+/+) mouse embryo fibroblasts (MEF), p53−/− and p53+/+ bone marrow stromal cells and 32D cl 3 murine IL-3 dependent hematopoietic progenitor cells were tested for CBZ mediated radiation protection and mitigation in clonagenic irradiation survival curves. We also measured CBZ effect on irradiation-induced apoptosis, and depletion of antioxidant stores in vitro and after (TBI) in vivo in control mice and in hind limb irradiated mice with orthotopic tumors. Results: CBZ was a significant radiation protector and mitigator for both ATG5−/− and ATG5 +/+ cell lines by an increased ñ (a measurement of the shoulder on the clonogenic survival curve). CBZ treated ATG5 +/+ cells has an increased ñ of 11.1 +/− 0.2 or 8.8 +/− 0.2 for CBZ added before or after irradiation respectively compared to 5.4 +/− 0.9 for irradiation control cells (p = 0.0287 or 0.0119,respectively). ATG −/− cells were also protected and mitigated by CBZ (ñ of 16.1 +/− 2.6 as a radioprotector or 9.8 +/− 1.5 as a mitigator compared to 4.6 +/− 0.7 for irradiated control cells (p = 0.0002 or 0.0037, respectively). Thus, CBZ functions independent of autophagy. Incubation of p53 +/+ and p53−/− cell lines in 10 μM CBZ for one hour before irradiation protected (ñ of 5.6 +/− 0.9 compared to 1.9 +/− 0.5 for control irradiated p53 +/+ cells (p = 0.0126) and 3.7 +/− 1.9 compared to 1.8 +/− 0.4 for irradiated control p53 −/− cells (p = 0.0018). Thus, CBZ functions in a p53 independent manner. Other pro-autophagy drugs, Valproic Acid and Lithium Carbonate, were ineffective radiation protectors or mitigators. CBZ treatment of 32D cl 3 cells for one hour before or immediately following irradiation had no effect on mitochondrial membrane depolarization, apoptosis or viability but was protective and mitigative in clonagenic survival curve assays. Irradiation initially decreased antioxidant stores in both ATG5+/+ and ATG5−/− cells 10 to 30 min following irradiation. However, cells treated with CBZ had a faster recovery of antioxidant stores to pre-irradiation levels by 90 min compared to 120 min for control irradiated cells. To determine if CBZ protected both normal tissues and tumors in vivo mice with 3LL Lewis Lung carcinoma hind limb xenografts were treated with 10 mg/kg CBZ before or after 20 Gy limb irradiation. There was no significant difference in tumor growth inhibition by 20 Gy in mice treated with CBZ before or after irradiation (p = 0.2431 or 0.5439, respectively). Conclusion: Thus, CBZ is an effective radiation protector and mitigator of normal but not tumor tissues by a novel mechanism independent of autophagy, p53 and apoptosis. Acknowledgments: This project was supported by U191A168021–06. Disclosures: No relevant conflicts of interest to declare.

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Conditionally immortalized human podocyte cell lines established from urine

AJP Renal Physiology ◽

10.1152/ajprenal.00509.2009 ◽

2010 ◽

Vol 298 (3) ◽

pp. F557-F567 ◽

Cited By ~ 42

Author(s):

Toru Sakairi ◽

Yoshifusa Abe ◽

Hiroshi Kajiyama ◽

Linda D. Bartlett ◽

Lilian V. Howard ◽

...

Keyword(s):

Cell Culture ◽

Healthy Volunteers ◽

Cell Lines ◽

Cell Biology ◽

Wilms Tumor ◽

T Antigen ◽

Sv40 Large T Antigen ◽

Glomerular Diseases ◽

Cell Culture Techniques

Evidence suggests that loss of podocytes into urine contributes to development of glomerular diseases; shed podocytes are frequently viable and proliferate in culture conditions. To determine the phenotypic characteristics of viable urinary cells derived from human subjects, we established long-term urinary cell culture from two patients with focal segmental glomerulosclerosis and two healthy volunteers, via transformation with the thermosensitive SV40 large T antigen (U19tsA58) together with human telomerase (hTERT). Characterization of arbitrarily selected two clonal cell lines from each human subject was carried out. mRNA expression for the podocyte markers synaptopodin, nestin, and CD2AP were detected in all eight clones. Podocin mRNA was absent from all eight clones. The expression of nephrin, Wilms' tumor 1 (WT1), and podocalyxin mRNA varied among the clones, which may be due to transformation and/or cloning. These results suggest that podocyte cell lines can be established consistently from human urine. The generation of podocyte cell lines from urine of patients and healthy volunteers is novel and will help to advance studies of podocyte cell biology. Further improvements in the approaches to cell transformation and/or cell culture techniques are needed to allow cultured podocytes to fully reproduce in vivo characteristics.

