The regulation of viral and cellular transcripts is of great importance, particularly with respect to latent viruses that persist in the human host for extended periods of time. In the case of adult T-cell leukemia, leukemic cells are infected with HTLV-I and contain the proviral genome, but there is extremely little expression of viral transcripts. Despite being a non-structural protein that localizes to the nucleolus, the HTLV-I accessory protein p30 is required for efficient infection in vivo [19
]. Recent studies by our group and others have since shown that p30 acts as a negative regulator of virus expression by inhibiting the nuclear export of tax/rex mRNA [17
]. Additional studies have reported that p30 is a transcriptional inhibitor of the viral LTR. In addition to regulating the export of tax/rex mRNA to the cytoplasm, p30 expression modifies a number of signalling pathways such as TLR4, CREB, and GSK3β [22
]. How p30 accomplishes these changes in the cell is currently unknown. p30 has, however, been documented to facilitate transcription from cellular and viral promoters in conjunction with the transcriptional coactivator p300/CBP [51
]. Thus, similar to another nucleolar protein, nucleolin, p30 possesses both transcriptional and posttranscriptional activities. The combination of these effects likely decrease Tax and other viral antigen expression, possibly permitting HTLV-I infected cells to remain hidden from the immune response.
In this study, we used a genome wide analysis to investigate the effect of p30 on host cell gene regulation and found a number of cellular transcripts to be increased or decreased (Tables , ; see Additional files 1
). These genes belonged to a variety of families, including transcriptional/translational control, cell cycle, DNA replication and repair, and cell signalling. While it is not yet known whether changes in expression of any of these genes identified here, alone or in combination, are required for cell transformation induced by HTLV-I, some have been shown to have altered expression patterns in acute ATL, such as PDCD4, 90 kDa heat-shock protein, RNA polymerase II (DNA directed), regulator of G-protein signalling, general transcription factor IIH, and Bcl-3 [34
]. Considering the possibility that p30 plays a role in the onset of ATL in HTLV-I-infected individuals, some of the genes regulated by p30 may be involved in the process of cell transformation.
Using a different experimental approach which relied upon long-term stable expression of p30 in Jurkat lymphocytes, it was previously shown that p30 alters the general abundance of a number of cellular genes [23
]. Several of the transcripts shown previously to be down-regulated were indeed seen in our array as also being negatively regulated by p30, but the difference was less than a 2.5-fold change in expression. The differences between these reports are not surprising given the differences in the methodology and cell lines used for these two experiments. While our study used a short-term lentiviral infection and co-transduction of peripheral blood mononuclear cells, Michael et al. used a long-term lentiviral transduction of Jurkat T-lymphocytes. It is also worthwhile to consider that long-term p30 expression has been documented to induce cell cycle alterations, which may also lead to different changes in gene expression [21
]. Considering the differences, both studies are beneficial to help understand the role that p30 plays in modulating gene expression.
The main objective of this study was to evaluate whether any cellular genes were regulated post-transcriptionally by p30, in much the same way that p30 regulates Tax expression by preventing tax/rex mRNA export to the cytoplasm. While it is not yet known exactly how p30 inhibits the export of tax/rex mRNA, we hypothesized that p30 would alter the cytoplasmic abundance of cellular transcripts. In fact, we observed a number of cellular transcripts that showed either a decreased or increased abundance in the cytoplasm (Tables , ). While none of these genes were previously identified as being regulated in ATL samples, this was to be expected since in our experimental approach the total abundance of the genes characterized in Tables and was unchanged and only cytoplasmic abundance was affected.
So how does p30 inhibit mRNA export into the cytoplasm? It is possible that p30 somehow modulates the activity of a cellular export mechanism. If this were the case, this might explain why a variety of cellular transcripts were altered in cytoplasmic abundance, indicative of a global effect. Alternatively, p30 may bind to mRNA transcripts to prevent their association with nuclear export proteins, and cellular transcripts that are inhibited may share sequence or secondary structural similarities with the tax/rex mRNA. It has been hypothesized that p30 may function by binding to both RNA and to Rex, another HTLV-I protein that is conversely responsible for up-regulating tax/rex mRNA export from the nucleus [20
]. Indeed, there is evidence to suggest that p30 does bind to RNA [17
], and that p30 might recognize a particular RNA sequence that is present in the tax-rex message, a short 150 base pair response element present specifically at the tax/rex splice junction [17
]. Whether these interactions are required for the inhibition of tax/rex mRNA export is not currently known. In the current study, Rex was not expressed, suggesting that p30 can function in the absence of Rex. As a result, it is more likely that p30 might have a broad mechanism of action that applies to a number of transcripts.
On the other hand it is also possible that p30 directly or indirectly alters regulation or function of RNA export machinery. In this way p30 may actually only alter the expression or export of a few genes. The altered expression of these, in turn, might then be required for the normal expression of remaining transcripts shown to be altered in the presence of p30. Future work is required to examine the effects of p30 at the level of protein expression of candidate proteins. Another possibility explaining the function of p30 involves the binding and alteration of known cellular proteins involved in RNA modification and transport. Again, if p30 alters a known cellular mRNA trafficking pathway, it would not come as a surprise to observe so many cellular RNA transcripts as having altered expression patterns, both in total RNA samples and in cytoplasmic fractions.
Following binding to RNA, p30 might then prevent the subsequent docking and function of splicing and/or mRNA export factors. Whether p30 recognizes the sequence of this region or recognizes a secondary RNA structure is not known. Indeed, such regulation has been reported for how the retroviral HTLV Rex protein and HIV Rev protein interact with their respective RNA binding sites [53
]. While there are no sequence similarities between the Rex responsive element (RexRE) and the Rev responsive element (RRE), HIV Rev functionally interacts with both sites while Rex is specific for the RexRE. Computer prediction of secondary structures of numerous mRNA transcripts using specific software is not an easy task but warrants future study.
Whether p30 interacts with RNA in a sequence-specific or secondary structure-specific manner, there may exist similarities between the tax/rex message and those transcripts down-regulated in the cytoplasm (Additional file 3
), and examining these similarities will likely be the subject of future work. In addition, such an analysis may lead to the identification of an RNA binding motif in the p30 protein. If such a motif were found, it could also possibly lead to identification of therapeutic agents that could target such a binding motif in p30. By impairing the function of p30, one could hope to break latency and increase Tax expression in HTLV-infected patients, leading to the increased detection of infected cells by the immune system and the eventual clearance of infected cells from the body. This could perhaps provide an effective therapy for HTLV-I-infected individuals, thereby protecting patients from developing ATL later in life.