Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of
viral gene products that recruit host cell
proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor
proteins to target regions of the cell (during de novo
infection, viruses co-opt
molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with
RNA genomes, coordinate the intracellular trafficking of both
protein and
RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of
RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important
biological information, and also underscored the importance of
RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their
RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles. FISH serves as a useful tool to identify changes in steady-state localization of
viral RNA. When combined with immunofluorescence (IF) analysis, FISH/IF co-analyses will provide information on the co-localization of
proteins with the
viral RNA. This analysis therefore provides a good starting point to test for
RNA-
protein interactions by other biochemical or biophysical tests, since co-localization by itself is not enough evidence to be certain of an interaction. In studying
viral RNA localization using a method like this, abundant information has been gained on both viral and cellular
RNA trafficking events. For instance, HIV-1 produces
RNA in the nucleus of infected cells but the
RNA is only translated in the cytoplasm. When one key
viral protein is missing (Rev), FISH of the
viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic
RNA in the nucleus. Here, we present the method for visual analysis of viral genomic
RNA in situ. The method makes use of a labelled
RNA probe. This probe is designed to be complementary to the viral genomic
RNA. During the in vitro synthesis of the
antisense RNA probe, the
ribonucleotide that is modified with
digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target
mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the
mRNA as well as
proteins of interest when performing FISH/IF.