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1.
J Gen Virol ; 93(Pt 4): 761-770, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22170634

ABSTRACT

Dengue virus (DENV) causes fever and severe haemorrhagic symptoms in humans. The DEN2 16681 strain, derived from a dengue haemorrhagic fever patient, has been widely used in studies related to DENV pathogenesis, such as mouse and non-human primate haemorrhagic models and human vascular endothelial-cell permeability. To clarify the entry mechanism of the 16681 strain, we characterized a novel cell receptor for this strain. Our two major findings were as follows: firstly, the SDC2 membrane protein was an effective DEN2 16681 receptor in a cloned K562 cell line. Secondly, a heparan sulfate (HS) glycochain (of four glycochains in SDC2) is the specific binding site of DENV and seems to be involved in tissue-culture adaptation. Our findings present an entry mechanism that could be implicated for DENV adaptation and HS-mediated DENV infection.


Subject(s)
Dengue Virus/physiology , Receptors, Virus/metabolism , Severe Dengue/virology , Syndecan-2/metabolism , Animals , Chlorocebus aethiops , Dengue Virus/metabolism , Disease Susceptibility/virology , Gene Expression , Gene Silencing , Heparitin Sulfate/metabolism , Humans , K562 Cells/virology , Severe Dengue/metabolism , Vero Cells , Virus Attachment , Virus Internalization
2.
Virology ; 405(2): 474-82, 2010 Sep 30.
Article in English | MEDLINE | ID: mdl-20638094

ABSTRACT

Adenovirus 11 prototype (Ad11p), belonging to species B, uses CD46 as an attachment receptor. CD46, a complement regulatory molecule, is expressed on all human nucleated cells. We show here that Ad11p virions downregulate CD46 on the surface of K562 cells as early as 5min p.i. Specific binding to CD46 by the Ad11p fiber knob was required to mediate downregulation. The complement regulatory factors CD55 and CD59 were also reduced to a significant extent as a consequence of Ad11p binding to K562 cells. In contrast, binding of Ad7p did not result in downregulation of CD46 early in infection. Thus, the presumed interaction between Ad7p and CD46 did not have the same consequences as the Ad11p-CD46 interaction, the latter virus (Ad11p) being a promising gene therapy vector candidate. These findings may lead to a better understanding of the pathogenesis of species B adenovirus infections.


Subject(s)
Adenoviruses, Human/pathogenicity , Capsid Proteins/metabolism , Down-Regulation , Membrane Cofactor Protein/metabolism , Receptors, Virus/metabolism , Adenoviruses, Human/classification , Adenoviruses, Human/genetics , CD55 Antigens/genetics , CD55 Antigens/metabolism , CD59 Antigens/genetics , CD59 Antigens/metabolism , Capsid Proteins/genetics , Cell Line, Tumor , Epithelial Cells/virology , Genetic Vectors , Humans , K562 Cells/virology , Lung/cytology , Lung/virology , Membrane Cofactor Protein/genetics , Receptors, Virus/genetics
3.
J Immunol Methods ; 264(1-2): 69-76, 2002 Jun 01.
Article in English | MEDLINE | ID: mdl-12191511

ABSTRACT

The study of the immunologic response to whole human immunodeficiency virus type 1 (HIV-1) antigen is limited by the presence of highly immunogenic human leukocyte antigen (HLA) alloantigens on the envelope of wild type virus. This paper outlines the production of HIV-1 infectious virions free of HLA for use as whole viral antigens in immunoassays. An infectious molecular clone of HIV-1 was transfected into the K-562 cell line, which does not express HLA on the cell surface. After a 30-day selection period, to ensure stable transfection, cells and culture supernatants were analyzed for productive HIV-1 infection and virion infectivity. An enzyme-linked immunosorbent assay (ELISA) confirmed the presence of p24 in the culture supernatants. Molecular confirmation of HIV-1 transfection was achieved by gene amplification. Flow cytometric analysis was used to identify gp120 on the surface of the infected cells. Viral supernatants were tested for HIV infectivity in peripheral blood mononuclear cells (PBMCs). The usefulness of this viral preparation as whole virus antigens was validated using PBMCs from HIV-infected individuals. These results indicate the successful production of HIV-1 infectious virions, which do not have HLA molecules on their viral envelope, and demonstrate their utility for immunoassays.