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A novel preclinical model of cholangiocarcinoma based on human aberrant FBXW7 expression.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e15624 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e15624-e15624

Author(s):

Jingxiao Wang ◽

Haichuan Wang ◽

Michele Peters ◽

Ning Ding ◽

Silvia Ribback ◽

...

Keyword(s):

Mouse Model ◽

Mrna Expression ◽

Cell Lines ◽

Inverse Correlation ◽

Tumor Burden ◽

Dominant Negative ◽

Preclinical Model ◽

Dominant Negative Form ◽

Clinical Models

e15624 Background: Pre-clinical models that mimic human genetic events occurring in intrahepatic cholangiocarcinoma (iCCA) are limited. The ubiquitin ligase F-box and WD repeat domain-containing 7 (FBXW7) is recognized as a tumor suppressor in many cancer types. Methods: Firstly, we determined the FBXW7 mutation frequency (n = 120) and mRNA expression (n = 87) in a collection of human iCCA. Based on the preliminary findings in human data, we generated a mouse model by hydrodynamic tail vein injection of activated/myristylated (myr-)AKT with Fbxw7ΔF, a dominant negative form of Fbxw7. Subsequently, we investigated the role of established targets of Fbxw7, namely Notch2, Yap, and c-Myc in this novel mouse model and in human CCA cell lines. Results: FBXW7 mRNA expression is almost ubiquitously downregulated (71/82; 86.6%) in human iCCA specimens, while only 0.8% of samples showed FBXW7 somatic mutations. In vivo, co-expression of AKT and Fbxw7ΔF triggered the development of iCCA lesions and mice were euthanized by 15 weeks post-injection due to high tumor burden. At the molecular level, a strong induction of FBXW7 canonical targets, including Yap, Notch2, and c-Myc oncoproteins, was detected. However, only c-Myc was consistently confirmed as a FBXW7 target in human CCA cell lines. Interestingly, selected ablation of c-Myc completely impaired iCCA formation in AKT/Fbxw7ΔF mice, whereas deletion of either Yap or Notch2 delayed cholangiocarcinogenesis in the same model. Furthermore, in human iCCA specimens, a strong, inverse correlation between the expression levels of FBXW7 and c-Myc was observed. Conclusions: Downregulation of FBXW7 is almost ubiquitous in human iCCA and cooperates with AKT to induce cholangiocarcinogenesis in mice. This pre-clinical mouse model could be used to test novel therapeutics targeting c-Myc, Notch2, and/or Yap.

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TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels

Cancers ◽

10.3390/cancers12051329 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1329 ◽

Cited By ~ 2

Author(s):

Wookyeom Yang ◽

Ha-Yeon Shin ◽

Hanbyoul Cho ◽

Joon-Yong Chung ◽

Eun-ju Lee ◽

...

Keyword(s):

Ovarian Cancer ◽

Cell Growth ◽

Cell Lines ◽

Therapeutic Option ◽

Disease Free Survival ◽

Tumor Burden ◽

Cancer Cell Growth ◽

Independent Manner ◽

First Line Therapy

TOM40 is a channel-forming subunit of translocase, which is essential for the movement of proteins into the mitochondria. We found that TOM40 was highly expressed in epithelial ovarian cancer (EOC) cells at both the transcriptional and translational levels; its expression increased significantly during the transformation from normal ovarian epithelial cells to EOC (p < 0.001), and TOM40 expression negatively correlated with disease-free survival (Hazard ratio = 1.79, 95% Confidence inerval 1.16–2.78, p = 0.009). TOM40 knockdown decreased proliferation in several EOC cell lines and reduced tumor burden in an in vivo xenograft mouse model. TOM40 expression positively correlated with intracellular adenosine triphosphate (ATP) levels. The low ATP and high reactive oxygen species (ROS) levels increased the activity of AMP-activated protein kinase (AMPK) in TOM40 knockdown EOC cells. However, AMPK activity did not correlate with declined cell growth in TOM40 knockdown EOC cells. We found that metformin, first-line therapy for type 2 diabetes, effectively inhibited the growth of EOC cell lines in an AMPK-independent manner by inhibiting mitochondria complex I. In conclusion, TOM40 positively correlated with mitochondrial activities, and its association enhances the proliferation of ovarian cancer. Also, metformin is an effective therapeutic option in TOM40 overexpressed ovarian cancer than normal ovarian epithelium.

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