Model for studying virus attachment: identification and quantitation of Epstein-Barr virus-binding cells by using biotinylated virus in flow cytometry.

Epstein-Barr virus (EBV) was purified and biotinylated without significant loss of its cell-transforming activity. The use of biotinylated virus in conjunction with antibodies specific for selected cell surface molecules and flow cytometric analysis allowed for the positive identification of the virus-binding lymphocytes among a heterogeneous mononuclear cell population. Biotinylated EBV efficiently bound to all B lymphocytes, including those bearing surface mu, delta, gamma, and alpha immunoglobulin heavy chains or the surface CD5 (Leu-1) marker, but not to T lymphocytes, natural killer cells, or monocytes. By using biotinylated EBV and specific monoclonal antibodies in competitive inhibition experiments, it was also found that the virus attaches to an epitope on the CR2 molecule (the receptor for C3d and EBV), which is close to or identical with the one recognized by OKB7 monoclonal antibody, and that cell surface structures other than CR2 cannot mediate attachment of EBV. Moreover, studies on the binding of the virus to induced B lymphocytes (cells in S through G2 phase), and this was associated with the disappearance of the surface CR2 molecule and the inability of the virus to attach to these cells. The approach described here should be useful in studying the attachment of other viruses, identifying the specific cell types involved, and analyzing the effect of the cell cycle on virus binding.     

Natural OX40L expressed on human T cell leukemia virus type-I-immortalized T cell lines interferes with infection of activated peripheral blood mononuclear cells by CCR5-utilizing human immunodeficiency virus

Background

Food allergy has been reported increasingly around the world during the past several decades. Epstein-Barr virus (EBV), a common herpesvirus with high infection rate, is now suspected to be a risk or protective factor in food allergy. The aim of the study was to investigate the possible role of EBV infection in IgE-mediated food allergy.

Methods

34 patients with an egg allergy and 34 healthy controls participated in this study. Egg allergy was confirmed by open-food challenge. Serum anti-viral capsid antigen (VCA), anti-Epstein-Barr nuclear antigen 1 (EBNA-1) IgG and egg specific (yolk and white)-IgE levels were evaluated by enzyme linked immunosorbent assay (ELISA). At the same time, EBV DNA as well as viral miRNAs in these samples was quantified by real-time PCR.

Results

The results showed that serum anti EBNA-1 IgG and two viral miRNAs (miR-BART1-5p and miR-BART7) were highly expressed in patients with egg allergy compared with healthy controls (p < 0.05, < 0.001 and < 0.01, respectively). Moreover, the expressions of anti EBNA-1 specific IgG, miR-BART1-5p and miR-BART7 positively correlated with the level of egg-specific IgE (p < 0.05, < 0.01 and < 0.01, respectively). The differences in anti VCA IgG concentration and EBV DNA copy number between the allergy patients and control individuals were not statistically significant.

Conclusions

The high expression of EBV-specific antibody and miRNAs indicated that EBV infection might play a promoting role in IgE-mediated egg food allergy, and viral miRNAs-related immunomodulatory pathway was likely involved in this allergy process.     

Spontaneous cytotoxicity of human peripheral blood mononuclear cells toward red blood cell targets. II. Time-dependent loss of suppressor cell activity.

In a previous paper we demonstrated that human peripheral blood mononuclear cells become strikingly cytotoxic toward a wide variety of red blood cell targets after 7 days of in vitro culture. The cell responsible for cytotoxicity does not rosette with SRBC and demonstrates both surface adherence and phagocytic properties. In this paper we wish to show that development of spontaneous cytotoxicity is due to a time-dependent loss of suppressor cell function. Fresh autologous lymphocytes, when added to cultured cells, abrogate the subsequent expression of spontaneous cytotoxicity toward RBC targets. The suppressor cell is radioresistant; requires 24 hr to suppress optimally; is inactivated by heating at 56 degrees C for 15 min, and is enriched in the non-T interface after SRBC rosette depletion over a discontinuous Ficoll-Hypaque gradient. Furthermore, the addition of a cell-free sonicate of fresh lymphocytes is capable of inhibiting spontaneous cytotoxicity toward RBC targets. However, if mononuclear cells are allowed to incubate in tissue culture medium for 7 days they are no longer suppressive after sonication. These data suggest that fresh mononuclear cells exert a potent negative regulatory influence on monocyte killing. Our culture conditions by removing this negative influence have produced a new model of spontaneous nonspecific killing by monocytes.     

A milk protein lactoferrin enhances human T cell leukemia virus type I and suppresses HIV-1 infection

Human T cell leukemia virus type I (HTLV-I) and HIV-1, causative agents of adult T cell leukemia/lymphoma and AIDS, respectively, are transmitted vertically via breast milk. Here we demonstrate that lactoferrin, a milk protein that has a variety of antimicrobial and immunomodulatory activities, facilitates replication of HTLV-I in lymphocytes derived from asymptomatic HTLV-I carriers and transmission to cord blood lymphocytes in vitro. Transient expression assays revealed that lactoferrin can transactivate HTLV-I long terminal repeat promoter. In contrast, lactoferrin inhibits HIV-1 replication, at least in part, at the level of viral fusion/entry. These results suggest that lactoferrin may have different effects on vertical transmission of the two milk-borne retroviruses.     

