Portis J L, Spangrude G J, McAtee F J
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840.
J Virol. 1994 Jun;68(6):3879-87. doi: 10.1128/JVI.68.6.3879-3887.1994.
Neonatal inoculation of the wild-mouse ecotropic retrovirus CasBrE (clone 15-1) causes a noninflammatory spongiform neurodegenerative disease with an incubation period of > or = 6 months. Introduction of sequences from Friend murine leukemia virus (clone FB29) into the genome of CasBrE results in a marked shortening of the incubation period. The FB29 sequences which influence the incubation period were previously localized to the 5' leader sequence of the viral genome (M. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 66:3298-3305, 1992). In the current study, we constructed a series of chimeric viruses consisting of the genome of CasBrE containing various segments of the leader sequence from FB29. A 41-nucleotide element (positions 481 through 521) near the 3' end of the leader was found to have a strong influence on the incubation period. This element influenced the kinetics of virus replication and/or spread in nonneuronal tissues, a property which was shown previously to determine the extent of central nervous system infection (M. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 66:3298-3305, 1992). Curiously, this sequence had no demonstrable effect on virus replication in vitro in a fibroblastic cell line from Mus dunni. This segment encodes 14 of the unique 88-amino-acid N terminus of pr75gag, the precursor of a glycosylated form of the gag polyprotein which is expressed at the cell surface. Previous in vitro studies of mutants of Moloney murine leukemia virus lacking expression of glycosylated Gag failed to reveal a function for this protein in virus replication. We mutated the Kozak consensus sequence around the initiation codon for this protein in the chimeric virus CasFrKP, a virus which induces neurologic disease with a short (18- to 23-day) incubation period. M. dunni cells infected with the mutants lacked detectable cell surface Gag, but, compared with CasFrKP, no effect on replication kinetics in vitro was observed. In contrast, there was a marked slowing of the replication kinetics in vivo and a dramatic attenuation of neurovirulence. These studies indicate that glycosylated Gag has an important function in virus replication and/or spread in the mouse and further suggest that the sequence of its N terminus is a critical, though likely indirect, determinant of neurovirulence.
新生野生小鼠嗜亲性逆转录病毒CasBrE（克隆15 - 1）接种可导致一种非炎性海绵状神经退行性疾病，潜伏期≥6个月。将Friend鼠白血病病毒（克隆FB29）的序列引入CasBrE基因组可显著缩短潜伏期。先前已将影响潜伏期的FB29序列定位到病毒基因组的5'前导序列（M. Czub、F. J. McAtee和J. L. Portis，《病毒学杂志》66:3298 - 3305，1992年）。在本研究中，我们构建了一系列嵌合病毒，其由含有来自FB29前导序列不同片段的CasBrE基因组组成。发现前导序列3'端附近一个41个核苷酸的元件（第481至521位）对潜伏期有强烈影响。该元件影响病毒在非神经组织中的复制和/或传播动力学，先前已证明这一特性可决定中枢神经系统感染的程度（M. Czub、F. J. McAtee和J. L. Portis，《病毒学杂志》66:3298 - 3305，1992年）。奇怪的是，该序列在来自邓氏小鼠的成纤维细胞系中对体外病毒复制没有明显影响。该片段编码pr75gag独特的88个氨基酸N端中的14个氨基酸，pr75gag是在细胞表面表达的糖基化形式的gag多蛋白的前体。先前对缺乏糖基化Gag表达的莫洛尼鼠白血病病毒突变体的体外研究未能揭示该蛋白在病毒复制中的功能。我们在嵌合病毒CasFrKP（一种潜伏期短（18至23天）可诱发神经疾病的病毒）中，对该蛋白起始密码子周围的科扎克共有序列进行了突变。感染突变体的邓氏小鼠细胞缺乏可检测到的细胞表面Gag，但与CasFrKP相比，未观察到对体外复制动力学有影响。相反，体内复制动力学明显减慢，神经毒力显著减弱。这些研究表明，糖基化Gag在病毒在小鼠体内的复制和/或传播中具有重要功能，并进一步表明其N端序列是神经毒力的关键决定因素，尽管可能是间接的。
