HIV-1 infection is facilitated in T cells by decreasing p56lck protein tyrosine kinase activity.

Several studies have suggested an important role for the protein tyrosine kinase p56lck (Lck) in HIV infection; however, the exact nature of this role remains unclear. Using a series of well characterized Jurkat-derived cell lines having a wide range of Lck kinase activity, our results showed that, while the entry of HIV-1 into these cell lines was similar, the kinetics of virus production by these cells were very different. Cells expressing a kinase-inactive Lck showed accelerated viral replication, whereas, cells expressing Lck with normal or elevated enzymatic activity showed a delay in virus replication that was proportional to the initial level of endogenous Lck activity. The cell line having the highest initial Lck kinase activity showed the slowest rate of productive HIV-1 infection. Analysis of 2-LTR circles revealed that this inhibitory effect of Lck was not due to inhibition of reverse transcription of HIV-1 genome or migration of the proviral DNA into the nuclei. This affect of Lck was confirmed in additional studies that used either the S1T cell line lacking completely Lck or where the Lck activity was altered in Jurkat cells prior to infection. S1T cells showed a 3- to 12-fold increase in the level of infection compared to Jurkat cells despite similar CD4 and chemokine coreceptor expression and cell doubling times. Pretreatment of Jurkat with an antisense lck oligodeoxynucleotide inhibited the synthesis of functional Lck and facilitated the viral replication by the cells as did expressing a dominant-negative mutant Lck which increased the productive infection>3-fold. Conversely, whereas IL-16 had no affect on productive infection in S1T cells that lack Lck, IL-16 pretreatment of Jurkat cells resulted in an immediate (within 5 min) and sustained and gradual (over 5 h) increase in Lck activity that resulted in a reduction of HIV-1 replication that paralleled the increasing Lck kinase activity. These results show that the enzymatic activity of Lck kinase can affect viral replication, that a lack of, or decreased Lck activity facilitates viral replication. Conversely, Lck can mediate a delay in HIV-1 infection that is proportional to the initial endogenous Lck enzyme activity.

Numerical modelling of the perturbation of HIV-1 during combination anti-retroviral therapy.

A competitive, chaos-free, implicit, finite-difference method is developed and used for a novel deterministic model for the perturbation of HIV by combination antiretroviral therapy. The compartmental model monitors the interaction between HIV and CD4(+) T cells, its principal target and site of replication in vivo, in the presence of reverse transcription inhibitors and protease inhibitors. The model exhibits two steady states, an uninfected (trivial) steady state (with no virus present) and an endemically infected state (with virus and infected T cells present). Stability and bifurcation analyses together with numerical simulations of the resulting dynamical system are reported.

Infrequent detection of human herpesvirus 6 DNA in peripheral blood mononuclear cells from multiple sclerosis patients.

Several studies have suggested an association between human herpesvirus 6 (HHV-6) infection and multiple sclerosis. As HHV-6 is predominantly a T-cell tropic virus, we examined the frequency of detection of HHV-6 genome in peripheral blood mononuclear cells from relapsing-remitting (n = 32) and chronic progressive (n = 14) patients and from healthy (n = 17) and neurological (n = 7) controls. Two sensitive polymerase chain reaction assays were used to target different regions within the HHV-6 genome. Depending on the polymerase chain reaction assay used, the detection of HHV-6 genome ranged from 11.7 to 23.5% (controls), 3.1 to 23.0% (relapsing-remitting), and 14.2 to 28.5% (chronic progressive). Although these observations do not exclude a pathogenic role for HHV-6 in multiple sclerosis, they indicate a lack of correlation between HHV-6 infection of peripheral blood mononuclear cells and the development of multiple sclerosis.

Activation-induced cell death in T cell hybridomas is due to apoptosis. Morphologic aspects and DNA fragmentation.

Some T cell hybridomas, upon activation via the TCR, rapidly undergo cell death. In this paper, we demonstrate that this activation-induced cell death (AICD) is accompanied by morphologic changes seen at the electron and light microscopy levels. The most striking changes are an extensive condensation of the chromatin and formation of membrane blebs. In addition to the morphologic changes, a significant portion of genomic DNA is broken at an interval of approximately 200 bp, producing a ladder of oligonucleosome-sized fragments after gel electrophoresis. Taken together, these observations indicate that AICD proceeds via apoptosis, or programmed cell death. This is additionally supported by the observation that AICD-associated phenomena are at least partially inhibited by cycloheximide or actinomycin D. Curiously, AICD and its associated DNA fragmentation are completely inhibited by aurintricarboxylic acid, a known nuclease inhibitor. The possible relationship between AICD in vitro, and the negative selection process (wherein selection may proceed via AICD of developing, autoreactive thymocytes) is discussed.

Cyclosporin A inhibits activation-induced cell death in T-cell hybridomas and thymocytes.

One mechanism by which the immune system develops the ability to discriminate self from nonself is the deletion of autoreactive T-cell clones during thymic maturation. The drug cyclosporin A (CsA) has been shown to interfere with this process, allowing the escape of normally 'forbidden' T-cell clones and the appearance of autoimmune disease. Recently, it has been demonstrated that immature thymocytes undergo programmed cell death (apoptosis) upon activation via the T-cell receptor. A similar phenomenon of activation-induced cell death (AICD) has been observed in T-cell hybridomas. Here we show that AICD in T-cell hybridomas in vitro and in thymocytes in vivo is blocked by CsA. Thus, clonal deletion may involve AICD when self-reactive, immature T cells are induced by self antigen, and CsA may cause autoimmunity by interfering with this process.

Specific inhibition of cell-surface T-cell receptor expression by antisense oligodeoxynucleotides and its effect on the production of an antigen-specific regulatory T-cell factor.

