Induction of endogenous mammary tumor virus in lymphocytes infected with murine acquired immunodeficiency syndrome virus.

Mice infected with murine acquired immunodeficiency syndrome (MAIDS) virus developed lymphoadenopathy and profound immunodeficiency. Concomitantly the expression of endogenous mammary tumor virus (MTV) mRNA increased significantly, especially for the 1.7-kb 3' open reading frame (ORF) mRNA encoding MTV superantigen. B cell lines that are established from MAIDS mice and exhibit superantigen activity also express a high level of 1.7-kb endogenous MTV and mRNA. Infection of a B cell tumor line in vitro with retrovirus containing the cloned MAIDS virus gene induced superantigen activity and this cell line also expressed the 1.7-kb superantigen coding MTV 3' ORF mRNA. These results strongly suggest a link between MAIDS virus infection and the induction of endogenous superantigen activity. This may play an important role in the pathogenesis of the MAIDS virus.

Sequence heterogeneity of murine acquired immunodeficiency syndrome virus: the role of endogenous virus.

A defective murine leukemia virus is the causative agent of murine acquired immunodeficiency syndrome (MAIDS). We have cloned cDNAs from both virus infected and non-infected cells using the PCR methods with primers corresponding to the franking sequence of the unique p12 gag gene. Sequence analysis of these cDNA clones revealed: (i) the presence of endogenous virus related to MAIDS virus in C57BL/6 mice, (ii) B cell lineage specific expression of endogenous virus and (iii) extensive heterogeneity of MAIDS virus recovered from virus infected cells due to the recombination of the related viruses (defective pathogenic virus, ecotropic virus and endogenous virus). These findings suggest that the creation of virus variants in infected cells may play an important role in virus pathogenesis and escape from immune attack during the development of MAIDS.

Functional and phenotypic change of T cells in murine acquired immune deficiency.

Infection of susceptible C57BL/6 mice with defective LP-BM5 murine leukemia virus causes disease termed murine acquired immune deficiency syndrome (MAIDS). The disease is characterized by lymphoadenopathy, hyperimmunoglobulinemia, and immune deficiency in both T and B cell functions. The development of disease requires the presence of mature T cells, especially CD4 T cells, and B cells. It has previously been shown that a B cell tumor line derived from MAIDS mouse stimulated a large fraction of unprimed T cells based on TCR V beta chain expression. This stimulatory activity was assumed to be mediated by a superantigen encoded by MAIDS virus. The stimulation of T cells by viral superantigen was thought to play a role in the development of the disease. To examine the role of T cell reactivity to MAIDS superantigen, we used TCR transgenic mice. There are two distinct T cell populations which can be distinguished based on their TCR expression and function in the TCR transgenic mice, one bearing the transgene derived alpha- and beta-chain TCR that is nonreactive to MAIDS superantigen and the other bearing an endogenous alpha- but transgene-derived beta-chain TCR that is reactive to superantigen. Unlike T cells found in noninfected TCR transgenic mice, anergic T cells expanding in virally infected TCR transgenic mice are homogeneous for the TCR phenotype, indicating the presence of a selection of T cells based on their TCR expression. T cell hybridomas established by fusing T cells from virus-infected transgenic mice to thymoma cell line are also anergic. We found mRNA of defective LP-BM5 virus in a majority of T cell hybridomas from virus-infected mice but not from noninfected mice. By using in vitro infection of T cell clones with recombinant virus containing LP-BM5 MAIDS virus gag gene, we have demonstrated that virus infection directly abrogated the Ag-specific reactivity of T cells. The establishment of anergic T cell hybridomas and the in vitro infection of T cells with recombinant viruses would be a useful tool in the analysis of biochemical and molecular mechanisms of T cell dysfunction in MAIDS.

Resistance of mice deficient in IL-4 to retrovirus-induced immunodeficiency syndrome (MAIDS)

The murine acquired immunodeficiency syndrome (MAIDS) is induced by a defective murine leukemia virus and has many symptoms similar to those found in patients infected with the human immunodeficiency virus. The presence of both B cells and CD4+ T cells is critical for the development of the disease. Furthermore, a Th2 cytokine response dominates during the progression of the disease. When interleukin-4 (IL-4)-deficient mice that are defective in Th2 cytokine responses were infected, there was no lethality, and the development of the T cell abnormalities associated with MAIDS was delayed. These data suggest that IL-4 or a Th2 response is involved in the development of retrovirus-induced immunodeficiency in mice.

Fully methylated oriC with negative superhelicity forms an oriC-membrane complex before initiation of chromosome replication.

In an in vitro assay, the oriC DNA has been shown to bind to the outer membrane fraction only when it is hemimethylated (G.B. Ogden et al., Cell, 54, 127-135,1988). In this report, however, we demonstrated that a significant amount of the oriC DNA was recovered from the cells just before initiation with the oriC DNA being fully methylated. Formation of this preinitiation oriC-membrane complex and following initiation of chromosome replication were strongly inhibited by novobiocin, a DNA gyrase B subunit inhibitor, which reduced the superhelicity of the reporter plasmid in the cells. On the other hand, both reactions proceeded in the presence of nalidixic acid, a DNA gyrase A subunit inhibitor, which did not have the effect of reducing the superhelicity. These results suggest that the negative superhelicity of the DNA is required for preinitiation oriC-membrane complex formation and following initiation event of replication.

Only oriC and its flanking region are recovered from the complex formed at the time of initiation of chromosome replication in Escherichia coli.

The oriC region of Escherichia coli constructs a specific complex to associate with the outer membrane fraction (oriC complex). The oriC complex was periodically formed before as well as in the short period after initiation of DNA replication. Using the DNA extracted from outer membrane fractions of the cells just after initiation as a probe, the whole E. coli genomic library was assayed by plaque hybridization. DNA regions that hybridized with the probe corresponded to about a 100-kb length of chromosome DNA which included oriC. In addition, clones located in a counterclockwise direction from oriC were more preferentially hybridized among these positive clones. Thus, we conclude that the oriC region is a unique locus of the chromosome that binds to the outer membrane at the time of initiation of chromosome replication.

Periodic formation of the oriC complex of Escherichia coli.

We examined formation of an oriC-membrane complex through the chromosome replication cycle by dot-blot hybridization using an oriC plasmid as a probe. In a wild-type culture synchronized for chromosome replication, oriC complex formation was observed periodically and transiently corresponding to the replication initiation event. Prior to initiation of replication the oriC complex was recovered in the outer membrane fraction as well as at the time of initiation of replication. Moreover, periodic formation of the oriC complex was observed even when further initiation of replication was suppressed by culturing an initiation ts mutant at the restrictive temperature. Similar periodic formation of the oriC complex was also observed when DNA elongation was inhibited by addition of nalidixic acid to the culture. However, the second periodic peak did not appear when rifampicin or chloramphenicol was added. Cells which formed the oriC complex at the restrictive temperature could immediately initiate chromosome replication when the cells were transferred to the permissive temperature. We conclude that the oriC region of Escherichia coli forms a specific complex periodically just before and at the time of initiation of chromosome replication and that oriC complex formation is a prerequisite for initiation of chromosome replication.