Electrical conductivity dispersion as a probe of membrane modifications in mouse polyomavirus infected cells in culture

1996 ◽  
Vol 16 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Adalberto Bonincontro ◽  
Anna Iacoangeli ◽  
Gianfranco Risuleo
Keyword(s):  
Electrical Conductivity ◽  
Dna Replication ◽  
Cell Membrane ◽  
Virus Titer ◽  
Experimental Conditions ◽  
Membrane Conductivity ◽  
Cells In Culture ◽  
Infected Cells ◽  
Murine Polyomavirus

In this report we investigate the inhibition of membrane conductivity, due to the murine polyomavirus infection in permissive cells in culture. We define experimental conditions to have reproducible results and demonstrate that the intensity of the effects on the cell membrane, depends upon the virus titer used in the infection. Finally, the virus dependent effects disappear if the infection is performed in the presence of a drug that inhibits polymavirus DNA replication.

RNA synthesis in polyoma virus-infected cells. I. Pattern of formation of polyribosome-associated messenger RNA during productive infection

1970 ◽  
Vol 48 (10) ◽  
pp. 1104-1112 ◽  
Author(s):  
William P. Cheevers ◽  
Rose Sheinin
Keyword(s):  
Dna Replication ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Polyoma Virus ◽  
Productive Infection ◽  
Mrna Synthesis ◽  
Viral Dna ◽  
Experimental Conditions ◽  
Embryo Cells ◽  
Infected Cells

Experimental conditions have been established for selective measurement of the synthesis of mRNA in mouse embryo cells. Using such conditions, it was found that productive infection of these cells by polyoma virus resulted in stimulation of mRNA synthesis. The pattern of induction of mRNA synthesis was biphasic, characterized by distinct "early" and "late" periods, as denoted by the time of initiation of progeny viral DNA replication. The formation of "early" mRNA was first detected at 9–11 h postinfection, 6 h prior to the time of onset of virus-induced synthesis of cell DNA and 9 h prior to initiation of polyoma DNA replication. The initiation of synthesis of "late" mRNA was approximately coincident with the onset of formation of viral DNA. Most of the newly synthesized "early" and "late" mRNA was of relatively small size (8–12 S) and was associated with polyribosomes which sedimented at less than 180 S. The proportion of the total "late" mRNA which was virus-specific was three times higher than that of the total "early" mRNA; however, the mRNA synthesized both "early" and "late" was predominantly cell-specific.

scholarly journals Antiviral Activity of the G-Quadruplex Ligand TMPyP4 against Herpes Simplex Virus-1

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 196
Author(s):  
Sara Artusi ◽  
Emanuela Ruggiero ◽  
Matteo Nadai ◽  
Beatrice Tosoni ◽  
Rosalba Perrone ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Antiviral Activity ◽  
Herpes Simplex Virus 1 ◽  
Tem Analysis ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways.

scholarly journals Bub1 in Complex with LANA Recruits PCNA To Regulate Kaposi's Sarcoma-Associated Herpesvirus Latent Replication and DNA Translesion Synthesis

2015 ◽  
Vol 89 (20) ◽  
pp. 10206-10218 ◽  
Author(s):  
Zhiguo Sun ◽  
Hem Chandra Jha ◽  
Erle S. Robertson
Keyword(s):  
Dna Replication ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Kinase Domain ◽  
Cellular Protein ◽  
Nuclear Antigen ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Molecular Bridge

ABSTRACTLatent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requirestrans-acting elements, both viral and cellular, such as ORCs, MCMs, and latency-associated nuclear antigen (LANA). However, how cellular proteins are recruited to the viral genome is not very clear. Here, we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with proliferating cell nuclear antigen (PCNA) via a highly conserved PIP box motif within the kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV-positive cells. This strongly indicated that Bub1 serves as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S phase and interacted with PCNA to promote its monoubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV-infected cells.IMPORTANCEDuring latency in KSHV-infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV LANA recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with PCNA, a critical protein for viral DNA replication. Furthermore, our findings suggest that Bub1, a spindle checkpoint protein, serves as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as monoubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells.