Subject(s)
HIV Antigens/immunology , HIV-1/immunology , HLA Antigens/physiology , Immunoassay/methods , Cell Culture Techniques/methods , Cell-Free System/immunology , Cells, Cultured , Gene Products, nef/biosynthesis , Gene Products, nef/genetics , HIV Antigens/genetics , HIV Core Protein p24/biosynthesis , HIV Core Protein p24/genetics , HIV Envelope Protein gp120/biosynthesis , HIV Envelope Protein gp120/genetics , HIV Infections/immunology , HIV-1/genetics , HLA Antigens/biosynthesis , HLA Antigens/genetics , HLA-DR Antigens/biosynthesis , HLA-DR Antigens/genetics , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/genetics , Humans , K562 Cells/immunology , K562 Cells/virology , Transfection , Transgenes , nef Gene Products, Human Immunodeficiency Virus
4.
FEBS Lett ; 500(1-2): 17-24, 2001 Jun 29.
Article in English | MEDLINE | ID: mdl-11434919

ABSTRACT

The mechanism of cell death induced by West Nile virus (WNV), a causative agent of human febrile syndrome and encephalitis, was investigated. WNV-infected K562 and Neuro-2a cells manifested the typical features of apoptosis, including cell shrinkage, chromatin condensation and subdiploid DNA content by flow cytometry. DNA fragmentation into nucleosomal size and changes in outer cell membrane phospholipid composition were also observed in K562 cells. UV-inactivated virus failed to induce the above-mentioned characteristics, suggesting that viral replication may be required for the induction of apoptosis by WNV. Additionally, signals involved in WNV-induced apoptosis are associated with the up-regulation of bax gene expression.


Subject(s)
Apoptosis/physiology , K562 Cells/cytology , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/biosynthesis , West Nile virus/physiology , Aedes , Animals , Cell Survival/physiology , Humans , K562 Cells/virology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , RNA, Messenger/metabolism , Tumor Cells, Cultured , Up-Regulation , bcl-2-Associated X Protein
5.
Cancer Res ; 61(7): 3009-15, 2001 Apr 01.
Article in English | MEDLINE | ID: mdl-11306480

ABSTRACT

Autologous hematopoietic stem cell transplantation after myelosuppressive chemotherapy is used for the treatment of high-risk breast cancer and other solid tumors. However, contamination of the autologous graft with tumor cells may adversely affect outcomes. Human hematopoietic bone marrow cells are resistant to herpes simplex virus type 1 (HSV-1) replication, whereas human breast cancer cells are sensitive to HSV-1 cytotoxicity. Therefore, we examined the utility of G207, a safe replication-competent multimutated HSV-1 vector, as a biological purging agent for breast cancer in the setting of stem cell transplantation. G207 infection of human bone marrow cells had no effect on the proportion or clonogenic capacity of CD34+ cells but did enhance the proliferation of bone marrow cells in culture and the proportion of CD14+ and CD38+ cells. On the other hand, G207 at a multiplicity of infection of 0.1 was able to purge bone marrow of contaminating human breast cancer cells. Because G207 also stimulates the proliferation of human hematopoietic cells, it overcomes a limitation of other purging methods that result in delayed reconstitution of hematopoiesis. The efficient infection of human bone marrow cells in the absence of detected toxicity suggests that HSV vectors may also prove useful for gene therapy to hematopoietic progenitor cells.


Subject(s)
Bone Marrow Cells/virology , Bone Marrow Purging , Breast Neoplasms/pathology , Breast Neoplasms/virology , Hematopoietic Stem Cell Transplantation , Herpesvirus 1, Human/physiology , Bone Marrow Cells/cytology , Hematopoietic Stem Cells/virology , Herpesvirus 1, Human/genetics , Humans , K562 Cells/virology , Virus Replication
6.
Virus Res ; 56(1): 41-51, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9784064

ABSTRACT

We used three-color fluorescent labeling and confocal microscopy to compare the permissive and the antibody-mediated, restricted replication of Aleutian mink disease parvovirus (ADV). In both permissive (CRFK cells) and restricted (K562 cells) situations, both ADV non-structural proteins (NS1 and NS2) concentrated at focal sites in the nucleus, which also contained viral DNA. Bromodeoxyuridine labeling demonstrated that these sites also supported active ADV single-strand DNA synthesis, indicating that they were replication compartments. ADV capsid proteins were located in intranuclear shells surrounding the replication compartments. At later time points, NS2 was readily detected in the cytoplasm of permissively infected CRFK cells, whereas the cytoplasmic presence of NS2 was much less pronounced in the K562 cells. These results showed that both permissive and restricted ADV replication are associated with a tight nuclear subcompartmentalization of viral products. Furthermore, differences between the permissive and restricted virus-cell interactions were noted, suggesting that there may be a morphological basis for examining the outcome of ADV infection.


Subject(s)
Aleutian Mink Disease Virus/physiology , Viral Proteins/metabolism , Virus Replication , Animals , Antibodies, Monoclonal , Antibody Specificity , Cell Line/virology , Cell Nucleus/virology , Cytoplasm/virology , DNA, Viral/metabolism , Fluorescent Antibody Technique, Indirect , Humans , K562 Cells/virology , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Rabbits , Time Factors
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