Membrane-protein traffic in pathogen-infected cells.

ABSTRACT All coronaviruses possess small open reading frames (ORFs) between structural genes that have been hypothesized to play important roles in pathogenesis. Infectious bronchitis virus (IBV) ORF 3a is one such gene. It is highly conserved among group 3 coronaviruses, suggesting that it has an important function in infection. IBV 3a protein is expressed in infected cells but is not detected in virions. Sequence analysis predicted that IBV 3a was a membrane protein; however, only a fraction behaved like an integral membrane protein. Microscopy and immunoprecipitation studies demonstrated that IBV 3a localized to the cytoplasm in a diffuse pattern as well as in sharp puncta in both infected and transfected cells. These puncta did not overlap cellular organelles or other punctate structures. Confocal microscopy demonstrated that IBV 3a puncta lined up along smooth endoplasmic reticulum (ER) tubules and, in a significant number of instances, were partially surrounded by these tubules. Our results suggest that IBV 3a is partially targeted to a novel domain of the smooth ER.

Download Full-text

Overexpression of the vaccinia virus A38L integral membrane protein promotes Ca2+ influx into infected cells.

Journal of Virology ◽

10.1128/jvi.70.2.905-914.1996 ◽

1996 ◽

Vol 70 (2) ◽

pp. 905-914 ◽

Cited By ~ 29

Author(s):

C M Sanderson ◽

J E Parkinson ◽

M Hollinshead ◽

G L Smith

Keyword(s):

Membrane Protein ◽

Vaccinia Virus ◽

Integral Membrane Protein ◽

Infected Cells

Download Full-text

The UL34 gene product of herpes simplex virus type 2 is a tail-anchored type II membrane protein that is significant for virus envelopment

Journal of General Virology ◽

10.1099/0022-1317-81-10-2397 ◽

2000 ◽

Vol 81 (10) ◽

pp. 2397-2405 ◽

Cited By ~ 63

Author(s):

C. Shiba ◽

T. Daikoku ◽

F. Goshima ◽

H. Takakuwa ◽

Y. Yamauchi ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Membrane Protein ◽

Herpes Simplex Virus Type ◽

Deletion Mutant ◽

Type Ii ◽

Infected Cells ◽

Simplex Virus ◽

Type Ii Membrane Protein

The UL34 gene of herpes simplex virus type 2 (HSV-2) is highly conserved in the herpesvirus family. The UL34 gene product was identified In lysates of HSV-2-infected cells as protein species with molecular masses of 31 and 32·5 kDa, the latter being a phosphorylated product. Synthesis of these proteins occurred at late times post-infection and was highly dependent on viral DNA synthesis. Immunofluorescence assays revealed that the UL34 protein was localized in the cytoplasm in a continuous net-like structure, closely resembling the staining pattern of the endoplasmic reticulum (ER), in both HSV-2-infected cells and in cells transiently expressing UL34 protein. Deletion mutant analysis showed that this colocalization required the C terminus of the UL34 protein. The UL34 protein associated with virions but not with A, B or C capsids. We treated virions, HSV-2-infected cells and cells expressing the UL34 protein with a protease in order to examine the topology of the UL34 protein. In addition, we constructed UL34 deletion mutant proteins and examined their intracellular localization. Our data strongly support the hypothesis that the UL34 protein is inserted into the viral envelope as a tail-anchored type II membrane protein and is significant for virus envelopment.

Download Full-text

Induction of protection in mice against a respiratory challenge by a vaccine formulated with exosomes isolated from Chlamydia muridarum infected cells

npj Vaccines ◽

10.1038/s41541-020-00235-x ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Sukumar Pal ◽

Yeva Mirzakhanyan ◽

Paul Gershon ◽

Delia F. Tifrea ◽

Luis M. de la Maza

Keyword(s):

