Ultrastructural study of type C virus particles in phytohemagglutinin-stimulated lymphocytes from healthy adults seropositive to adult T-cell leukemia-associated antigens

PHA-stimulated peripheral lymphocytes from six healthy adults consisting of four family members of an adult T-cell leukemia (ATL) patient and two blood bank donors, seropositive to ATL-associated antigens (ATLA), were all positive for the expression of ATLA and ATL-associated virus (ATLV). These results revealed that anti-ATLA-positive persons were healthy carriers of ATLV and that the numbers of ATLV observed in each culture were more proportional to the percentage of ATLA-positive lymphocytes than anti-ATLA titers of each person. Virus particles detected were C-type in morphology, and essentially similar to those observed in MT-2 and other ATL-related cells, but rather uniform in size, mostly 120-130 nm in diameter. Furthermore, it is of interest that tubuloreticular structures and striated fusiform structures, which were found in fresh or short term cultured ATL cells, were observed in some cultures of lymphocytes from anti-ATLA-positive healthy persons.     

Immunoelectron microscopic study on the cross-reactivity of antibodies to adult T-cell leukemia-associated antigens in human and monkey sera

The cross-reactivity of antibodies to adult T-cell leukemia (ATL)-associated antigens (ATLA) in human and monkey sera was investigated by indirect immunoperoxidase and immunoferritin methods using a human cell line (MT-2) carrying a type C virus (HTLV), two monkey cell lines (Si-1 and Si-3) carrying HTLV, and a monkey cell line (Si-2) carrying a type C virus isolated from an anti-ATLA-positive monkey. Anti-ATLA-positive but not-negative human and monkey sera gave positive immunoperoxidase reaction with all four virus-positive cell lines when studied by light microscopy. Electron microscopic findings revealed ferritin or peroxidase labeling of virus particles and plasma membranes of these four cell lines with antibody-positive but not-negative human and monkey sera. These results clearly indicate the cross-reactivity of anti-ATLA antibodies in human and monkey sera at light and electron microscopic levels, and the presence of antigenic determinants common to the surface of type C virus particles of human and monkey origin.     

Entry of adult T-cell leukemia-associated virus into human peripheral blood leukocytes

Summary Within 10 minutes after tritium-labeled adult T-cell leukemia-associated virus (ATLV) inoculation, silver grains were found over human lymphocytes. At the time of entry of ATLV, the viral envelope was observed to fuse with the cell membrane.With 3 Figures     

Seropositivity of a blood recipient from a donor with positive adult T-cell leukemia-associated antigens

A blood recipient, aged 66, was found to have positive adult T-cell leukemia-associated antigens (ATLA), approximately half a year after a transfusion. The donor's ATLA-antibody titer was 1: 640. Routine screening of blood donors for ATLA antibody was proposed.     

Ultrastructural study on type C virus particles in a human cord T-cell line established by co-cultivation with adult T-cell leukemia cells

Summary A human cord T-cell line etablished by co-cultivation with adult T-cell leukemia cells contained numerous type C virus particles, which were mostly mature, with scarce budding. Negative staining showed no surface spikes on their surface.With 4 Figures     

Detection of HLA-D-region-associated antigens on the surface of adult T-cell leukemia virus particles by immunoelectron microscopy

To search for lymphocyte marker antigens on the surface of human T-cell leukemia virus (HTLV), an immunoelectron microscopic study was performed on a HTLV-producing human T-cell line, MT-2, using monoclonal antibodies, such as anti-Leu-1, -Leu-2b, -Leu-3a, -Leu-5, -Leu-10 and -HLA-DR and OKIal. The reactivity of each antibody with MT-2 cells was tested by the immunoperoxidase method at the light microscopic level. OKIal, anti-HLA-DR and -Leu-10 gave positive results. At the ultrastructural level, the surface of HTLV as well as the plasma membranes of MT-2 cells were labeled with ferritin by the monoclonal antibodies OKIal, anti-HLA-DR and -Leu-10, but not by anti-Leu-1 and -Leu-3a. These findings suggest that HLA-D region -associated antigens are common antigenic determinants shared by the surface of HTLV and the plasma membranes of MT-2 cells. These antigens on the virus surface are probably picked up selectively from the plasma membranes and may play an important role in the interaction of HTLV and target T-cells.     

Chromosome studies of murine T-cell lymphoid leukemia and derived cell lines.