Analysis of potential phosphorylation sites in human T cell leukemia virus type 1 Tax

The human T cell leukemia virus type 1 (HTLV-1) Tax is a phosphoprotein, however, the contribution of phosphorylation to Tax activity is unknown. Previous studies have shown that phosphorylation of Tax occurs on serine residue(s), within one tryptic fragment, in response to 4-phorbol-12-myristate-13-acetate, in both mouse and human cells. Studies were conducted in multiple cell lines to identify the specific phosphorylated serines as a prelude to functional analysis. The phosphorylation pattern of Tax was found to be different in 293T and COS-7 cells in comparison with MT-4 and Px-1 cells. However, one tryptic fragment remained consistent in comigration analyses among all cell lines. Using selected Tax serine mutants a tryptic fragment containing a serine at residue 113 believed to be the site of phosphorylation of Tax did not comigrate with the common phosphorylated tryptic fragment. Analysis of selected Tax mutants for ability totrans-activate the cytomegalovirus promoter demonstrated mutation of serine 77 to alanine reducedtrans-activation by 90% compared to wild-type Tax. However, examination of the phosphorylation pattern of the serine 77 mutant demonstrated that it is not the site of phosphorylation. These studies demonstrate the importance of using relevant cell lines to characterize the role of phosphorylation in protein function.     

Identification of an 80-kilodalton membrane glycoprotein important for human T-cell leukemia virus type I and type II syncytium formation and infection.

Human T-cell leukemia virus type I and type II (HTLV-I and HTLV-II, respectively) infect certain sublines of the BJAB human B-cell line. We observed that the WH subline, but not the CC/84 subline, of BJAB cells were infectible by cell-free HTLV-I or HTLV-II and formed syncytia with cells infected by these retroviruses. This suggests that the BJAB-CC/84 cells possibly lack a membrane molecule(s) important for syncytium formation and infectibility. In order to identify this antigen, we generated polyclonal anti-BJAB-WH antisera which were adsorbed on BJAB-CC/84 cells. The adsorbed antisera bound only BJAB-WH and BJAB-CC/79 cells as demonstrated by complement-dependent cytotoxicity and flow cytometric assays. Furthermore, this adsorbed antisera bound several human T-cell clones, including SupT-1, as determined by flow cytometric assays. The adsorbed antiserum was monospecific as it immunoprecipitated only one 78- to 80-kDa protein from lysates of metabolically labeled BJAB-WH, BJAB-CC/79, and SupT-1, but not BJAB-CC/84, cells. The monospecific antisera detected a glycoprotein composed of a 64- to 66-kDa core protein containing tunicamycin-sensitive N-linked oligosaccharides. This membrane glycoprotein appears to be involved in HTLV-I- and HTLV-II-induced fusion and infection, as the monospecific antisera were capable of inhibiting both of these processes. The monospecific antisera diluted 1:50 and 1:90 inhibited 85 to 90% of syncytium formation induced in BJAB-WH, BJAB-CC/79, and SupT-1 cells cultured with HTLV-I- or HTLV-II-infected MT2, MoT, or FLW human T- or B-cell lines. At the same dilution, antisera inhibited 70 to 80% of infection of BJAB-WH cells by cell-free HTLV-I or HTLV-II. Thus, these studies indicate a role for a 78- to 80-kDa glycoprotein in HTLV-I or HTLV-II infection and syncytium formation.     

Ubiquitin-specific peptidase 20 targets TRAF6 and human T cell leukemia virus type 1 tax to negatively regulate NF-kappaB signaling

NF-κB plays a key role in innate and acquired immunity. Its activity is regulated through intricate signaling networks. Persistent or excessive activation of NF-κB induces diseases, such as autoimmune disorders and malignant neoplasms. Infection by human T cell leukemia virus type 1 (HTLV-1) causes a fatal hematopoietic malignancy termed adult T cell leukemia (ATL). The HTLV-1 viral oncoprotein Tax functions pivotally in leukemogenesis through its potent activation of NF-κB. Recent findings suggest that protein ubiquitination is crucial for proper regulation of NF-κB signaling and for Tax activity. Here, we report that ubiquitin-specific peptidase USP20 deubiquitinates TRAF6 and Tax and suppresses interleukin 1β (IL-1β)- and Tax-induced NF-κB activation. Our results point to USP20 as a key negative regulator of Tax-induced NF-κB signaling.     

