In vivo analysis of highly conserved Nef activities in HIV-1 replication and pathogenesis.
In vivo analysis of highly conserved Nef activities in HIV-1 replication and pathogenesis.
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DOI：
10.1186/1742-4690-10-125
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发表时间：
2013-10-30
期刊：
影响因子：
3.3
通讯作者：
Garcia JV
中科院分区：
文献类型：
--
作者：
Watkins RL;Zou W;Denton PW;Krisko JF;Foster JL;Garcia JV
HIV-1的辅助蛋白Nef对艾滋病的发展具有决定性作用。该蛋白的体外表征已经描述了许多体内意义未知的Nef活性，包括CD 4下调和许多依赖于Nef与宿主SH 3结构域蛋白相互作用的活性。在这里，我们使用BLT人源化小鼠模型的HIV-1感染，以评估其对病毒复制和发病机制的影响和选择压力，以恢复这些活动，使用强制体内进化。我们跟踪了BLT小鼠感染期间HIV-1 LAI（LAI）与移码nef（LAINeffs）的演变。LAINeffs在血液中被nef中具有短缺失的病毒迅速取代，恢复了开放阅读框架（LAINeffs-1和LAINeffs-13）。随后，LAINeffs β-1经常被野生型LAI取代。出乎意料的是，LAINeffs NF_1和LAINeffs NF_13 Nef对于CD 4下调活性是特异性缺陷的。使用具有这些突变nef的病毒感染BLT小鼠。LAINeffs E10 - 1和LAINeffs E10 - 13表现出病毒复制减少三倍（与LAI相比）和系统性CD 4 + T细胞减少50%（对于LAI>90%），证明了CD 4下调的重要性。这些结果还表明，与LAINeffs相比，除了CD 4下调以外的功能增强了LAINeffs E11 - 1和LAINeffs E11 - 13的病毒复制和发病机制。为了深入了解这些活动的性质，我们构建了双突变体P72 A/P75 A。通过将SH 3结构域结合位点（P72 Q73 V74 P75 L76 R77）突变为P72 A/P75 A可以否定多种Nef活性，并且该突变不影响CD 4下调。nef突变为P72 A/P75 A的病毒在体内与野生型病毒非常相似，因为病毒复制和发病机制没有显著改变。与上述LAINeff不同，P72 A/P75 A突变具有非常弱的回复至野生型序列的倾向。Nef的体内表型显著依赖于CD 4下调，但最低限度地依赖于需要完整SH 3结构域结合基序的众多Nef活性。这些结果表明，CD 4下调加上一个或多个未知的Nef活动有助于增强病毒复制和发病机制，是抗HIV治疗的合适靶点。在BLT小鼠中的强制进化研究将极大地促进这些关键活动的鉴定。
The HIV-1 accessory protein, Nef, is decisive for progression to AIDS. In vitro characterization of the protein has described many Nef activities of unknown in vivo significance including CD4 downregulation and a number of activities that depend on Nef interacting with host SH3 domain proteins. Here, we use the BLT humanized mouse model of HIV-1 infection to assess their impact on viral replication and pathogenesis and the selection pressure to restore these activities using enforced in vivo evolution. We followed the evolution of HIV-1LAI (LAI) with a frame-shifted nef (LAINeffs) during infection of BLT mice. LAINeffs was rapidly replaced in blood by virus with short deletions in nef that restored the open reading frame (LAINeffs∆-1 and LAINeffs∆-13). Subsequently, LAINeffs∆-1 was often replaced by wild type LAI. Unexpectedly, LAINeffs∆-1 and LAINeffs∆-13 Nefs were specifically defective for CD4 downregulation activity. Viruses with these mutant nefs were used to infect BLT mice. LAINeffs∆-1 and LAINeffs∆-13 exhibited three-fold reduced viral replication (compared to LAI) and a 50% reduction of systemic CD4+ T cells (>90% for LAI) demonstrating the importance of CD4 downregulation. These results also demonstrate that functions other than CD4 downregulation enhanced viral replication and pathogenesis of LAINeffs∆-1 and LAINeffs∆-13 compared to LAINeffs. To gain insight into the nature of these activities, we constructed the double mutant P72A/P75A. Multiple Nef activities can be negated by mutating the SH3 domain binding site (P72Q73V74P75L76R77) to P72A/P75A and this mutation does not affect CD4 downregulation. Virus with nef mutated to P72A/P75A closely resembled the wild-type virus in vivo as viral replication and pathogenesis was not significantly altered. Unlike LAINeffs described above, the P72A/P75A mutation had a very weak tendency to revert to wild type sequence. The in vivo phenotype of Nef is significantly dependent on CD4 downregulation but minimally on the numerous Nef activities that require an intact SH3 domain binding motif. These results suggest that CD4 downregulation plus one or more unknown Nef activities contribute to enhanced viral replication and pathogenesis and are suitable targets for anti-HIV therapy. Enforced evolution studies in BLT mice will greatly facilitate identification of these critical activities.
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影响因子：
64.5
作者：
Hanna, Z;Kay, DG;Jolicoeur, P
通讯作者：
Jolicoeur, P
影响因子：
5.4
作者：
Chateau, Morgan;Swanson, Michael D.;Garcia, J. Victor
通讯作者：
Garcia, J. Victor
影响因子：
32.4
作者：
BAUR, AS;SAWAI, ET;PETERLIN, BM
通讯作者：
PETERLIN, BM
影响因子：
5.4
作者：
GOLDSMITH, MA;WARMERDAM, MT;GREENE, WC
通讯作者：
GREENE, WC
影响因子：
5.4
作者：
Denton, Paul W.;Olesen, Rikke;Garcia, J. Victor
通讯作者：
Garcia, J. Victor