Tsai W P, Conley S R, Kung H F, Garrity R R, Nara P L
Laboratory of Biochemical Physiology, NCI-FCRDC, Frederick, Maryland 21702, USA.
Virology. 1996 Dec 15;226(2):205-16. doi: 10.1006/viro.1996.0648.
Recent interest focused on the dynamics of HIV-1 replication in primary monocytes/macrophages and T-lymphocytes of the immune system, as well as the standardization of virological and immunological in vitro assays with primary isolates, provided the impetus for these studies. These types of studies have never been performed as they would occur in vivo, i.e., where the envelope of the virus and cell membranes of the two cell types of the same host origin. Therefore, the biological and physicochemical properties of an uncloned, primary dual-tropic isolate HIV-1ADA during the initial lag, log, and stationary phases of viral replication were studied in an autologous donor cell assay in peripheral blood mononuclear cells (PBMC) and blood monocyte-derived macrophages (MDM). Similar total numbers (10(9) virus particles/ml) were produced by both cell types during the stationary period. On a per cell per day basis, during peak stationary periods, 0.92 x 10(3) virions/day for MDMs and 5.31 x 10(3) virions/day for PBMCs were produced. Interestingly, virus replicating from MDMs during the log-growth phase demonstrated the greatest infectious fraction which was 3 logs greater than virus replicating in PBMCs. Despite constant virus particle production in MDMs, the infectious fraction was found to fall 3 to 4 logs over a 10-day period. Due to an infectious fraction less than 1 (0.053 infectious unit/cell/24 hr), virus spread in PBMCs during the rapid log phase could only have occurred by cell-to-cell contact, whereas in MDMs with an infectious fraction of about one infectious particle (approximately 1/cell/24 hr), cell-free transmission could account for the observed results. Most of the MDMs (> 90%) became productively infected, whereas only 5-10% of the total PBMCs were found replicating virus. The period of peak stationary virus production (i.e., stationary phase) was at minimum 4 to 5 times longer in MDMs than PBMCs. Whereas the majority of p24, RT, and gp 120 found to be associated with MDM-derived virions, no increased dissociation of these components was observed in PBMC-derived virions. The virion-associated gp 120 was 3 to 4 times more stable on both PBMC- and MDM-derived virus (> 96 hr) and present at 10-25 times the concentration per virion than that observed for a T-cell-line-adapted laboratory strain of HIV-1 replicating in T-cell lines. These in vitro results suggest that important differences exist between MDMs and PBMCs with regard to the viral dynamics of infection and replication which should provide for a qualitative and quantitative basis to estimate virus replication on a per-cell basis for other known cellular targets of HIV-1. Studying the multiple biophysicochemical characteristics and viral replication dynamics as described herein provides an autologous in vitro model of additional quantifiable parameters for analysis and understanding of virus/host factor(s) and/or antivirals which influence them.
近期，对HIV-1在免疫系统的原代单核细胞/巨噬细胞和T淋巴细胞中的复制动态的关注，以及使用原代分离株进行病毒学和免疫学体外检测的标准化，为这些研究提供了动力。这类研究从未像在体内那样进行过，即在同一宿主来源的两种细胞类型的病毒包膜和细胞膜的情况下。因此，在一项外周血单核细胞（PBMC）和血液单核细胞衍生巨噬细胞（MDM）的自体供体细胞检测中，研究了未克隆的原代双嗜性分离株HIV-1ADA在病毒复制的初始延迟期、对数期和稳定期的生物学和物理化学特性。在稳定期，两种细胞类型产生的病毒总数相似（10⁹个病毒颗粒/毫升）。在稳定高峰期，MDM每天每细胞产生0.92×10³个病毒体，PBMC每天每细胞产生5.31×10³个病毒体。有趣的是，在对数生长期从MDM复制的病毒显示出最大的感染比例，比在PBMC中复制的病毒高3个对数。尽管MDM中病毒颗粒持续产生，但在10天内发现感染比例下降了3至4个对数。由于感染比例小于1（0.053感染单位/细胞/24小时），在快速对数期PBMC中的病毒传播只能通过细胞间接触发生，而在感染比例约为一个感染性颗粒（约1/细胞/24小时）的MDM中，游离病毒传播可以解释观察到的结果。大多数MDM（>90%）被有效感染，而在总共PBMC中仅发现5-10%在复制病毒。稳定期病毒产生高峰期（即稳定期）在MDM中比PBMC至少长4至5倍。虽然发现与MDM衍生病毒体相关的p24、RT和gp120大部分，但在PBMC衍生病毒体中未观察到这些成分的解离增加。病毒体相关的gp120在PBMC和MDM衍生的病毒上都更稳定（>96小时），并且每个病毒体的浓度比在T细胞系中复制的HIV-1实验室适应株高10-25倍。这些体外结果表明，MDM和PBMC在感染和复制的病毒动态方面存在重要差异，这应为按细胞计算估计HIV-1其他已知细胞靶点的病毒复制提供定性和定量基础。研究本文所述的多种生物物理化学特征和病毒复制动态，为分析和理解影响病毒/宿主因子和/或抗病毒药物的额外可量化参数提供了一个自体体外模型。
