Single-Cell and Single-Cycle Analysis of HIV-1 Replication.
Single-Cell and Single-Cycle Analysis of HIV-1 Replication.
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DOI：
10.1371/journal.ppat.1004961
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发表时间：
2015-06
期刊：
影响因子：
6.7
通讯作者：
Bieniasz PD
中科院分区：
文献类型：
--
作者：
Holmes M;Zhang F;Bieniasz PD
HIV-1生命周期晚期的动态记录很少。病毒复制动力学通常在感染细胞群体中测量，但在HIV-1复制的早期步骤中引入的病毒复制使后续步骤进展的测量复杂化，并且可以掩盖复制动力学及其在单个感染细胞中的变化。我们建立了基于显微镜的方法来动态测量HIV-1编码的报告基因和抗病毒基因在单个感染细胞中的表达。我们将这些测量与常规分析相结合，以量化HIV-1复制周期中的延迟，这是由HIV-1基因表达的双相性和Gag基质结构域的组装抑制特性造成的。我们进一步将限制因子（APOBEC 3G）去除的动力学与单个细胞中HIV-1复制的动力学联系起来。这些研究为HIV-1复制周期中的关键事件提供了时间轴，并揭示了早期和晚期HIV-1基因表达开始之间的间隔仅为~ 3 h，但基质导致细胞外病毒体产生延迟~6- 12 h。有趣的是，基质将颗粒组装延迟到APOBEC 3G已经从细胞中大量去除的时间。因此，需要准备感染的细胞是有效的生产者的感染性HIV-1可能会提供一个动力，程序延迟HIV-1病毒粒子的发生。我们的研究结果还强调了同质细胞群中HIV-1复制周期长度的显著异质性，并表明典型的感染细胞在48小时寿命结束时仅在几个小时内产生新的病毒体。因此，感染细胞寿命的微小变化可能对单个周期中的病毒产量和感染个体的总体临床过程产生很大影响。HIV-1的复制周期由几个连续的步骤组成。虽然HIV-1复制的早期步骤的时间是相当好理解的，但由于在早期步骤中将HIV-1引入感染细胞群体中，因此测量后期步骤的持续时间是复杂的。我们设计了成像方法来测量单个感染细胞中HIV-1复制后期步骤的持续时间，避免了与异步感染细胞群体中测量相关的问题。通过将这些测量与细胞群体中HIV-1复制的常规分析相结合，我们得出了HIV-1生命周期后期关键事件的时间线。我们发现早期和晚期基因表达之间的延迟很小，但随后病毒组装的程序性延迟使HIV-1能够在新病毒体产生之前从感染细胞中去除宿主抗病毒蛋白。这样做，HIV-1可能会阻止无效的病毒体产生。
The dynamics of the late stages of the HIV-1 life cycle are poorly documented. Viral replication dynamics are typically measured in populations of infected cells, but asynchrony that is introduced during the early steps of HIV-1 replication complicates the measurement of the progression of subsequent steps and can mask replication dynamics and their variation in individual infected cells. We established microscopy-based methods to dynamically measure HIV-1-encoded reporter gene and antiviral gene expression in individual infected cells. We coupled these measurements with conventional analyses to quantify delays in the HIV-1 replication cycle imposed by the biphasic nature of HIV-1 gene expression and by the assembly-inhibiting property of the matrix domain of Gag. We further related the dynamics of restriction factor (APOBEC3G) removal to the dynamics of HIV-1 replication in individual cells. These studies provide a timeline for key events in the HIV-1 replication cycle, and reveal that the interval between the onset of early and late HIV-1 gene expression is only ~3h, but matrix causes a ~6–12h delay in the generation of extracellular virions. Interestingly, matrix delays particle assembly to a time at which APOBEC3G has largely been removed from the cell. Thus, a need to prepare infected cells to be efficient producers of infectious HIV-1 may provide an impetus for programmed delays in HIV-1 virion genesis. Our findings also emphasize the significant heterogeneity in the length of the HIV-1 replication cycle in homogenous cell populations and suggest that a typical infected cell generates new virions for only a few hours at the end of a 48h lifespan. Therefore, small changes in the lifespan of infected cells might have a large effect on viral yield in a single cycle and the overall clinical course in infected individuals. The HIV-1 replication cycle is composed of several sequential steps. While the timing of the early steps of HIV-1 replication is quite well understood, measuring the duration of later steps is complicated by the fact that asynchrony is introduced into populations of infected cells during early steps. We devised imaging methods for measuring the duration of late steps in HIV-1 replication in single infected cells, circumventing the problems associated with measurements in populations of asynchronously infected cells. By combining these measurements with conventional analyses of HIV-1 replication in populations of cells, we derived a time-line of key events during the late steps of the HIV-1 life cycle. We find that the delay between early and late gene expression is small but that a subsequent programmed delay in virus assembly enables HIV-1 to remove a host antiviral protein from infected cells before new virions are generated. In so doing, HIV-1 may prevent futile virion production.
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影响因子：
32.4
作者：
Blanco-Melo D;Venkatesh S;Bieniasz PD
通讯作者：
Bieniasz PD
影响因子：
64.5
作者：
Fornerod, M;Ohno, M;Mattaj, IW
通讯作者：
Mattaj, IW
DOI：
10.1073/pnas.89.6.2223
发表时间：
1992-03-15
影响因子：
11.1
作者：
FERNANDEZLARSSON, R;SRIVASTAVA, KK;ROBINSON, HL
通讯作者：
ROBINSON, HL
DOI：
10.1073/pnas.86.15.5781
发表时间：
1989-08-01
影响因子：
11.1
作者：
GOTTLINGER, HG;SODROSKI, JG;HASELTINE, WA
通讯作者：
HASELTINE, WA
影响因子：
4.9
作者：
Donahue, Daniel A.;Sloan, Richard D.;Wainberg, Mark A.
通讯作者：
Wainberg, Mark A.