scholarly journals Vaccinia Virus Entry into Cells via a Low-pH-Dependent Endosomal Pathway

2006 ◽  
Vol 80 (18) ◽  
pp. 8899-8908 ◽  
Author(s):  
Alan C. Townsley ◽  
Andrea S. Weisberg ◽  
Timothy R. Wagenaar ◽  
Bernard Moss
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Virus Entry ◽  
Low Ph ◽  
Neutral Ph ◽  
Concanamycin A ◽  
Endosomal Acidification ◽  
Wide Range ◽  
Endosomal Pathway ◽  
Ph Dependent

ABSTRACT Previous studies established that vaccinia virus could enter cells by fusion with the plasma membrane at neutral pH. However, low pH triggers fusion of vaccinia virus-infected cells, a hallmark of viruses that enter by the endosomal route. Here, we demonstrate that entry of mature vaccinia virions is accelerated by brief low-pH treatment and severely reduced by inhibitors of endosomal acidification, providing evidence for a predominant low-pH-dependent endosomal pathway. Entry of vaccinia virus cores into the cytoplasm, measured by expression of firefly luciferase, was increased more than 10-fold by exposure to a pH of 4.0 to 5.5. Furthermore, the inhibitors of endosomal acidification bafilomycin A1, concanamycin A, and monensin each lowered virus entry by more than 70%. This reduction was largely overcome by low-pH-induced entry through the plasma membrane, confirming the specificities of the drugs. Entry of vaccinia virus cores with or without brief low-pH treatment was visualized by electron microscopy of thin sections of immunogold-stained cells. Although some virus particles fused with the plasma membrane at neutral pH, 30 times more fusions and a greater number of cytoplasmic cores were seen within minutes after low-pH treatment. Without low-pH exposure, the number of released cores lagged behind the number of virions in vesicles until 30 min posttreatment, when they became approximately equal, perhaps reflecting the time of endosome acidification and virus fusion. The choice of two distinct pathways may contribute to the ability of vaccinia virus to enter a wide range of cells.

scholarly journals Two Distinct Low-pH Steps Promote Entry of Vaccinia Virus

2007 ◽  
Vol 81 (16) ◽  
pp. 8613-8620 ◽  
Author(s):  
Alan C. Townsley ◽  
Bernard Moss
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Low Ph ◽  
Surface Proteins ◽  
Step Model ◽  
Endosomal Acidification ◽  
Membrane Attachment ◽  
Endosomal Pathway ◽  
Rate Limiting ◽  
Virion Surface

ABSTRACT Entry of vaccinia virus into cells occurs by an endosomal route as well as through the plasma membrane. Evidence for an endosomal pathway was based on findings that treatment at a pH of <6 of mature virions attached to the plasma membrane enhances entry, whereas inhibitors of endosomal acidification reduce entry. Inactivation of infectivity by low-pH treatment of virions prior to membrane attachment is characteristic of many viruses that use the endosomal route. Nevertheless, we show here that the exposure of unattached vaccinia virus virions to low pH at 37°C did not alter their infectivity. Instead, such treatment stably activated virions as indicated by their accelerated entry upon subsequent addition to cells, as measured by reporter gene expression. Moreover, the rate of entry was not further enhanced by a second low-pH treatment following adsorption to the plasma membrane. However, the entry of virions activated prior to adsorption remained sensitive to inhibitors of endosomal acidification, whereas virions treated with low pH after adsorption were resistant. Activation of virions by low pH was closely mimicked by proteinase digestion, suggesting that the two treatments operate through a related mechanism. Although proteinase cleavage of the virion surface proteins D8 and A27 correlated with activation, mutant viruses constructed by individually deleting these genes did not exhibit an activated phenotype. We propose a two-step model of vaccinia virus entry through endosomes, in which activating or unmasking the fusion complex by low pH or by proteinase is rate limiting but does not eliminate a second low-pH step mediating membrane fusion.

scholarly journals Vaccinia Virus A25 and A26 Proteins Are Fusion Suppressors for Mature Virions and Determine Strain-Specific Virus Entry Pathways into HeLa, CHO-K1, and L Cells

2010 ◽  
Vol 84 (17) ◽  
pp. 8422-8432 ◽  
Author(s):  
Shu-Jung Chang ◽  
Yu-Xun Chang ◽  
Roza Izmailyan ◽  
Yin-Liang Tang ◽  
Wen Chang
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Membrane Fusion ◽  
Virus Entry ◽  
Low Ph ◽  
Viral Envelope ◽  
Viral Fusion ◽  
Entry Pathway ◽  
L Cells ◽  
Wide Range

