Martin Jessica L, Cao Sheng, Maldonado Jose O, Zhang Wei, Mansky Louis M
Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA Pharmacology Graduate Program, University of Minnesota, Minneapolis, Minnesota, USA.
Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA Department of Diagnostic and Biological Sciences, University of Minnesota, Minneapolis, Minnesota, USA.
J Virol. 2016 Aug 26;90(18):8074-84. doi: 10.1128/JVI.00666-16. Print 2016 Sep 15.
The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of virus-like particles (VLPs). In this study, we sought to investigate-in parallel comparative analyses-Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP, where YFP is yellow fluorescent protein) created from all representative retroviral genera: Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, and Spumavirus. We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thin-section transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative Beta- and Epsilonretrovirus Gag proteins. This is the first report of Epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative Beta- and Epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed Deltaretrovirus-like particles with flat regions of electron density that did not follow viral membrane curvature, Lentivirus-like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and Spumavirus-like particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway.
Comparative analysis among retroviruses has been critically important in enhancing our understanding of retroviral replication and pathogenesis, including that of important human pathogens such as human T-cell leukemia virus type 1 (HTLV-1) and HIV-1. In this study, parallel comparative analyses have been used to study Gag expression and virus-like particle morphology among representative retroviruses in the known retroviral genera. Distinct differences were observed, which enhances current knowledge of the retroviral assembly pathway.
Gag蛋白是主要的逆转录病毒结构蛋白，仅其表达通常就足以产生病毒样颗粒（VLP）。在本研究中，我们试图通过平行比较分析，利用从所有代表性逆转录病毒属（α逆转录病毒属、β逆转录病毒属、δ逆转录病毒属、ε逆转录病毒属、γ逆转录病毒属、慢病毒属和泡沫病毒属）构建的Gag表达构建体（Gag或Gag-YFP，其中YFP是黄色荧光蛋白），研究Gag在细胞中的分布、VLP大小和基本形态特征。我们通过共聚焦显微镜分析Gag在细胞中的分布，通过超薄切片透射电子显微镜（TEM）分析VLP出芽情况，并通过低温透射电子显微镜（cryo-TEM）分析VLP的一般形态特征。除了代表性的β和ε逆转录病毒Gag蛋白外，在所有测试的Gag构建体中，均在质膜附近观察到点状Gag。这是关于ε逆转录病毒Gag定位于HeLa细胞核的首次报道。虽然代表性的β和ε逆转录病毒Gag蛋白未产生VLP，但其他Gag蛋白产生了VLP，TEM证实了这一点，并且cryo-TEM观察到了形态学差异。特别是，我们观察到具有扁平电子密度区域且不遵循病毒膜曲率的δ逆转录病毒样颗粒、具有窄范围且一致电子密度的慢病毒样颗粒，这表明Gag晶格紧密堆积，以及具有大的包膜蛋白刺突且没有与Gag晶格相关的可见电子密度的泡沫病毒样颗粒。综上所述，这些平行比较分析首次证明了逆转录病毒样颗粒之间存在的独特形态特征。对这些差异的研究将为逆转录病毒组装途径提供更深入的见解。
逆转录病毒之间的比较分析对于增强我们对逆转录病毒复制和发病机制的理解至关重要，包括对重要人类病原体如1型人类T细胞白血病病毒（HTLV-1）和HIV-1的理解。在本研究中，平行比较分析已用于研究已知逆转录病毒属中代表性逆转录病毒之间的Gag表达和病毒样颗粒形态。观察到了明显差异，这增强了我们目前对逆转录病毒组装途径的认识。
