Mechanisms of Retroviral RNA Trafficking and Packaging

逆转录病毒科在病毒家族中是独一无二的，因为一个病毒粒子包装了其基因组的两个全长副本。然而，每个感染事件只产生一个前病毒；因此，逆转录病毒是假二倍体。我们还不完全了解伪二倍体的好处，我们建议研究将两个RNA包装在一个病毒粒子中的好处。此外，逆转录病毒用来包装两个RNA的确切机制目前还不清楚。我们已经进行了实验，研究了共包装RNA伙伴选择的机制，并证明了DIS序列在这一过程中发挥着重要作用。在我们的实验系统中，我们可以通过操纵两种病毒的DIS序列来增加或减少杂合病毒的形成。这些结果表明，GAG识别并包装了一个RNA二聚体，而不是两个单体。我们开发了一种直接检查病毒颗粒中RNA的成像方法(单病毒粒子分析)。通过这种方法，我们已经确定大多数HIV-1颗粒含有一个RNA二聚体。使用不同的方法，我们也得出结论，HIV-1Gag主要包装二聚体RNA，它在细胞质中引发二聚化；复制RNA伙伴选择的一个主要决定因素是二聚化起始信号(DIS)；尽管通过CRM-1和Nxf1途径运输到细胞质的RNA可以被有效包装，但这两个RNA的复制包装并不受欢迎。我们将继续研究病毒RNA包装和RNA贩运的机制，包括对包装RNA拷贝数和合作伙伴选择的调节，以及HIV-1 RNA核出口和贩运。此外，我们将修改和重新定向RNA包装特异性和RNA运输，以更好地了解这些过程及其如何影响逆转录病毒复制。这些研究将阐明逆转录病毒复制的最基本特征之一，并将说明假二倍体的优势和局限性。[对应于2007年4月HIV耐药计划实地考察报告中的HU项目2]

Retroviridae are unique among virus families in that one virion packages two full-length copies of its genome. However, each infectious event generates only one provirus; thus, retroviruses are pseudodiploid. We do not fully understand the benefit of pseudodiploidy, and we propose to examine the benefit of packaging two RNAs in one virion. Additionally, the exact mechanisms that retroviruses use to package two RNAs are not currently understood. We have performed experiments examining the mechanisms of copackaged RNA partner selection and have demonstrated that the DIS sequence plays an important role in this process. In our experimental system, we can increase or decrease heterozygous virus formation by manipulating the DIS sequences of the two viruses. These results suggest that Gag recognizes and packages an RNA dimer rather than two monomers. We have developed an imaging method to directly examine RNA in the viral particles (single-virion analysis). By this method, we have determined that most HIV-1 particles contain one RNA dimer. Using various approaches, we have also concluded that HIV-1 Gag mostly packages dimeric RNA, which initiates dimerization in the cytoplasm; a major determinant for copackaged RNA partner selection is the dimerization initiation signal (DIS); and although RNA transported to the cytoplasm using either the CRM-1 and NXF1 pathway can be packaged efficiently, copackaging of these two RNAs is not favored. We will continue to investigate the mechanisms of viral RNA packaging and RNA trafficking, including the regulation of packaged RNA copy number and partner selection, and HIV-1 RNA nuclear export and trafficking. Additionally, we will modify and redirect RNA packaging specificity and RNA trafficking to gain a better understanding of these processes and how they affect retroviral replication. These studies will shed light on one of the most fundamental features of retroviral replication and will illustrate both the advantages and the limitations of pseudodiploidy. [Corresponds to Hu Project 2 in the April 2007 site visit report of the HIV Drug Resistance Program]