REPLICATION AND XC-FUSION DEFICIENCY OF ENDOGENOUS ECOTROPIC C3H/HE PROVIRUS

这些差异的分子基础已经被确定。 内源性生态型小鼠的感染性和Xc表型 低白血病小鼠株C3H/He的白血病病毒(MuLV)； 其在高白血病小鼠品系AKR中的亲缘关系； 体外筛选出具有感染性、XC阳性的C3H病毒变异株。 碘脱氧尿嘧啶核苷诱导的内源性生态型C型病毒 未转化的C3H/10T1/2细胞系为XC阴性 复制不足。相比之下，病毒在晚些时候产生 化学转化C3H/10T1/2细胞中碘脱氧尿苷的诱导 细胞(MCA5)呈XC阳性，具有感染性。XC阴性病毒 在某些情况下生长后可转化为XC阳性病毒 转化的细胞系。我们已经克隆了内源生态型 从MCA5细胞中分离出C3H/He前病毒，XC阴性， 复制缺陷，以及两个XC阳性的C3H前病毒 通过体外转化获得。核苷酸测序已建立 XC阴性的C3Hp110整合在R 内源性VL30长末端重复序列的反向区域 定向，不同于感染性AKR p623前病毒 通过点突变将Lys取代潜在的Arg Gp70和p15E的前体裂解位点。体外转化的 XC阳性的C3H前病毒克隆C1 3211和4211具有Arg at 因此，这个位点和正常的切割位点在这些 克隆人。我们已经在蛋白水解物中将赖氨酸残基改变为Arg 通过定点突变p110的裂解位点，我们已经 重建了前病毒。来自此结构的DNA，在 转染，产生XC阳性，复制能力强 普罗鲁斯。因此，我们已经确定了单点突变 在包膜前体蛋白gp85的加工位置是 传染性和XC表型差异的原因 从C3H/He和AKR小鼠体内分离到内源性亲生小鼠病毒，并 体外转化的基础是这个位点的突变。

The molecular basis has been determined for differences in the infectivity and XC phenotype of the endogenous ecotropic murine leukemia virus (MuLV) of the low leukemia mouse strain, C3H/He; its relative in the high leukemia mouse strain AKR; and highly infectious, XC-positive C3H virus variants selected in vitro. Endogenous ecotropic type C virus induced by iododeoxyuridine from the nontransformed C3H/10T1/2 cell line is XC negative and replication deficient. In contrast, viruses produced late after iododeoxyuridine induction in chemically transformed C3H/10T1/2 cells (MCA5) are XC positive and infectious. XC-negative viruses can be converted to XC-positive viruses upon growth in certain transformed cell lines. We have cloned the endogenous ecotropic provirus of C3H/He from MCA5 cells, which is XC negative and replication deficient, as well as two XC-positive C3H proviruses derived by in vitro conversion. Nucleotide sequencing established that the XC-negative C3H p110 was integrated within the R region of an endogenous VL30 long terminal repeat in reverse orientation, and differed from the infectious AKR p623 provirus by a point mutation substituting Lys for Arg at the potential precursor cleavage site for gp70 and p15E. The in vitro-converted XC-positive C3H proviral clones, C1 3211 and 4211, have Arg at this site and the normal cleavage site is thus regenerated in these clones. We have altered the Lys residue to Arg at the proteolytic cleavage site of p110 by site-directed mutagenesis and we have reconstructed the provirus. DNA from this construct, upon transfection, gave rise to XC-positive, replication-competent provirus. Thus, we have established that a single point mutation at the processing site of the envelope precursor protein, gp85, is responsible for the difference in the infectivity and XC phenotype of endogenous ecotropic MuLV from C3H/He and AKR mice, and that the basis for in vitro conversion is a mutation at this site.