Hout David R, Gomez Lisa M, Pacyniak Erik, Miller Jean-Marie, Hill M Sarah, Stephens Edward B
Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, 66160, USA.
Virology. 2006 May 10;348(2):449-61. doi: 10.1016/j.virol.2005.12.025. Epub 2006 Feb 3.
Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIV(KU-1bMC33) in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIV(M2)) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M., Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIV(KU-1bMC33)) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIV(VpuA19H) replicated with similar kinetics as the parental SHIV(KU-1bMC33) and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIV(KU-1bMC33). This SHIV(VpuA19H) virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIV(M2). Electron microscopic examination of SHIV(VpuA19H)-infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIV(M2)-infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel with the alteration of one amino acid and provide additional evidence that drugs targeting the Vpu TM/ion channel can be effective anti-HIV-1 drugs.
我们实验室之前的研究表明，人类免疫缺陷病毒1型（HIV-1）的Vpu蛋白的跨膜结构域（TM）在猕猴体内对猿猴免疫缺陷病毒（SHIV(KU-1bMC33)）的发病机制有影响，并且Vpu的TM结构域可以被甲型流感病毒的M2蛋白病毒孔蛋白取代。最近，我们发现用甲型流感病毒的M2蛋白的TM结构域替换Vpu的TM结构域后产生了一种对金刚烷胺敏感的病毒（SHIV(M2)）[Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M., Powers, M.F., Wong, S.W., Stephens, E.B., 2006. 用甲型流感病毒M2的跨膜结构域替换猿猴免疫缺陷病毒（SHIV(KU-1bMC33)）中Vpu的跨膜结构域，产生一种对M2离子通道抑制剂敏感且对食蟹猴有致病性的病毒。病毒学344, 541 - 558]。基于之前对M2蛋白的研究表明M2内的His-X-X-X-Trp基序对M2质子通道的功能至关重要，我们构建了一种新型的SHIV，其中TM结构域第19位的丙氨酸被组氨酸残基取代，形成了His-Ile-Leu-Val-Trp基序。SHIV(VpuA19H)的复制动力学与亲本SHIV(KU-1bMC33)相似，脉冲追踪分析表明病毒蛋白的加工过程与SHIV(KU-1bMC33)相似。发现这种SHIV(VpuA19H)病毒比SHIV(M2)对M2离子通道阻滞剂金刚烷胺更敏感。对用金刚烷胺处理的SHIV(VpuA19H)感染细胞进行电子显微镜检查发现，病毒颗粒在细胞表面和细胞内囊泡中积累，这与之前观察到的用金刚烷胺处理的SHIV(M2)感染细胞的情况相似。这些数据表明，HIV-1的Vpu蛋白通过改变一个氨基酸就可以转化为对金刚烷胺敏感的离子通道，并提供了额外的证据，证明靶向Vpu TM/离子通道的药物可以成为有效的抗HIV-1药物。