Several cell lines were previously established from a spontaneous murine T-cell leukemia (LB). The aim of this study was to analyze the G- and C-banded karyotypes of the parental LB tumor cells and the derived cell lines. A sensitive cell line (LBL) from which two sublines originated, as well as Vincristine (LBR-V160) and Doxorubicin (LBR-D160) resistant cell lines, were used. Our results showed that LB cells had a pseudo-diploid karyotype with 40 acrocentric chromosomes in which trisomy of chromosome 14 was the most relevant alteration. The sensitive cell line showed this alteration in all metaphases studied; no changes in karyotypes were observed in either subline, despite their dissimilar morphology and growth patterns. In contrast, both resistant lines displayed a more heterogeneous karyotype with no common markers, except for the finding that chromosome 5 was involved in a trisomy in LBR-V160 and in a translocation with chromosome 12 in LBR-D160. Taking into account that the mdr genes are located in chromosome 5, these results suggest a possible association between such alterations and the acquisition of drug resistance.     

Expression of human T-cell leukaemia virus type I and associated antigens,and interleukin-2 and receptor in lymph nodes of adult T-cell leukaemia/lymphoma

To examine the relationship between the expression of human T-cell leukaemia virus type (HTLV-I) mRNA and associated antigens and clinicopathological features, we studied 31 lymph nodes of patients with adult T-cell leukaemia/lymphoma (ATLL) and related diseases, using in situ hybridization and immunohistochemistry. We classified the patients into four types on the basis of their clinicopathological features (HTLV-I associated lymphadenitis, incipient ATLL, ATLL with complete HTLV-I provirus, and ATLL with defective HTLV-I provirus. The expression of HTLV-I mRNA was detected in all 3 patients with incipient ATLL, in 5 of 10 patients with defective-provirus ATLL, in 5 of 11 patients with complete-provirus ATLL, and 3 of 7 with HTLV-I associated lymphadenitis, but the amounts were very small; approximately 1 in 10000–200000 lymph node cells express the viral genomes. This suggests that expression of viral genomes may not be important for immortalization, but it is important that to note the capacity for HTLV-I infection is preserved in each group of non-neoplastic and neoplastic states. HTLV-I mRNA was detected only in lymphocytes and/or lymphoma cells, but the HTLV-I associated antigens (env, gag and pX) were found in histiocytes and endothelial cells, as well as in lymphocytes and/or lymphoma cells. Anti-interleukin 2 receptor (IL-2R) antibody reacted with the giant cells of incipient ATLL and with the transformed lymphocytes and immunoblast-like cells of the HTLV-I-associated lymphadenitis but not with the lymphocytes in the background. Of the typical ATLL, IL-2R was found in both lymphoma cells and giant cells. IL-2 was rarely detected.     

Immunocytochemical and ultrastructural characterization of human T-lymphotropic virus type I (HTLV-I)-producing rabbit lymphoid cell lines

Summary Fine structural and immunocytochemical characterization of rabbit lymphoid cell lines transformed by human T-lymphotropic virus type I (HTLV-I) was carried out. All nine cell lines tested were reactive with anti-HTLV-I-positive human, monkey, and rabbit sera and monoclonal antibody to HTLV-Ip 19, but not with anti-HTLV-I-negative sera and monoclonal antibodies to human Ia and pan-T antigens. All cell lines were strongly positive for monoclonal antibodies to rabbit Ia and pan-T antigens. Ultrastructurally, each cell line contained C-type virus particles in varying numbers in the extracellular space. These particles showed replication patterns similar to those in HTLV-I or simian T-lymphotropic virus type I (STLV-I)-producing human or monkey cells. In addition, anti-HTLV-I-positive rabbit serum gave positive immunoreactivity to HTLV-I or STLV-I by indirect immunoferritin method.These results indicate that the ultramorphology and replication patterns of HTLV-I in rabbit cell lines are indistinguishable from those of HTLV-I in human and monkey cell lines, HTLV-I in rabbit cells shares the common surface antigenic determinants with HTLV-I or STLV-I in human or monkey cells, and that these cells are definitely rabbit T cells bearing their own Ia antigens.     

