Production of infectious human immunodeficiency virus type 1 does not require depletion of APOBEC3G from virus-producing cells.
Production of infectious human immunodeficiency virus type 1 does not require depletion of APOBEC3G from virus-producing cells.
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DOI：
10.1186/1742-4690-1-27
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发表时间：
2004-09-17
期刊：
影响因子：
3.3
通讯作者：
Strebel, Klaus
中科院分区：
文献类型：
--
作者：
Kao, Sandra;Miyagi, Eri;Strebel, Klaus
背景技术背景：人免疫缺陷病毒Vif蛋白通过阻止APOBEC3G胞苷脱氨酶包装到病毒体中来克服其抑制活性。结果：在单细胞水平上，Vif对APOBEC3G表达的影响主要表现在细胞内APOBEC3G水平的降低，这可能与Vif诱导的APOBEC3G蛋白酶体依赖性降解有关。用Vif和APOBEC3G表达载体双重转染的HeLa细胞显示两种蛋白质的有效共表达。在最佳染色条件下，约80%的转染细胞的Vif和APOBEC3G评分为双阳性。然而，在相同培养物中观察到的双阳性细胞的比例根据固定方案和所用抗体的选择而变化，范围从低至40%到高达80%的转染细胞。重要的是，在野生型Vif和生物学上无活性的Vif变体中均观察到表达Vif或APOBEC3G的单阳性细胞。因此，在一些Vif表达细胞中缺乏APOBEC3G不能归因于Vif诱导的APOBEC3G降解。这些发现与我们的免疫印迹分析结果一致，免疫印迹分析显示Vif对APOBEC3G稳态水平仅具有中度影响。值得注意的是，在这种条件下产生的病毒是完全感染性的，这表明在我们的分析中使用的Vif蛋白是功能性的，并在饱和level.CONCLUSIONS：我们的研究结果表明，Vif和APOBEC3G可以有效地共表达。因此，如前所述，从Vif表达细胞中耗尽APOBEC3G不是Vif的普遍性质，因此对于感染性病毒体的产生不是必需的。
BACKGROUND: The human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions. Inhibition of APOBEC3G encapsidation is paralleled by a reduction of its intracellular level presumably caused by the Vif-induced proteasome-dependent degradation of APOBEC3G.RESULTS: In this report we employed confocal microscopy to study the effects of Vif on the expression of APOBEC3G on a single cell level. HeLa cells dually transfected with Vif and APOBEC3G expression vectors revealed efficient co-expression of the two proteins. Under optimal staining conditions approximately 80% of the transfected cells scored double-positive for Vif and APOBEC3G. However, the proportion of double-positive cells observed in identical cultures varied dependent on the fixation protocol and on the choice of antibodies used ranging from as low as 40% to as high as 80% of transfected cells. Importantly, single-positive cells expressing either Vif or APOBEC3G were observed both with wild type Vif and a biologically inactive Vif variant. Thus, the lack of APOBEC3G in some Vif-expressing cells cannot be attributed to Vif-induced degradation of APOBEC3G. These findings are consistent with our results from immunoblot analyses that revealed only moderate effects of Vif on the APOBEC3G steady state levels. Of note, viruses produced under such conditions were fully infectious demonstrating that the Vif protein used in our analyses was both functional and expressed at saturating levels.CONCLUSIONS: Our results suggest that Vif and APOBEC3G can be efficiently co-expressed. Thus, depletion of APOBEC3G from Vif expressing cells as suggested previously is not a universal property of Vif and thus is not imperative for the production of infectious virions.