scholarly journals Subcellular Localization of Hepatitis C Virus Structural Proteins in a Cell Culture System That Efficiently Replicates the Virus

2006 ◽  
Vol 80 (6) ◽  
pp. 2832-2841 ◽  
Author(s):  
Yves Rouillé ◽  
François Helle ◽  
David Delgrange ◽  
Philippe Roingeard ◽  
Cécile Voisset ◽  
...  
Keyword(s):  
Cell Culture ◽  
Subcellular Localization ◽  
Capsid Protein ◽  
Lipid Droplets ◽  
Structural Proteins ◽  
Intracellular Compartments ◽  
A Cell ◽  
Infected Cells ◽  
Infectious Cycle ◽  
Hcv Structural Proteins

ABSTRACT Due to the recent development of a cell culture model, hepatitis C virus (HCV) can be efficiently propagated in cell culture. This allowed us to reinvestigate the subcellular localization of HCV structural proteins in the context of an infectious cycle. In agreement with previous reports, confocal immunofluorescence analysis of the subcellular localization of HCV structural proteins indicated that, in infected cells, the glycoprotein heterodimer is retained in the endoplasmic reticulum. However, in contrast to other studies, the glycoprotein heterodimer did not accumulate in other intracellular compartments or at the plasma membrane. As previously reported, an association between the capsid protein and lipid droplets was also observed. In addition, a fraction of labeling was consistent with the capsid protein being localized in a membranous compartment that is associated with the lipid droplets. However, in contrast to previous reports, the capsid protein was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Surprisingly, no colocalization was observed between the glycoprotein heterodimer and the capsid protein in infected cells. Electron microscopy analyses allowed us to identify a membrane alteration similar to the previously reported “membranous web.” However, no virus-like particles were found in this type of structure. In addition, dense elements compatible with the size and shape of a viral particle were seldom observed in infected cells. In conclusion, the cell culture system for HCV allowed us for the first time to characterize the subcellular localization of HCV structural proteins in the context an infectious cycle.

scholarly journals Replication of Salmonella enterica serovar Typhimurium in RAW264.7 Phagocytes Correlates With Hypoxia and Lack of iNOS Expression

2020 ◽  
Vol 10 ◽  
Author(s):  
Marie Wrande ◽  
Kim Vestö ◽  
Speranta Puiac Banesaru ◽  
Naeem Anwar ◽  
Johan Nordfjell ◽  
...  
Keyword(s):  
Cell Culture ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Inos Expression ◽  
Raw264.7 Cells ◽  
Serovar Typhimurium ◽  
A Cell ◽  
Infected Cells ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Salmonella infection associates with tissue hypoxia, while inducible nitric oxide synthase (iNOS), relying for its activity on molecular oxygen, stands as a central host defence measure in murine salmonellosis. Here, we have detailed hypoxia and iNOS responses of murine macrophage-like RAW264.7 cells upon infection with Salmonella enterica serovar Typhimurium. We noted that only a proportion of the infected RAW264.7 cells became hypoxic or expressed iNOS. Heavily infected cells became hypoxic, while in parallel such cells tended not to express iNOS. While a proportion of the infected RAW264.7 cells revealed shutdown of protein synthesis, this was only detectable after 12 h post infection and after iNOS expression was induced in the cell culture. Our data implicate an intrinsic heterogeneity with regard to hypoxia and iNOS expression in a cell culture-based infection setting.

scholarly journals The Human Cytomegalovirus UL82 Gene Product (pp71) Accelerates Progression through the G1 Phase of the Cell Cycle

2003 ◽  
Vol 77 (6) ◽  
pp. 3451-3459 ◽  
Author(s):  
Robert F. Kalejta ◽  
Thomas Shenk
Keyword(s):  
Cell Cycle ◽  
Human Cytomegalovirus ◽  
Cell Cycle Progression ◽  
Protein Product ◽  
S Phase ◽  
Quiescent Cells ◽  
A Cell ◽  
Infected Cells ◽  
The One ◽  
Infectious Cycle

