Specialization to cell-free or cell-associated spread by BAC-cloned HCMV strains not determined by the UL128-131 and RL13 loci
Specialization to cell-free or cell-associated spread by BAC-cloned HCMV strains not determined by the UL128-131 and RL13 loci
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不由 UL128-131 和 RL13 位点决定的 BAC 克隆 HCMV 毒株的无细胞或细胞相关传播的专门化
DOI：
10.1101/760611
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Brent J. Ryckman
中科院分区：
文献类型：
--
作者：
E. Schultz;J. Lanchy;L. Z. Day;Q. Yu;Christopher Peterson;Jessica Preece;Brent J. Ryckman
一种广泛持有的观点是，人巨细胞病毒（HCMV）的临床分离株是细胞相关的，并且影响UL 128 -131和RL 13基因座的突变在随后的培养传代过程中出现，并导致无细胞扩散表型的出现。为了区分影响细胞相关和无细胞传播的因素，我们分析了三种HCMV BAC克隆的传播特征; Merlin（ME），其表达高水平的UL 128 -131并在RL 13内携带移码突变，以及TB 40/e（TB）和TR，其在UL 128 -131中均为低水平，在RL 13处为完整的。通过流式细胞术对12天内新感染细胞的数量进行定量，发现菌株间成纤维细胞的扩散效率非常相似。然而，通过比较中和抗体对传播的抑制以及子代病毒的数量和感染性，表明TB和TR传播主要是无细胞的，而ME传播主要是细胞相关的。虽然UL 128 -131的转录抑制大大增强了ME的无细胞扩散，但细胞相关模式的效率不受影响。上皮细胞培养物中的传播是高度相关的所有菌株，ME是最有效的。UL 128 -131的抑制降低了ME在上皮细胞中传播的效率，但不影响传播的主导模式。所有菌株在表达RL 13的细胞中的扩散均显著减少，并且在成纤维细胞中比在上皮细胞中更明显。RL 13的影响不能通过子代病毒的生产、释放或感染性的变化来清楚地解释，并且菌株无细胞或细胞相关传播的倾向没有变化。总之，HCMV毒株对无细胞传播的特化与无细胞后代的数量和感染性有关，这可能受到UL 128 -131水平的影响，但细胞相关的专门表型可能由UL 128 -131或RL 13基因座以外的因素决定。HCMV的无细胞和细胞相关传播模式之间的实验区别在很大程度上是相对的。当无细胞扩散被中和抗体抑制时，或者如果特定病毒株在无细胞扩散方面较差，则观察到的扩散可以简单地定义为“细胞相关”。然而，这种观点并不容易有助于分析细胞相关扩散的效率或所涉及的因素。在我们的研究中，我们测量了HCMV无细胞和细胞相关传播作为独立过程的动力学，并显示HCMV毒株可以高度专化于一种或另一种传播模式的证据。决定传播模式特化的遗传因素尚不清楚，但考虑到人群中传播的HCMV的遗传多样性，似乎两种模式都有代表性。干预措施的有效性很可能受到传播方式的影响。例如，中和抗体对限制细胞相关扩散的效果较差。我们的研究结果提供了一个概念性的方法来评估干预方法，如中和抗体提出的候选疫苗和药物化合物的目标病毒复制过程的能力，以限制无细胞或细胞相关的传播模式作为独立的过程。
A widely held view is that clinical isolates of human cytomegalovirus (HCMV) are cell-associated and that mutations affecting the UL128-131 and RL13 loci arise during subsequent passage in culture and lead to the appearance of a cell-free spread phenotype. To distinguish the factors influencing cell-associated and cell-free spread, we analyzed the spread characteristics of three HCMV BAC-clones; Merlin (ME), which expresses high levels of UL128-131 and harbors a frameshift mutation within RL13, and TB40/e (TB) and TR, which are both low in UL128-131 and intact at RL13. Quantitation of the number of newly infected cells over 12 days by flow cytometry revealed remarkably similar spread efficiencies in fibroblasts among strains. However, comparing the inhibition of spread by neutralizing antibodies and the quantities and infectivity of progeny virus indicated that TB and TR spread was predominately cell-free, whereas spread of ME was predominantly cell-associated. While transcriptional repression of UL128-131 greatly enhanced cell-free spread by ME, the efficiency of the cell-associated mode was not affected. Spread in epithelial cultures was highly cell-associated for all strains, and ME was the most efficient. Repression of UL128-131 reduced the efficiency of ME spread in epithelial cells, but did not affect the predominate mode of spread. Spread in RL13-expressing cells was comparably reduced for all strains, and more pronounced in fibroblasts than in epithelial cells. RL13 effects could not be clearly explained by changes in production, release, or infectivity of progeny virus, and there were no changes to the proclivity of strains for cell-free or cell-associated spread. In sum, the specialization of HCMV strains to cell-free spread is linked to the quantity and infectivity of cell-free progeny, which can be influenced by UL128-131 levels, but the cell-associated specialist phenotype is likely determined by factors beyond the UL128-131 or RL13 loci. AUTHOR SUMMARY Experimental distinctions between cell-free and cell-associated modes of spread for HCMV have been largely relativistic. When cell-free spread is inhibited by neutralizing antibodies, or if particular strain of virus is poor at cell-free spread, then the observed spread may be simply defined as “cell-associated”. However, such a view does not easily lend towards analysis of the efficiency of cell-associated spread or the factors involved. In our study, we measured the kinetics of HCMV cell-free and cell-associated spread as independent processes and show evidence that HCMV strains can be highly specialized to one or the other mode of spread. The genetic factors that determine specialization for mode of spread are unclear, but given the genetic diversity of HCMV circulating in human populations, it seems likely that both modes of are represented. The efficacy of intervention approaches is likely affected by the mode of spread. For example, neutralizing antibodies are less effective to limit cell-associated spread. Our results provide a conceptual approach to evaluating intervention approaches such as neutralizing antibodies raised by vaccine candidates and drug compounds that target viral replication processes for their ability to limit cell-free or cell-associated modes of spread as independent processes.
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DOI：
10.1126/science.1227919
发表时间：
2012-11-23
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Stern-Ginossar N;Weisburd B;Michalski A;Le VT;Hein MY;Huang SX;Ma M;Shen B;Qian SB;Hengel H;Mann M;Ingolia NT;Weissman JS
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影响因子：
6.7
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DOI：
10.1056/nejm198304213081603
发表时间：
1983
期刊：
The New England journal of medicine
影响因子：
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DOI：
10.1007/978-1-4939-1875-1_3
发表时间：
2015
期刊：
Methods in molecular biology (Clifton, N.J.)
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Rooney, John P;Ryde, Ian T;Sanders, Laurie H;Howlett, Evan H;Colton, Meryl D;Germ, Kaylyn E;Mayer, Greg D;Greenamyre, J Timothy;Meyer, Joel N
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鼠巨细胞病毒糖蛋白O促进体内上皮细胞感染
影响因子：
5.4
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