RNa-polymerase,type I: Activity in rat brain cell nuclei and peripheral blood mononuclear cells after Venezuelan Equine Encephalomyelitis virus infection

Rats inoculated intraperitoneally with 100 LD₅₀ of Venezuelan Equine Encephalomyelitis virus (VEE), showed a significant decrease of DNA-dependent RNA polymerase type I activity of brain nuclei of 29.7% and 59.3% at 24 and 48 hours after infection, respectively, while the animals had no clinical symptoms of illness. No alterations were observed in the nuclei of mononuclear cells at any time. VEE virus titer was higher in the serum than in the brain. The results suggest that viral infection produced a modification in the activity of this enzyme only in brain, even with a low amount of virus, and had no effect on enzyme activity of mononuclear cells.     

Isolation and characterization of a human T cell leukemia virus type II from a hemophilia-A patient with pancytopenia.

Human T cell leukemia virus (HTLV) type I has been isolated from the cultured T cells of several patients with adult T cell leukemia (ATL) and has been etiologically linked to ATL. However, HTLV-type II has been isolated only once, from the T cells of a patient with a T cell variant of hairy-cell leukemia. We report here the isolation of HTLV-II-related virus from the cultured T cells of a hemophilia-A patient with pancytopenia. The T cell line (CM) grows in the absence of T cell growth factor. Cord blood T cells were rapidly transformed when co-cultivated with irradiated CM cells. Heterologous competition radioimmunoassays using purified HTLV-I p24 showed the expression of HTLV-IIMO-related protein in these cells. Electron microscopy of the CM cells showed the presence of intracellular and extracellular type C viral particles. Comparison of the proviral genome in the CM cell line and the prototype HTLV-IIMO-containing cell line (MO) by molecular hybridization with probes specific for HTLV-IIMO indicated that restriction cleavage sites were identical. The fresh peripheral blood leukocytes of the patient contained two complete copies of the proviral genome, despite the lack of HTLV-II p24 expression. The virus from the cell line CM is designated as HTLV-IICM to distinguish it from the original HTLV-IIMO isolate.     

Binding sites for angiotensin II in human mononuclear leucocytes.

Angiotensin II binding sites were demonstrated in human mononuclear leucocytes by use of [125I]angiotensin II. The binding of [125I]angiotensin II to mononuclear leucocytes was rapid and reversible. The abilities of unlabeled compounds to displace [125I]angiotensin II were proportional to their abilities to displace labeled hormone in adrenal and smooth muscle membrane preparations. The Scatchard plot revealed two apparent orders of binding sites. The affinity constants were comparable with those for binding sites in other main target tissues of angiotensin II.     

Damaged DNA and miscounted chromosomes: human T cell leukemia virus type I tax oncoprotein and genetic lesions in transformed cells

Genetic instability is a recurring theme in human cancers. Although the molecular mechanisms mediating this effect commonly observed in transformed cells are not completely understood, it has been proposed to involve either the loss of DNA repair capabilities or the loss of chromosomal stability. The transforming retrovirus human T cell leukemia virus type I (HTLV-I) encodes a viral oncoprotein Tax, which is believed to cause the genomic instability characteristic of HTLV-I-infected cells. This review focuses on the ability of HTLV-I Tax to disrupt the cellular processes of DNA repair and chromosomal segregation. The consequences of these effects as well as the evolutionary advantage this may provide to HTLV-I are discussed.     

Recent advances in detection of human T-cell leukemia viruses type I and type II infection

The human T-cell leukemia viruses type I (HTLV-I) and type II (HTLV-II) have been implicated in the pathogenesis of a variety of neoplastic and neurological disorders. Classical techniques for detection involve assay of serum for antibodies by Western blotting or ELISA, which do not discriminate between infection with HTLV-I and HTLV-II. In order to provide appropriate prognostic information to infected individuals and to obtain an accurate assessment of the prevalence of both retroviruses in the United States, we and others have applied the technique of enzymatic DNA amplification to detect HTLV-I and HTLV-II. These techniques allow rapid detection of viral nucleic acids in freshly isolated peripheral blood samples. Recent studies indicate an unusually high rate of HTLV-II infection among seropositive individuals in a sampling of New Orleans intravenous drug users, indicating a need for combined serological and molecular genetic screening of high-risk populations.     

Human T cell leukemia virus type I induces DNA synthesis and immunoglobulin secretion in human lymphocyte cultures

Viral particles obtained from HTLV-I (human T cell leukemia virus, type I)-transformed T cell lines induced immunoglobulin production by normal peripheral blood lymphocytes. Conversely, no immunoglobulin could be detected in the supernatant medium in purified B cells cultivated with HTLV-I, suggesting that the presence of T cells is mandatory for HTLV-I to induce B cell polyclonal activation. The T cell help was mediated by soluble factors, as indicated in experiments showing that cell-free conditioned medium from T lymphocytes activated by HTLV-I was able to induce B cell proliferation and differentiation. Furthermore, a direct effect of HTLV-I on B cell proliferation was demonstrated when viral particles were added to purified B cells together with suboptimal doses of Staphylococcus aureus Cowan strain I (SAC). These observations show that an immediate early effect of HTLV-I infection was exerted on B cells, mainly in a T cell-dependent manner. Such an effect may account for the hypergammaglobulinemia observed in HTLV-I seropositive individuals, and in patients with HTLV-I-associated neurological disorders.