Mass Spectrometry ◽

Body Weight ◽

Membrane Protein ◽

Immune Responses ◽

Hela Cells ◽

Outer Membrane Protein ◽

Chlamydia Muridarum ◽

Infected Cells ◽

Respiratory Challenge ◽

Negative Controls

Abstract The goal of this study was to determine if exosomes, isolated from Chlamydia muridarum infected HeLa cells (C. muridarum-exosomes), induce protective immune responses in mice following vaccination using CpG plus Montanide as adjuvants. Exosomes, collected from uninfected HeLa cells and PBS, formulated with the same adjuvants, were used as negative controls. Mass spectrometry analyses identified 113 C. muridarum proteins in the C. muridarum-exosome preparation including the major outer membrane protein and the polymorphic membrane proteins. Vaccination with C. muridarum-exosomes elicited robust humoral and cell-mediated immune responses to C. muridarum elementary bodies. Following vaccination, mice were challenged intranasally with C. muridarum. Compared to the negative controls, mice immunized with C. muridarum-exosomes were significantly protected as measured by changes in body weight, lungs’ weight, and number of inclusion forming units recovered from lungs. This is the first report, of a vaccine formulated with Chlamydia exosomes, shown to elicit protection against a challenge.

Download Full-text

Subcellular localization of the latent membrane protein LMP-1 in Epstein-Barr virus infected cells by immunogold EM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168979 ◽

1994 ◽

Vol 52 ◽

pp. 248-249

Author(s):

D. N. Misra ◽

R. M. Agostini ◽

E. J. Yunis

Keyword(s):

Membrane Protein ◽

Subcellular Localization ◽

Cell Lines ◽

Epstein Barr Virus ◽

Hodgkin’S Disease ◽

Burkitt’S Lymphoma ◽

Latent Membrane Protein ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr

Epstein-Barr virus (EBV) infection, prevalent in all human populations, is clinically silent in general, but causes infectious mononucleosis in some adolescents and B-lymphocyte proliferative disorders (LPDs) in immunocompromised individuals (e.g. AIDS infected; allograft recipients). EBV is also etiologically associated with African Burkitt’s lymphoma, nasopharyngeal carcinoma, and Hodgkin’s disease. The virus infects B lymphocytes and transforms them into lymphoblastoid cells which proliferate indefinitely in culture. The latently infected cells express an array of EBV gene products including 6 nuclear antigens (EBNAs), terminal proteins LMP-2A and LMP-2B, latent membrane protein LMP-1, and untranslated RNAs EBER 1 and EBER 2. These components are being extensively studied since they are involved in latency or proliferative transformation; LMP-1 has also shown oncogenic properties. In this work, we have used immunogold electron microscopy for precise subcellular localization of LMP-1 in EBV infected cell lines.Two human cell lines, P3HR-1 (Burkitt’s lymphoma) and CCL-113 (Hodgkin’s disease), obtained from ATCC, were grown in RPMI 1640 containing 20% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin.

Download Full-text

Neurobeachin, a protein implicated in membrane protein traffic and autism, is required for the formation and functioning of central synapses

The Journal of Physiology ◽

10.1113/jphysiol.2009.178236 ◽

2009 ◽

Vol 587 (21) ◽

pp. 5095-5106 ◽

Cited By ~ 46

Author(s):

Lucian Medrihan ◽

Astrid Rohlmann ◽

Richard Fairless ◽

Johanna Andrae ◽

Markus Döring ◽

...

Keyword(s):

Membrane Protein ◽

Protein Traffic ◽

Central Synapses

Download Full-text

Identification and characterization of Inc766, an inclusion membrane protein in Chlamydophila abortus-infected cells

Microbial Pathogenesis ◽

10.1016/j.micpath.2008.06.007 ◽

2008 ◽

Vol 45 (4) ◽

pp. 265-272 ◽

Cited By ~ 8

Author(s):

Evangelia Vretou ◽

Evangelia Katsiki ◽

Evgenia Psarrou ◽

Kostantinos Vougas ◽

George Th. Tsangaris

Keyword(s):

Membrane Protein ◽

Inclusion Membrane ◽

Chlamydophila Abortus ◽

Infected Cells ◽

Identification And Characterization

Download Full-text

Adenovirus E3 protein causes constitutively internalized epidermal growth factor receptors to accumulate in a prelysosomal compartment, resulting in enhanced degradation.