scholarly journals Natural Cytotoxicity Receptors: Pattern Recognition and Involvement of Carbohydrates

2005 ◽  
Vol 5 ◽  
pp. 151-154 ◽  
Author(s):  
Angel Porgador
Keyword(s):  
Pattern Recognition ◽  
Cell Membrane ◽  
Cell Surface ◽  
Innate Immune ◽  
Target Cells ◽  
Natural Cytotoxicity ◽  
Tumor Cell Membrane ◽  
Infected Cells ◽  
Natural Cytotoxicity Receptors ◽  
New Evidence

Natural cytotoxicity receptors (NCRs), expressed by natural killer (NK) cells, trigger NK lysis of tumor and virus-infected cells on interaction with cell-surface ligands of these target cells. We have determined that viral hemagglutinins expressed on the surface of virus-infected cells are involved in the recognition by the NCRs, NKp44 and NKp46. Recognition of tumor cells by the NCRs NKp30 and NKp46 involves heparan sulfate epitopes expressed on the tumor cell membrane. Our studies provide new evidence for the identity of the ligands for NCRs and indicate that a broader definition should be applied to pathological patterns recognized by innate immune receptors. Since nonmicrobial endogenous carbohydrate structures contribute significantly to this recognition, there is an imperative need to develop appropriate tools for the facile sequencing of carbohydrate moieties.

scholarly journals The Early Gene hhi1 Reactivates Heliothis zea Nudivirus 1 in Latently Infected Cells

2009 ◽  
Vol 84 (2) ◽  
pp. 1057-1065 ◽  
Author(s):  
Yueh-Lung Wu ◽  
Carol P. Wu ◽  
Song-Tay Lee ◽  
Han Tang ◽  
Chi-Hua Chang ◽  
...  
Keyword(s):  
Viral Infection ◽  
Critical Role ◽  
Heliothis Zea ◽  
Virus Titer ◽  
Viral Reactivation ◽  
Early Gene ◽  
Mechanistic Study ◽  
Early Genes ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT Heliothis zea nudivirus 1 (HzNV-1), previously known as Hz-1 virus, is an insect virus able to establish both productive and latent infections in several lepidopteran insect cells. Here, we have cloned and characterized one of the HzNV-1 early genes, hhi1, which maps to the HindIII-I fragment of the viral genome. During the productive viral infection, a 6.2-kb hhi1 transcript was detectable as early as 0.5 h postinfection (hpi). The level of transcript reached a maximum at 2 hpi and gradually decreased after 4 hpi. The transcript was not detectable during the latent phase of viral infection. Upon cycloheximide treatment, much higher levels of hhi1 transcript were detected throughout the productive viral infection cycle, suggesting that newly synthesized proteins are not needed for the expression of hhi1. Nevertheless, viral coinfection can further stimulate the expression of transfected hhi1 promoter in a plasmid. Transient hhi1 expression in latently infected cells resulted in a significant increase in virus titer and viral DNA propagation, suggesting that hhi1 plays a critical role in viral reactivation. Additional experiments showed that six early genes, which possibly function in transcription or DNA replication, were activated in the latent cells upon hhi1 transfection. Among these six genes, orf90 and orf121 expression could be induced by hhi1 alone without the need for other viral genes. Our discovery should be useful for future mechanistic study of the switches of latent/productive HzNV-1 viral infections.

scholarly journals The Boron & De Weer Model of Intracellular pH Regulation

2020 ◽  
Author(s):  
Rossana Occhipinti ◽  
Soroush Safaei ◽  
Peter J. Hunter ◽  
Walter F. Boron
Keyword(s):  
Cell Membrane ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Quantitative Model ◽  
Historical Background ◽  
Acid Base ◽  
Experimental Conditions ◽  
Subsequent Work ◽  
Energy Dependent ◽  
Parameter Values

