Extensive biochemical studies both in vitro and in vivo indicate interaction and utilization of cellular machinery by human immunodeficiency retroviruses. However, there is little information available about where, when and how these events take place within the cell nucleus. Moreover, there is no information at the ultrastructural level about these particular viral/cellular processes and their relationship to the structure and organization of the nucleus. Understanding of essential virus-host interactions may provide a basis for development of therapeutic interventions. We were interested in visualizing the viral protein Rev using both confocal light microscopy and intermediate high voltage electron microscopy (IVBM). We investigated mechanisms of regulation and utilization of HIV mRNAs, specifically post-transcriptional regulation. Rev, interacting with a Rev dependent element in the viral mRNA (RRE), plays a critical role in the expression of viral proteins. Our work on understanding of virus-cell interaction in the nucleus was correlated with the known 3-dimensional organization of transcription and splicing machinery within the eukaryotic cell nucleus. A study using 3-dimensional confocal imaging has been published on the distribution of the Rev protein in transfected cells in the presence or absence of its target sequence, RRE (Luznik et al., 1995). In this study, it was found that the pattern of Rev labeling changed depending upon whether or not RREcontaining RNA was present, going from a primarily nucleolar distribution to concentrated foci within the nucleoplasm. These foci were seen in three dimensions to form tracks reminiscient of those observed for specific mRNAs within the nucleus. Thus, these results were consistent with a role for Rev in mRNA transport out of the nucleus. In addition, the localization of Rev foci was compared to that of the nuclear speckles, which represent nuclear domains (spliceosomes) specialized for mRNA splicing. A the light microscopic level, the Rev foci became more closely associated with the speckles over time. The speckles also became larger and fewer in number at later time points after transfection.
NIH ExPorter
NIH ExPorter