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.3695 ◽

1994 ◽

Vol 14 (6) ◽

pp. 3695-3706 ◽

Cited By ~ 29

Author(s):

P Hoffman ◽

C Carlin

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Membrane Protein ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Intracellular Localization ◽

Tyrosine Kinase Activity ◽

Down Regulation ◽

Infected Cells ◽

Epidermal Growth

We have previously identified and characterized an integral membrane protein coded for by the early transcription region 3 (E3) of human group C adenoviruses that down-regulates the epidermal growth factor receptor (EGFR). The goal of this study was to characterize the early receptor trafficking events leading to enhanced EGFR degradation in adenovirus-infected cells. Specifically, we wished to determine whether adenovirus increases the rate of EGFR internalization or alters the subcellular compartmentalization of internalized EGFRs. Once the optimal time for measuring early trafficking events was determined, surface EGFRs were labeled with a cleavable biotin reagent to measure internalization rates and with a receptor-specific monoclonal antibody (MAb) conjugated to colloidal gold for intracellular localization studies. We first showed that the rate of EGFR internalization in adenovirus-infected cells is indistinguishable from the constitutive internalization rate for unoccupied EGFRs. The possibility that the E3 protein can affect trafficking of EGFRs internalized at a low constitutive rate was further supported by studies showing that adenovirus-mediated down-regulation occurs independently of EGFR oligomerization and intrinsic EGFR tyrosine kinase activity, which are required for efficient ligand-induced internalization. Other tyrosine kinases inhibited by genistein are also not required for adenovirus-induced down-regulation. When the intracellular localization of EGFRs during adenovirus-mediated down-regulation was examined by electron microscopy, there was a threefold increase in the number of EGFRs localized to multivesicular bodies. The multivesicular body has been proposed to be important for regulating intracellular membrane protein sorting, since trafficking patterns for receptors that recycle and receptors that are degraded diverge in this organelle. These data therefore suggest that adenovirus may enhance EGFR degradation by causing constitutively internalized EGFRs to accumulate in a prelysosomal compartment. This is the first example of a mechanism that efficiently down-regulates EGFR without significantly increasing the rate of internalization or that does not require EGFR tyrosine kinase activity. Since viral proteins often mimic or modify a host counterpart, this suggests that there are normal physiological conditions when receptor destruction without tyrosine signalling is beneficial.

Download Full-text

Identification of Chlamydia pneumoniae-Derived Mouse CD8 Epitopes

Infection and Immunity ◽

10.1128/iai.70.7.3336-3343.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3336-3343 ◽

Cited By ~ 21

Author(s):

Anne Sarén ◽

Steve Pascolo ◽

Stefan Stevanovic ◽

Tilman Dumrese ◽

Mirja Puolakkainen ◽

...

Keyword(s):

T Cell ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Chlamydia Pneumoniae ◽

Cell Function ◽

Target Cells ◽

Heat Shock Protein 60 ◽

T Cell Epitopes ◽

Infected Cells

ABSTRACT Chlamydia pneumoniae is a common intracellular human pathogen that has been associated with several severe pathological conditions, including coronary heart disease and atherosclerosis. There is no vaccine against C. pneumoniae infection, but CD8+ T cells have been shown to be crucial for protection during experimental infection. However, the effector functions and epitope specificity of the protective CD8+ T cell remain unknown. The aim of this study was to identify C. pneumoniae-derived mouse CD8 epitopes by using a recent epitope prediction method. Of four C. pneumoniae proteins (the major outer membrane protein, outer membrane protein 2, polymorphic outer membrane protein 5, and heat shock protein 60), 53 potential CD8+ T-cell epitopes were predicted by H-2 class I binding algorithms. Nineteen of the 53 peptides were identified as CD8 epitopes by testing for induction of a cytotoxic response after immunization. To test whether the predicted epitopes are naturally processed and presented by C. pneumoniae-infected cells, we generated a panel of seven peptide-specific cytotoxic T lymphocyte lines that were subsequently tested for recognition of C. pneumoniae-infected target cells. By using this strategy, we were able to identify three C. pneumoniae CD8 epitopes that were, indeed, processed and presented on infected cells. Identification of these natural CD8 epitopes provides tools for characterization of CD8+ T-cell function in vivo and generation of epitope-specific prevention strategies.

Download Full-text