ABSTRACT Mature vaccinia virus enters cells through either fluid-phase endocytosis/macropinocytosis or plasma membrane fusion. This may explain the wide range of host cell susceptibilities to vaccinia virus entry; however, it is not known how vaccinia virus chooses between these two pathways and which viral envelope proteins determine such processes. By screening several recombinant viruses and different strains, we found that mature virions containing the vaccinia virus A25 and A26 proteins entered HeLa cells preferentially through a bafilomycin-sensitive entry pathway, whereas virions lacking these two proteins entered through a bafilomycin-resistant pathway. To investigate whether the A25 and A26 proteins contribute to entry pathway specificity, two mutant vaccinia viruses, WRΔA25L and WRΔA26L, were subsequently generated from the wild-type WR strain. In contrast to the WR strain, both the WRΔA25L and WRΔA26L viruses became resistant to bafilomycin, suggesting that the removal of the A25 and A26 proteins bypassed the low-pH endosomal requirement for mature virion entry. Indeed, WRΔA25L and WRΔA26L virus infections of HeLa, CHO-K1, and L cells immediately triggered cell-to-cell fusion at a neutral pH at 1 to 2 h postinfection (p.i.), providing direct evidence that viral fusion machinery is readily activated after the removal of the A25 and A26 proteins to allow virus entry through the plasma membrane. In summary, our data support a model that on vaccinia mature virions, the viral A25 and A26 proteins are low-pH-sensitive fusion suppressors whose inactivation during the endocytic route results in viral and cell membrane fusion. Our results also suggest that during virion morphogenesis, the incorporation of the A25 and A26 proteins into mature virions may help restrain viral fusion activity until the time of infections.

pH-dependent proton permeability of the plasma membrane is a regulating mechanism of polar transport through the submerged leaves of Potamogeton lucens

1991 ◽  
Vol 69 (5) ◽  
pp. 1116-1122 ◽  
Author(s):  
H. Miedema ◽  
H. B. A. Prins
Keyword(s):  
Plasma Membrane ◽  
Cell Types ◽  
Low Ph ◽  
Proton Pumps ◽  
Lower Side ◽  
Proton Permeability ◽  
Dependent Change ◽  
Ph Dependent ◽  
Ph Buffer ◽  
Set Up

Recently it has been hypothesized that light-induced polarity in leaves of the submerged angiosperms Potamogeton and Elodea results in part from a pH-dependent change in the permeability of the plasma membrane for protons. It is assumed that the proton permeability increases at high pH. In this paper we studied the effects on polarity of pH buffers, applied to just one side of the leaf. An experimental set up was used in which the solutions in contact with either side of the leaf surface were separated. The above hypothesis was partly confirmed, in that acidification, normally observed at the morphological lower side, was strongly suppressed by the application of a low-pH buffer at the upper side. Apparently, the low pH at the upper epidermis reduced the proton permeability, and as a result the proton pumps at the lower side were inactivated by a lack of substrate or by an effect on cytoplasmic pH. However, increasing the apoplastic pH at the lower side indicated that the proton permeability of the plasma membrane of these ceils did not change significantly. Hence both cell types seem to behave differently concerning the pH dependent proton permeability of the plasma membrane. This may explain why in the polar leaves of the angiosperms acidification always occurs at the lower and alkalinization always at the upper side. Key words: plasma membrane, proton permeability, pH polarity, Potamogeton, Elodea.

scholarly journals Vaccinia Virus H2 Protein Is an Essential Component of a Complex Involved in Virus Entry and Cell-Cell Fusion

2005 ◽  
Vol 79 (8) ◽  
pp. 4744-4754 ◽  
Author(s):  
Tatiana G. Senkevich ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Cell Fusion ◽  
Essential Role ◽  
Transmembrane Domain ◽  
Virus Entry ◽  
Recombinant Vaccinia Virus ◽  
Low Ph ◽  
Microscopic Appearance ◽  
Recombinant Vaccinia ◽  
Virus Morphogenesis

ABSTRACT The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.

scholarly journals The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion

2021 ◽  
Author(s):  
Ruben M. Markosyan ◽  
Mariana Marin ◽  
You Zhang ◽  
Fredric S. Cohen ◽  
Gregory B. Melikyan
Keyword(s):  
Cell Fusion ◽  
New World ◽  
Virus Entry ◽  
Low Ph ◽  
Host Cells ◽  
Fusion Pore ◽  
Lassa Virus ◽  
Anionic Lipid ◽  
Late Endosomes ◽  
Ph Dependent

AbstractArenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH in cells expressing human LAMP1 compared to cells lacking this cognate receptor, but this receptor is not absolutely required for virus entry. GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins – a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion is specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. We show that BMP promotes late steps of LASV fusion downstream of hemifusion – the formation and enlargement of fusion pores. This lipid also specifically promotes cell fusion mediated by GPC of the unrelated New World Junin arenavirus. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to direct virus entry to late endosomes.Author SummaryPathogenic arenaviruses pose a serious health threat. The viral envelope glycoprotein GPC mediates attachment to host cells and drives virus entry via endocytosis and low pH-dependent fusion within late endosomes. Understanding the host factors and processes that are essential for arenavirus fusion may identify novel therapeutic targets. To delineate the mechanism of arenavirus entry, we examined cell-cell fusion induced by the Old World Lassa virus GPC proteins at low pH. Lassa virus fusion was augmented by the LAMP1 receptor and progressed through lipid curvature-sensitive intermediates, such as hemifusion (merger of contacting leaflets of viral and cell membrane without the formation of a fusion pore). We found that most GPC-mediated fusion events were off-path hemifusion structures and that the transition from hemifusion to full fusion and fusion pore enlargement were specifically promoted by an anionic lipid, bis(monoacylglycero)phosphate, which is highly enriched in late endosomes. This lipid also specifically promotes fusion of unrelated New World Junin arenavirus. Our results imply that arenaviruses evolved to use bis(monoacylglycero)phosphate to enter cells from late endosomes.

A study of field drain ochre deposits. 2. The distribution of micro-organisms

1987 ◽  
Vol 108 (2) ◽  
pp. 347-351 ◽  
Author(s):  
I. Ap Dbwi ◽  
D. B. Johnson ◽  
W. I. Kelso
Keyword(s):  
Thiobacillus Ferrooxidans ◽  
Heterotrophic Bacteria ◽  
Drainage Water ◽  
Low Ph ◽  
Filamentous Bacteria ◽  
England And Wales ◽  
Neutral Ph ◽  
Wide Range ◽  
Water Ph ◽  
Micro Organisms

SummarySheathed filamentous bacteria,Leptothrixspp. andGallionellaspp., were observed in ochre samples from sites in England and Wales.Thiobacillus ferrooxidanswas found in acidic samples (pH < 4·0) and in ochre from drainage water of near neutral pH suggesting that it can contribute to ochre formation over a wide range of drainage water pH, Heterotrophic bacteria capable of growing in artificial media of low pH and complexdegrading heterotrophic bacteria were also isolated. Some ochre deposits could be described as either pyritic or filamentous but the majority of samples fell between these extremes and had various combinations ofT. ferrooxidans, sheathed filamentous bacteria and other heterotrophic bacteria.

scholarly journals Receptor-Mediated Entry by Equine Infectious Anemia Virus Utilizes a pH-Dependent Endocytic Pathway

2005 ◽  
Vol 79 (23) ◽  
pp. 14489-14497 ◽  
Author(s):  
Sha Jin ◽  
Baoshan Zhang ◽  
Ora A. Weisz ◽  
Ronald C. Montelaro
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Low Ph ◽  
Endocytic Pathway ◽  
Target Cells ◽  
Dependent Manner ◽  
Entry Pathway ◽  
Concanamycin A ◽  
Equine Infectious Anemia ◽  
Ph Dependent ◽  
Anemia Virus

ABSTRACT Previous studies of human and nonhuman primate lentiviral entry mechanisms indicate a predominant use of pH-independent pathways, although more recent studies of human immunodeficiency virus type 1 entry appear to reveal the use of a low-pH-dependent entry pathway in certain target cells. To expand the characterization of the specificity of lentiviral entry mechanisms, we have in the current study examined the entry pathway of equine infectious anemia virus (EIAV) during infection of its natural target, equine macrophages, permissive equine fibroblastic cell lines, and an engineered mouse cell line expressing the recently defined equine lentivirus receptor-1. The specificity of EIAV entry into these various cells was determined by assaying the effects of specific drug treatments on the level of virus entry as measured by quantitative real-time PCR assay of early reverse transcripts or by measurements of virion production. The results of these studies demonstrated that EIAV entry into all cell types was substantially inhibited in a dose-dependent manner by treatment with the vacuolar H+-ATPase inhibitors concanamycin A and bafilomycin A1 or the lysosomotropic weak base ammonium chloride. In contrast, treatments with sucrose to block clathrin-mediated endocytosis or with chloroquine to block organelle acidification failed to inhibit EIAV entry into the same target cells. The observed inhibition of EIAV entry was shown not to be related to cytotoxicity. Taken together, these experiments reveal for the first time that EIAV receptor-mediated entry into target cells is via a low-pH-dependent endocytic pathway.

scholarly journals Analysis of the pH Requirement for Membrane Fusion of Different Isolates of the Paramyxovirus Parainfluenza Virus 5