ABSTRACT As viruses are reliant upon their host cell to serve as proper environments for their replication, many have evolved mechanisms to alter intracellular conditions to suit their own needs. For example, human cytomegalovirus induces quiescent cells to enter the cell cycle and then arrests them in late G1, before they enter the S phase, a cell cycle compartment that is presumably favorable for viral replication. Here we show that the protein product of the human cytomegalovirus UL82 gene, pp71, can accelerate the movement of cells through the G1 phase of the cell cycle. This activity would help infected cells reach the late G1 arrest point sooner and thus may stimulate the infectious cycle. pp71 also induces DNA synthesis in quiescent cells, but a pp71 mutant protein that is unable to induce quiescent cells to enter the cell cycle still retains the ability to accelerate the G1 phase. Thus, the mechanism through which pp71 accelerates G1 cell cycle progression appears to be distinct from the one that it employs to induce quiescent cells to exit G0 and subsequently enter the S phase.

scholarly journals Robust production of infectious viral particles in Huh-7 cells by introducing mutations in hepatitis C virus structural proteins

2007 ◽  
Vol 88 (9) ◽  
pp. 2495-2503 ◽  
Author(s):  
David Delgrange ◽  
André Pillez ◽  
Sandrine Castelain ◽  
Laurence Cocquerel ◽  
Yves Rouillé ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Potential Effect ◽  
Structural Proteins ◽  
Wild Type ◽  
Genotype 1A ◽  
Viral Particles ◽  
A Cell

Recently, the characterization of a cell culture system allowing the amplification of an authentic virus, named hepatitis C virus cell culture (HCVcc), has been reported by several groups. To obtain higher HCV particle productions, we investigated the potential effect of some amino acid changes on the infectivity of the JFH-1 isolate. As a first approach, successive infections of naïve Huh-7 cells were performed until high viral titres were obtained, and mutations that appeared during this selection were identified by sequencing. Only one major modification, N534K, located in the E2 glycoprotein sequence was found. Interestingly, this mutation prevented core glycosylation of E2 site 6. In addition, JFH-1 generated with this modification facilitated the infection of Huh-7 cells. In a second approach to identify mutations favouring HCVcc infectivity, we exploited the observation that a chimeric virus containing the genotype 1a core protein in the context of JFH-1 background was more infectious than wild-type JFH-1 isolate. Sequence alignment between JFH-1 and our chimera, led us to identify two major positions, 172 and 173, which were not occupied by similar amino acids in these two viruses. Importantly, higher viral titres were obtained by introducing these residues in the context of wild-type JFH-1. Altogether, our data indicate that a more robust production of HCVcc particles can be obtained by introducing a few specific mutations in JFH-1 structural proteins.

scholarly journals Leader of the Capsid Protein in Feline Calicivirus Promotes Replication of Norwalk Virus in Cell Culture

2008 ◽  
Vol 82 (19) ◽  
pp. 9306-9317 ◽  
Author(s):  
Kyeong-Ok Chang ◽  
David W. George ◽  
John B. Patton ◽  
Kim Y. Green ◽  
Stanislav V. Sosnovtsev
Keyword(s):  
Cell Culture ◽  
Capsid Protein ◽  
Recombinant Plasmid ◽  
Fluorescent Protein ◽  
Feline Calicivirus ◽  
Norwalk Virus ◽  
Human Noroviruses ◽  
Infected Cells ◽  
Green Fluorescent ◽  
In Cells

ABSTRACT The inability to grow human noroviruses in cell culture has greatly impeded the studies of their pathogenesis and immunity. Vesiviruses, in the family Caliciviridae, grow efficiently in cell culture and encode a unique protein in the subgenomic region designated as leader of the capsid protein (LC). We hypothesized that LC might be associated with the efficient replication of vesiviruses in cell culture and promote the replication of human norovirus in cells. To test this hypothesis, a recombinant plasmid was engineered in which the LC region of feline calicivirus (FCV) was placed under the control of the cytomegalovirus promoter (pCI-LC) so that the LC protein could be provided in trans to replicating calicivirus genomes bearing a reporter gene. We constructed pNV-GFP, a recombinant plasmid containing a full-length NV genome with a green fluorescent protein (GFP) in the place of VP1. The transfection of pNV-GFP in MVA-T7-infected cells produced few GFP-positive cells detected by fluorescence microscopy and flow cytometry analysis. When pNV-GFP was cotransfected with pCI-LC in MVA-T7-infected cells, we observed an increase in the number of GFP-positive cells (ca. 3% of the whole-cell population). Using this cotransfection method with mutagenesis study, we identified potential cis-acting elements at the start of subgenomic RNA and the 3′ end of NV genome for the virus replication. We conclude that LC may be a viral factor which promotes the replication of NV in cells, which could provide a clue to growing the fastidious human noroviruses in cell culture.