The classic Boron & De Weer (1976) paper provided the first evidence of active regulation of pH} in cells by an energy-dependent acid-base transporter. These authors also developed a quantitative model --- comprising passive fluxes of acid-base equivalents across the cell membrane, intracellular reactions, and an active transport mechanism in the cell membrane (modelled as a proton pump) --- to help interpret their measurements of intracellular pH under perturbations of both extracellular CO2/HCO3- and extracellular NH3/NH4+. This Physiome paper seeks to make that model, and the experimental conditions under which it was developed, available in a reproducible and well-documented form, along with a software implementation that makes the model easy to use and understand. We have also taken the opportunity to update some of the units used in the original paper, and to provide a few parameter values that were missing in the original paper. Finally, we provide an historical background to the Boron & De Weer (1976) proposal for active pH regulation and a commentary on subsequent work that has enriched our understanding of this most basic aspect of cellular physiology.

Patents for the Physiological Quality in Seeds of Peach Rootstock Classified by Weight and Stored for Different Periods

2019 ◽  
Vol 10 (2) ◽  
pp. 124-130 ◽  
Author(s):  
Aline G. Souza ◽  
Oscar J. Smiderle ◽  
Renata D. Menegatti ◽  
Marcos Aurélio C. de Lima ◽  
Tainá R. das Neves ◽  
...  
Keyword(s):  
Electrical Conductivity ◽  
Experimental Design ◽  
Growth Analysis ◽  
Prunus Persica ◽  
Stone Fruit ◽  
Germination Test ◽  
Experimental Conditions ◽  
Physiological Quality

Background: Among stone fruit, the peach (Prunus persica (L) Batsch) is one of the most widely grown species in Brazil, in both area cultivated and in production. Objective: The aim of this study was to evaluate the physiological quality of heavy and light seeds of four cultivars of Prunus persica for two storage periods, from tests of electrical conductivity, germination, and an analysis of initial plantlets growth. Methods: The Electrical Conductivity test (EC) was conducted in a Completely Randomised Design (CRD), in a 4 x 2 x 5 factorial scheme with five replications. The germination test was carried out in CRD, in a 4 x 2 factorial scheme with eight replications. The physiological quality of the seeds was determined at zero and twelve month’s storage. For the growth analysis, the experimental design was in CRD, in a 4 x 2 factorial scheme with four replications. Results: Under the conditions of the present study, it was found that the tests of germination and electrical conductivity were complementary in evaluating physiological quality in seeds of Prunus persica rootstock, suggesting that independent of the weight of the seeds, in ‘Capdeboscq’, ‘Aldrighi’, ‘Okinawa’ and ‘Okinawa Roxo’, there is a loss of quality and viability when the seeds are stored for a period of 12 months. Conclusion: Under the experimental conditions of the present study, it was concluded that storage for a period of 12 months in Recent patents is not rather recommendable for maintaining quality and viability in seeds of Prunus persica of the Capdeboscq, Aldrighi, Okinawa and Okinawa Roxo cultivars.

scholarly journals A Role for Single-Stranded Templates in Cell-Free Adeno-Associated Virus DNA Replication

2000 ◽  
Vol 74 (2) ◽  
pp. 744-754 ◽  
Author(s):  
Peter Ward ◽  
R. Michael Linden
Keyword(s):  
Dna Replication ◽  
Infected Cell ◽  
Dna Binding Protein ◽  
Strand Displacement ◽  
Dna Templates ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Cell Extracts ◽  
Infected Cells

ABSTRACT Assays have been described in which duplex adeno-associated virus (AAV) DNA can be replicated in HeLa cell extracts with exogenous AAV Rep protein. These assays appear to mimic the AAV DNA replication that occurs in the cell, including the ability of extracts from adenovirus (Ad)-infected cells to replicate duplex AAV DNA templates more efficiently than extracts from uninfected cells can. We showed previously that the Ad-infected extract was able to support a more processive replication than the uninfected extract. When the Ad single-stranded DNA binding protein (Ad-DBP) was added to an uninfected extract, DNA replication became processive. Based on a strand displacement replication model, we hypothesized that the Ad-DBP was stabilizing the displaced single-stranded DNA during strand displacement replication. In this report, we show that in Ad-infected extracts most of the newly replicated duplex DNA is converted into a single-stranded form shortly after synthesis. Using the results of assays for the replication of single-stranded AAV DNA, we show that these single-stranded molecules serve as templates for additional replication. In addition, we identify a class of molecules which are likely to be intermediates of replication on single-stranded templates. We discuss a possible role for replication of single-stranded molecules in the infected cell.