Canine distemper virus in primary and continuous cell lines of human and monkey origin

Summary Canine distemper virus produced characteristic cytopathic effects in primary and continuous cell cultures of human and monkey origin. The importance of selection of variants for successful adaptation to a given cell type was indicated by the marked differences in growth and CPE demonstrated by CDV lines of varying background. The Onderstepoort strain which had previously been passaged in ferret kidney cell cultures readily induced lesions in the AV3 human amnion cell line and virus yields of 10⁵ to 10⁶ TCD₅₀ per ml. were obtained. On serial passage in AV3 cells, a rapid CPE variant of this strain was obtained which induced lesions within 24 hrs. of inoculation. A chick embryo cell culture passaged line of the Onderstepoort strain also induced cytopathic effects in AV3 cells. However, the lesions developed slower and were not as extensive as those obtained with the ferret kidney propagated virus. The following additional cell types were also sensitive to canine distemper virus: primary human amnion, human fibroblasts, HeLa cells, primary rhesus and tamarin kidney cells, and continuous cell lines derived from rhesus and green monkeys. The Rockborn and 4856 strains of canine distemper also produced cytopathic effects in one or more of the above cell types. The lesions induced by canine distemper virus were compared to those produced by measles virus in a number of the above cell types. In all cases, similar, if not identical cytopathic effects were observed in a given cell type. The two viruses produced a similar type of cytopathic effect in human fibroblast cells which differed from that induced in epithelial cells.This investigation was supported in part by Public Health Service research grants (AI-03873 and AI-02396) from the National Institute of Allergy and Infectious Diseases.This investigation was supported in part by a Public Health Service research career program award (AI-K6-1136) from the National Institute of Allergy and Infectious Diseases.     

Atomic force microscopy investigation of Mason-Pfizer monkey virus and human immunodeficiency virus type 1 reassembled particles

Particles of DeltaProCANC, a fusion of capsid (CA) and nucleocapsid (NC) protein of Mason-Pfizer monkey virus (M-PMV), which lacks the amino terminal proline, were reassembled in vitro and visualized by atomic force microscopy (AFM). The particles, of 83-84 nm diameter, exhibited ordered domains based on trigonal arrays of prominent rings with center to center distances of 8.7 nm. Imperfect closure of the lattice on the spherical surface was affected by formation of discontinuities. The lattice is consistent only with plane group p3 where one molecule is shared between contiguous rings. There are no pentameric clusters nor evidence that the particles are icosahedral. Tubular structures were also reassembled, in vitro, from two HIV fusion proteins, DeltaProCANC and CANC. The tubes were uniform in diameter, 40 nm, but varied in length to a maximum of 600 nm. They exhibited left handed helical symmetry based on a p6 hexagonal net. The organization of HIV fusion proteins in the tubes is significantly different than for the protein units in the particles of M-PMV DeltaProCANC.     

Characterization of human T-cell lines harboring defective human immunodeficiency virus type 1

Defective HIV-producing T-cell lines were subcloned from MT-4/HIV_HTLV-IIIB MOLT-4/HIV_HTLV-IIIB, and H9/HIV_HTLV-IIIB cell lines chronically infected with HIV. The NY-M10 cell line derived from MOLT-4/HIV_HTLV-IIIB and the NY-H6 cell line derived from H9/HIV_HTLV-IIIB produce defective HIV, which lacks the ability to infect human T-cell lines. NY-M10 cells retain the capacity to form multinucleated giant cells in cocultivation with HIV-uninfected CD4-positive cells. However, NY-H6 cells failed to fuse with CD4-positive cells. Electron microscopic analysis indicated that the defective HIV produced from NY-M10, like those reported previously, lacked the structure of the nucleocapsid, and the virion released from NY-H6 was indistinguishable from those of authentic HIV particles. Southern and Northern blotting analyses of NY-M10 and NY-H6 cleared that the genome of those defective viruses was not significantly deleted, suggesting minor mutation(s) should take place on the viral genome.     

Spontaneous interferon production and Epstein-Barr virus antigen expression in human lymphoid cell lines.

Established human lymphoid cell lines, many of which spontaneously produce interferon, differ in the efficiency by which they allow expression of Epstein-Barr virus (EBV) lytic functions. Six EBV carrying lymphoid cell lines, selected to either be extremely susceptible or very refractory to EBV superinfection, were tested for spontaneous interferon production. Only the three cell lines which were poorly superinfectable with EBV were found to produce interferon. These same three lines could not be induced to express EBV-specific early antigens from intrinsic EBV genomes. It is suggested that interferon acts as a negative control factor affecting a cell's susceptibility to EBV.