In vitro synthesis of infectious DNA of murine leukaemia virus.

DNA synthesised in vitro by purified virions of murine leukaemia virus is infectious. Neither RNA nor protein is required for infectivity. Transfection with reverse trancriptase product shows a single-hit dose response and results in the production of complete, infectious virus.     

Abelson murine leukaemia virus protein is phosphorylated in vitro to form phosphotyrosine

The Abelson murine leukaemia virus protein (P120) can become phosphorylated in vitro by [gamma-32P]ATP. The protein has been purified from cell membranes to the point that in specific conditions virtually all of the incorporated 32P is in P120. The reaction is stimulated by Mn2+ and Mg2+ but not Ca2+ and is very rapid even at 0 degrees C. The phosphate is linked to P120 at tyrosine, a linkage not previously reported for a phosphorylation reaction. Phosphorylation may be involved in the transforming activity of viruses that cause leukaemia as well as sarcomas.     

The transforming gene of Moloney murine sarcoma virus

A cleavage map of the Moloney murine sarcoma viral DNA was constructed and compared with that of a spontaneously occurring deletion mutant. By restriction enzyme analysis, it was shown that a region encompassing over 40% of the viral information was not essential for transformation or rescue of the deletion mutant. The transforming region was further localised by analysis of the transforming activity in tissue culture of isolated restriction fragments of linear duoble-stranded sarcoma viral DNA. In each case, DNA fragments that retained transforming activity preserved the cell-derived insertion sequences of the viral genome. Moreover, such transformants invariably expressed RNA specific to this region. By these two approaches, it was possible to demonstrate that the transforming region of the viral genome begins very near or within the cell-derived insertion sequences. Thus, the transforming gene of this mammalian sarcoma virus originates from within the mouse cell genome.     

Isolation of a replication-defective murine leukaemia virus from cultured AKR leukaemia cells.

Mice of the AKR strain are characterised by a high incidence of spontaneous thymic lymphomas. AKR chromosomes contain the genomes of ecotropic murine leukaemia virus (MuLV) at two loci, termed Akv-1 and Akv-2 (refs 2-6). Shortly after birth, the normal tissues of AKR mice begin to produce high levels of this XC-positive MuLV (ref. 7) (that is, one that forms XC plaques). A second class of MuLV, termed mink cell focus-inducing virus (MCF), is produced specifically by preleukaemic and leukaemic AKR thymocytes. Nowinski et al. have established a series of tissue culture lines from AKR leukaemias and reported that the resulting cell lines produce virus particles, but that these particles, surprisingly, do not give rise to XC plaques. We have analysed the virus particles produced by one of these cell lines, termed AKRSL2. We show here that, unlike most or all of the nonmalignant tissues in the AKR mouse, these cultured lymphoma cells produce very little non-defective ecotropic MuLV; however, they do produce replication-defective ecotropic MuLV.     

Mature murine B lymphocytes immortalized by Kirsten sarcoma virus

Clonal, antigen-specific, functionally responsive cell populations have proved critical for the analysis of the activation and regulation of lymphocytes. Such studies with B lymphocytes, the precursors of antibody-secreting cells, are hampered by the difficulty in generating phenotypically mature, antigen-reactive lines from defined cell populations. One method is to use acutely transforming retroviruses, which can transform B-lineage lymphocytes in vitro. However, Abelson murine leukaemia virus (A-MuLV) infection of murine bone marrow cells in vitro yields mostly immature B-cell lines, and infection of murine bone marrow cells with murine sarcoma viruses carrying ras related genes produces only immature lymphoid cell lines. Retroviruses which contain ras can immortalize nonlymphoid cells without causing loss of mature phenotypic characteristics. We used ras-containing Kirsten sarcoma virus (KiSV) pseudotyped with an amphotropic MuLV helper virus, to infect a purified population of mature, hapten-binding murine splenic B lymphocytes, aiming to generate mature B-cell lines to use as models for the study of B-cell growth and differentiation physiology. Immortalized B-cell lines which retain the same mature phenotype as the starting population, including hapten-specific binding, were produced. This is the first demonstration of a method for immortalizing selected antigen-binding B lymphocytes, and the first example of immortalization of mature B cells in vitro with an acutely transforming ras-containing retrovirus.     

清开灵注射液体外抑制HIV-1作用

目的:研究清开灵注射液(QKL)体外抑制HIV-1的作用.方法:用MTT比色法检测QKL对H9/HIV-1ⅢB细胞(慢性感染HIV-1ⅢB的H9细胞)和MT-2细胞的毒性;采用荧光染料Calcein-AM标记的H9/HIV-1ⅢB细胞和系列稀释的QKL作用后.与MT-2细胞混合培养,2 h后在荧光下计数融合细胞数目,评价QKL对两种细胞早期融合的影响;用MTT法检测QKL对HIV-1ⅢB急性感染的MT-2细胞的保护作用;用HIVp24抗原试剂盒检测HIV-1ⅢB感染的MT-2细胞的培养上清p24抗原的含量,分析QKL对HIV-1复制的影响.结果:QKL对H9/HIV-1ⅢB细胞和MT-2细胞的CC50分别为1/50.76和1/36.97;抑制H9/HIV-1ⅢB细胞和MT-2细胞早期融合作用的EC50=1/235.29,SI=6.36;对HIV-1ⅢB感染的MT-2细胞保护作用的EC50=1/144.93,SI=3.92;抑制p24抗原产生作用的EC50=1/175.44,SI=4.75.结论:QKL体外有抗HIV-1活性,作用机制可能是多靶点的,可抑制病毒进入细胞和胞内复制.     

Severe immunodeficiency disease induced by a defective murine leukaemia virus

Different classes of retroviruses have been shown to induce immunodeficiency diseases in various animal species. These animal models may provide an insight into our understanding of AIDS but, with the exception of one strain of feline leukaemia virus, the determinants of pathogenicity have not yet been mapped to these viral genomes. The immunodeficiency-inducing feline leukaemia virus is replication-defective, harbouring the determinant of pathogenicity within its env sequences. We have studied the Duplan strain of murine leukaemia virus which induces, in C57BL/6 mice, a severe immunodeficiency disease with striking similarities to human AIDS. We have identified the aetiological agent of this murine immunodeficiency disease as another defective retrovirus, with a genome of 4.8 kilobases. Molecular cloning and sequencing of this DNA showed that the pol and env genes have been deleted, but that the complete gag region has been conserved and has a novel sequence encoding the p12 protein. As with the feline leukaemia virus, these results provide evidence for the role of defective retroviruses in inducing immunodeficiency and facilitate the study of the mechanisms underlying the pathogenesis of retrovirus-induced immunodeficiency syndromes, including AIDS.