2006 ◽  
Vol 80 (6) ◽  
pp. 3071-3077 ◽  
Author(s):  
Mei Lin Z. Bissonnette ◽  
Sarah A. Connolly ◽  
Daniel F. Young ◽  
Richard E. Randall ◽  
Reay G. Paterson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Low Ph ◽  
Activation Mechanism ◽  
Parainfluenza Virus ◽  
Neutral Ph ◽  
Biophysical Studies ◽  
Parainfluenza Virus 5 ◽  
Receptor Binding Protein

ABSTRACT Paramyxoviruses enter cells by fusing their envelopes with the plasma membrane, a process that occurs at neutral pH. Recently, it has been found that there is an exception to this dogma in that a porcine isolate of the paramyxovirus parainfluenza virus 5 (PIV5), known as SER, requires a low-pH step for fusion (S. Seth, A. Vincent, and R. W. Compans, J. Virol. 77: 6520-6527, 2003). As a low-pH activation mechanism for fusion would greatly facilitate biophysical studies of paramyxovirus-mediated membrane fusion, we have reexamined the triggering of the PIV5 SER fusion protein. Using multiple assays, we could not find a requirement for low-pH triggering of PIV5 SER fusion. The challenge of discovering how the paramyxovirus receptor binding protein (HN, H, or G) activates the metastable fusion protein to cause membrane fusion at neutral pH remains.

scholarly journals Global Role of Cyclic AMP Signaling in pH-Dependent Responses in Candida albicans

mSphere ◽  
2016 ◽  
Vol 1 (6) ◽  
Author(s):  
Jeffrey M. Hollomon ◽  
Nora Grahl ◽  
Sven D. Willger ◽  
Katja Koeppen ◽  
Deborah A. Hogan
Keyword(s):  
Candida Albicans ◽  
Cyclic Amp ◽  
Hyphal Growth ◽  
Filamentous Growth ◽  
Low Ph ◽  
Camp Signaling ◽  
Content Type ◽  
Neutral Ph ◽  
Ph Dependent

ABSTRACT Candida albicans is a human commensal and the causative agent of candidiasis, a potentially invasive and life-threatening infection. C. albicans experiences wide changes in pH during both benign commensalism (a common condition) and pathogenesis, and its morphology changes in response to this stimulus. Neutral pH is considered an activator of hyphal growth through Rim101, but the effect of low pH on other morphology-related pathways has not been extensively studied. We sought to determine the role of cyclic AMP signaling, a central regulator of morphology, in the sensing of pH. In addition, we asked broadly what cellular processes were altered by pH in both the presence and absence of this important signal integration system. We concluded that cAMP signaling is impacted by pH and that cAMP broadly impacts C. albicans physiology in both pH-dependent and -independent ways. Candida albicans behaviors are affected by pH, an important environmental variable. Filamentous growth is a pH-responsive behavior, where alkaline conditions favor hyphal growth and acid conditions favor growth as yeast. We employed filamentous growth as a tool to study the impact of pH on the hyphal growth regulator Cyr1, and we report that downregulation of cyclic AMP (cAMP) signaling by acidic pH contributes to the inhibition of hyphal growth in minimal medium with GlcNAc. Ras1 and Cyr1 are generally required for efficient hyphal growth, and the effects of low pH on Ras1 proteolysis and GTP binding are consistent with diminished cAMP output. Active alleles of ras1 do not suppress the hyphal growth defect at low pH, while dibutyryl cAMP partially rescues filamentous growth at low pH in a cyr1 mutant. These observations are consistent with Ras1-independent downregulation of Cyr1 by low pH. We also report that extracellular pH leads to rapid and prolonged decreases in intracellular pH, and these changes may contribute to reduced cAMP signaling by reducing intracellular bicarbonate pools. Transcriptomics analyses found that the loss of Cyr1 at either acidic or neutral pH leads to increases in transcripts involved in carbohydrate catabolism and protein translation and glycosylation and decreases in transcripts involved in oxidative metabolism, fluconazole transport, metal transport, and biofilm formation. Other pathways were modulated in pH-dependent ways. Our findings indicate that cAMP has a global role in pH-dependent responses, and this effect is mediated, at least in part, through Cyr1 in a Ras1-independent fashion. IMPORTANCE Candida albicans is a human commensal and the causative agent of candidiasis, a potentially invasive and life-threatening infection. C. albicans experiences wide changes in pH during both benign commensalism (a common condition) and pathogenesis, and its morphology changes in response to this stimulus. Neutral pH is considered an activator of hyphal growth through Rim101, but the effect of low pH on other morphology-related pathways has not been extensively studied. We sought to determine the role of cyclic AMP signaling, a central regulator of morphology, in the sensing of pH. In addition, we asked broadly what cellular processes were altered by pH in both the presence and absence of this important signal integration system. We concluded that cAMP signaling is impacted by pH and that cAMP broadly impacts C. albicans physiology in both pH-dependent and -independent ways.

Sign in / Sign up

Export Citation Format

Share Document