Production of monoclonal antibodies against hepatitis E virus capsid protein and evaluation of their neutralizing activity in a cell culture system

2008 ◽  
Vol 153 (4) ◽  
pp. 657-666 ◽  
Author(s):  
Masaharu Takahashi ◽  
Yu Hoshino ◽  
Toshinori Tanaka ◽  
Hideyuki Takahashi ◽  
Tsutomu Nishizawa ◽  
...  
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibodies ◽  
Capsid Protein ◽  
Culture System ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Cell Culture System ◽  
Virus Capsid ◽  
A Cell ◽  
Virus Capsid Protein

scholarly journals Intracellular localization and effects of individually expressed human parechovirus 1 non-structural proteins

2007 ◽  
Vol 88 (3) ◽  
pp. 831-841 ◽  
Author(s):  
Camilla Krogerus ◽  
Olga Samuilova ◽  
Tuija Pöyry ◽  
Eija Jokitalo ◽  
Timo Hyypiä
Keyword(s):  
Endoplasmic Reticulum ◽  
Complex Formation ◽  
Golgi Apparatus ◽  
Lipid Droplets ◽  
Intracellular Localization ◽  
Structural Proteins ◽  
Human Parechovirus ◽  
Replication Complex ◽  
Infected Cells ◽  
Cellular Secretion

Human parechovirus 1 (HPEV-1) has many unique features compared with other picornaviruses and it has been shown that the replication complex formed during HPEV-1 infection is different from that of other picornaviruses. Here, the intracellular localization and functional effects of individually expressed HPEV-1 non-structural proteins were studied. The 2A and 3D proteins were found diffusely in the cytoplasm and nucleus of the cell. The 3A and 3AB proteins were observed to co-localize with the markers for the Golgi apparatus, whereas 2B co-localized with markers for the endoplasmic reticulum and the 2C and 2BC proteins were observed mainly on the surface of lipid droplets. The 2C protein, which has been implicated in replication-complex formation in enterovirus-infected cells, was not able to induce vesicles similar to those seen in HPEV-1-infected cells when expressed individually. However, in superinfected cells, the fusion protein was able to relocate to the virus replication complexes. Similar to other picornaviruses, HPEV-1 was found to interfere with cellular secretion, but this function could not be ascribed to any of the individually expressed non-structural proteins.

Automated cell counting of astrocytes on patterned substrates containing aliphatic and charged properties

1995 ◽  
Vol 53 ◽  
pp. 974-975
Author(s):  
W. Shain ◽  
H. Ancin ◽  
H.C. Craighead ◽  
M. Isaacson ◽  
L. Kam ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Attachment ◽  
Culture Method ◽  
Cell Counting ◽  
Data Sets ◽  
Nuclear Staining ◽  
Double Positive ◽  
A Cell ◽  
Wafer Test ◽  
Cell Densities

Neural protheses have potential to restore nervous system functions lost by trauma or disease. Nanofabrication extends this approach to implants for stimulating and recording from single or small groups of neurons in the spinal cord and brain; however, tissue compatibility is a major limitation to their practical application. We are using a cell culture method for quantitatively measuring cell attachment to surfaces designed for nanofabricated neural prostheses.Silicon wafer test surfaces composed of 50-μm bars separated by aliphatic regions were fabricated using methods similar to a procedure described by Kleinfeld et al. Test surfaces contained either a single or double positive charge/residue. Cyanine dyes (diIC18(3)) stained the background and cell membranes (Fig 1); however, identification of individual cells at higher densities was difficult (Fig 2). Nuclear staining with acriflavine allowed discrimination of individual cells and permitted automated counting of nuclei using 3-D data sets from the confocal microscope (Fig 3). For cell attachment assays, LRM5 5 astroglial cells and astrocytes in primary cell culture were plated at increasing cell densities on test substrates, incubated for 24 hr, fixed, stained, mounted on coverslips, and imaged with a 10x objective.

Sign in / Sign up

Export Citation Format

Share Document