scholarly journals Cyclin-Dependent Kinase Activity Is Required for Efficient Expression and Posttranslational Modification of Human Cytomegalovirus Proteins and for Production of Extracellular Particles

2006 ◽  
Vol 80 (12) ◽  
pp. 5886-5896 ◽  
Author(s):  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Steady State ◽  
Human Cytomegalovirus ◽  
Virus Titer ◽  
State Level ◽  
Early Gene ◽  
Tegument Protein ◽  
Cyclin Dependent Kinase ◽  
Viral Dna ◽  
Infected Cells ◽  
Steady State Levels

ABSTRACT We have previously shown that the addition of the cyclin-dependent kinase (cdk) inhibitor Roscovitine at the beginning of infection of cells with human cytomegalovirus (HCMV) significantly disrupts immediate-early gene expression and the progression of the infection. In the present study, we have examined the effects of cdk inhibition on late viral events by delaying addition of Roscovitine until 24 h postinfection. Although viral DNA replication was inhibited two- to threefold by treatment of infected cells with Roscovitine, the drop did not correspond to the 1- to 2-log-unit decrease in virus titer. Quantification of viral DNA in the supernatant from cells revealed that there was a significant reduction in the production or release of extracellular particles. We observed a lag in the expression of several viral proteins but there was a significant decrease in the steady-state levels of IE2-86. Likewise, the steady-state level of the essential tegument protein UL32 (pp150) was reduced. The levels of pp150 and IE2-86 mRNA were not greatly affected by treatment with Roscovitine and thus did not correlate with the reduced levels of protein. In contrast, the expression of the tegument protein ppUL69 was higher in drug-treated samples, and the protein accumulated in a hyperphosphorylated form. ppUL69 localized to intranuclear aggregates that did not overlap with viral replication centers in cells treated with Roscovitine. Taken together, these data indicate that cdk activity is required at multiple steps during HCMV infection, including the expression, modification, and localization of virus-encoded proteins.

scholarly journals STUDIES ON PNEUMONIA VIRUS OF MICE (PVM) IN CELL CULTURE

1967 ◽  
Vol 126 (2) ◽  
pp. 267-276 ◽  
Author(s):  
Richard W. Compans ◽  
Donald H. Harter ◽  
Purnell W. Choppin
Keyword(s):  
Cell Culture ◽  
Cell Membrane ◽  
Cesium Chloride ◽  
Virus Particles ◽  
Internal Component ◽  
Infected Cells

Pneumonia virus of mice (PVM) particles are spheres 80–120 mµ in diameter, or filaments of similar diameter with lengths up to 3 µ. The particles possess an outer spike-covered envelope and helical internal component 120–150 A in diameter. Virus particles acquire their envelope by a budding process at the cell membrane; mature particles are seen only extracellularly. Dense inclusions are prominent in the cytoplasm of PVM-infected BHK21 cells by 48 hr after inoculation. The inclusions appear to consist of aggregates of the internal component of PVM, and the helical component has been isolated in a cesium chloride gradient from extracts of osmotically shocked cells. Murine erythrocytes, which are agglutinated by PVM, adsorb to the surface of infected cells and to budding and extracellular PVM particles. On the basis of its structure and morphogenesis, PVM appears to be a myxovirus; however, it does not fit into either of the established subgroups of myxoviruses. The 120–150 A diameter of the PVM internal component differs from the diameters of the internal components of the two established subgroups of myxoviruses, and suggests that a third subgroup of these viruses may exist.

Sign in / Sign up

Export Citation Format

Share Document