Fujisawa R, McAtee F J, Wehrly K, Portis J L
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840, USA.
J Virol. 1998 Jul;72(7):5619-25. doi: 10.1128/JVI.72.7.5619-5625.1998.
The tempo and intensity of retroviral neuropathogenesis are dependent on the capacity of the virus to invade the central nervous system. For murine leukemia viruses, an important determinant of neuroinvasiveness is the virus-encoded protein glycosylated Gag, the function of which in the virus life cycle is not known. While this protein is dispensable for virus replication, mutations which prevent its expression slow the spread of virus in vivo and restrict virus dissemination to the brain. To further explore the function of this protein, we compared two viruses, CasFrKP (KP) and CasFrKP41 (KP41), which differ dramatically in neurovirulence. KP expresses high early viremia titers, is neuroinvasive, and induces clinical neurologic disease in 100% of neonatally inoculated mice, with an incubation period of 18 to 23 days. In contrast, KP41 expresses early viremia titers 100- fold lower than those of KP, exhibits attenuated neuroinvasiveness, and induces clinical neurologic disease infrequently, with a relatively long incubation period. The genomes of these two viruses differ by only 10 nucleotides, resulting in differences at five residues, all located within the N-terminal cytoplasmic tail of glycosylated Gag. In this study, using KP as the parental virus, we systematically mutated each of the five amino acid residues to those of KP41 and found that substitution mutation of two membrane-proximal residues, E53 and L56, to K and P, respectively produced the greatest effect on early viremia kinetics and neurovirulence. These mutations disrupted the KP sequence E53FLL56, the leucine dipeptide of which suggests the possibility that it may represent a sorting signal for glycosylated Gag. Supporting this idea was the finding that alteration of this sequence motif increased the level of cell surface expression of the protein, which suggests that analysis of the intracellular trafficking of glycosylated Gag may provide further clues to its function.
逆转录病毒神经发病机制的节奏和强度取决于病毒侵入中枢神经系统的能力。对于鼠白血病病毒而言，神经侵袭性的一个重要决定因素是病毒编码的糖基化Gag蛋白，其在病毒生命周期中的功能尚不清楚。虽然这种蛋白对于病毒复制并非必需，但阻止其表达的突变会减缓病毒在体内的传播，并限制病毒向脑部的扩散。为了进一步探究这种蛋白的功能，我们比较了两种病毒，CasFrKP（KP）和CasFrKP41（KP41），它们在神经毒力上有显著差异。KP在早期病毒血症中表达的滴度很高，具有神经侵袭性，并且在100%新生接种的小鼠中诱发临床神经疾病，潜伏期为18至23天。相比之下，KP41表达的早期病毒血症滴度比KP低100倍，神经侵袭性减弱，很少诱发临床神经疾病，潜伏期相对较长。这两种病毒的基因组仅相差10个核苷酸，导致五个残基存在差异，所有这些残基都位于糖基化Gag的N端细胞质尾巴内。在本研究中，我们以KP作为亲本病毒，将五个氨基酸残基中的每一个系统地突变为KP41的相应残基，发现将两个靠近膜的残基E53和L56分别替换为K和P，对早期病毒血症动力学和神经毒力产生的影响最大。这些突变破坏了KP序列E53FLL56，其中的亮氨酸二肽表明它可能代表糖基化Gag的分选信号。支持这一观点的是，该序列基序的改变增加了该蛋白的细胞表面表达水平，这表明对糖基化Gag细胞内运输的分析可能为其功能提供进一步线索。