We have used antisense oligodeoxynucleotides corresponding to genes encoding the variable (V) region of the T-cell receptor (TCR) alpha and beta chains (V alpha and V beta) to control TCR expression in T-cell hybridomas. Two hybridomas, A1.1 and B1.1, recognize a synthetic polypeptide antigen designated poly 18 (poly[Glu-Tyr-Lys-(Glu-Tyr-Ala)5]) together with I-Ad. We have found that TCR function (production of lymphokines in response to antigen) and T3 expression were removed after protease treatment of the cells and were fully recovered 48 hr later. However, when antisense oligodeoxynucleotides corresponding to the appropriate TCR V genes were present after protease treatment, little or no recovery of TCR function or T3 expression was observed. This effect was specific for the TCR V genes utilized by the T cell: antisense oligodeoxynucleotides corresponding to the TCR V regions of A1.1 had no effect on TCR expression in B1.1 and vice versa. Thus, antisense oligodeoxynucleotides can be used to temporarily block expression of a TCR gene in a T-cell hybridoma. This technique was then applied to a paradoxical phenomenon in A1.1 cells. We had observed previously that A1.1 releases an antigen-specific immunoregulatory activity that shows the same antigenic fine specificity as is displayed by the TCR of A1.1. We now report that antisense oligodeoxynucleotides corresponding to the A1.1 V alpha gene blocked the production of this soluble antigen-specific activity by the cell. Antisense oligodeoxynucleotides corresponding to A1.1 V beta, on the other hand, had no effect on the production of this antigen-specific activity. We discuss these observations in the context of recent findings on the nature of T cell-derived antigen-specific regulatory factors.

Structural and evolutionary implications of the packaging of DNA for differentiation and proliferation in the lymphocyte.

During the differentiation of the clonally distributed lymphocytes of mouse and man into mature resting B and T cells, their DNA becomes tightly packed into dense heterochromatin masses and exhibits very little transcriptional activity; it also becomes extensively nicked, containing some 3000-4000 single-strand breaks per diploid genome. The nuclear matrix is sparse and poorly organized and there are but trace amounts of the matrix-linked enzyme DNA topoisomerase II; the nucleus of these small cells is surrounded by a thin rim of cytoplasm. The resting cell can thus be considered (by analogy to a sperm cell) as a vector for transporting tightly packed and relatively inert genetic information to all parts of the body. When the lymphocyte is stimulated to enter a proliferative cycle by binding of appropriately presented antigen or mitogen to relevant membrane receptors, the cell enlarges, due to increased synthesis of protein; the dense heterochromatin is pulled out into very small clumps, as a result of an enormous growth in size as well as complexity of the nuclear matrix, and a great increase in transcriptional activity occurs. We have identified four nuclear matrix antigens that are very widely conserved in the evolution of eucaryotes and that occupy distinctive domains in interphase nuclei. Of particular interest is antigen P1, detected in organisms ranging from algae to mammals. By virtue of its location at the interface between nuclear envelope and chromatin, we propose that it plays a major and evolutionarily conserved role in chromatin organization and orientation in all eucaryotic cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative decatenation assay for type II topoisomerases.

Type II topoisomerases catalyze decatenation of the catenated network of kinetoplast DNA [J. C. Marini, K. G. Miller, and P. T. Englund (1980) J. Biol. Chem. 255, 4976-4979]. The individual DNA circles and small catenanes produced during the decatenation reaction can be separated from the large network of substrate DNA by 5 min centrifugation at 13,000g and quantitated. The appearance of these decatenated DNA molecules which appear in the supernatant first showed a lag, whose duration depended on the enzyme concentration, and then increased linearly with time until it reached a plateau. The slope of the linear part of the kinetic curve was directly proportional to the enzyme concentration, whether a purified or crude preparation of type II topoisomerase from mammalian cells was used. These findings led us to a rapid quantitative assay of type II topoisomerases not involving electrophoresis. The method was developed with purified enzyme but was also useful for assay of the activity in crude extracts. Surprisingly, the type I topoisomerase, even when present in large excess, failed to decatenate the nicked DNA circles often present in the kinetoplast DNA. This renders the assay virtually free from interference by type I enzyme. The method is sensitive and allowed quantitative estimation of the enzyme activity present in the crude extracts corresponding to that derived from 500 to 700 cultured mammalian cells. Since various type II topoisomerases from procaryotic, eucaryotic, and viral sources decatenate kinetoplast DNA and generate similar DNA products, the assay method is likely to be generally applicable.

DNA topoisomerase I and II activities during cell proliferation and the cell cycle in cultured mouse embryo fibroblast (C3H 10T1/2) cells.

We have used C3H 10T1/2 cells to examine the regulation of topoisomerase activities during cell proliferation and the cell cycle. The specific activity of topoisomerase I was about 4-fold greater in proliferating (log phase) cells than in non-proliferating (confluent) cells. In synchronized cells, the bulk of the increased activity occurred during or just prior to S phase, depending upon the method of synchronization. A smaller increase in activity also occurred during G1 phase. The increase in activity during S phase was not altered by a hydroxyurea block at the G1/S phase boundary indicating that it is not directly coupled to DNA synthesis and is not the result of topoisomerase I gene dosage. The increase was inhibited by blocking cells at mid-G1 phase using isoleucine deprivation. Thus, the increase in activity during S phase is dependent on events occurring during mid- to late G1 phase. In contrast to the changes in topoisomerase I levels, the specific activity of topoisomerase II showed no detectable difference in proliferating vs non-proliferating cells. In addition, no detectable difference in topoisomerase II specific activity was seen in G1, S and M phases of the cell cycle. The differences in the activity profiles of the topoisomerases I and II during the cell cycle suggest that the two activities are regulated independently and may be required for different functions.