In this study, we present experiments demonstrating efficient inhibition of HIV-1 by unmodified antisense RNA. Our data clearly show that the expression of an antisense RNA targeting a region in env
is able to significantly reduce particle production, even at very low ratios of antisense to target HIV-1 RNA. Although this was an unexpected finding, based on many previous studies showing the inefficiency of similar approaches to target HIV and other viruses, as well as cellular genes (26
), it confirms the recent studies of Lu et al. targeting HIV-1 using a similar RRE-containing antisense RNA (42
). Although our study was performed in 293T cells, the studies of Lu et al. (42
) were performed in CD4+
lymphocytes. It is thus clear that efficient inhibition of HIV by antisense also occurs in the primary cells that are the natural host for HIV.
Our data specifically show that efficient targeting requires trafficking of the antisense RNA along the Rev/RRE pathway. In support of this conclusion, we demonstrated that the inhibition of HIV-1 was virtually abolished when the antisense RNA lacked an RRE or when Rev was not provided. In addition, the efficiency of antisense inhibition was significantly reduced when the RRE-containing antisense RNA was redirected to the Tap/Nxf1 pathway that normally is used to export CTE-containing RNA and many cellular mRNAs. We conclude that the efficient inhibition achieved using HIV-1-derived lentiviral vectors can be explained by the specific RNA trafficking pathway utilized by HIV-1.
Cotargeting of the antisense and HIV-1 target RNA to the same cellular compartment in the nucleus has recently been proposed as a possible mechanism for the efficiency of the vector that is currently in clinical trials (61
). It was proposed that this would trigger extensive adenosine deamination of the HIV-1-antisense duplex, resulting in nuclear retention of the resulting dsRNA complexes (36
). However, our results show that, although cotargeting may contribute to antisense efficiency, it is clearly not essential, since an HIV-1 proviral clone that was altered to use an MPMV CTE, rather than an RRE, was also efficiently inhibited by the RRE-driven antisense RNA in the presence of Rev. In this case, the antisense RNA used the Rev/RRE pathway, whereas the HIV-1 target RNA was exported through the Tap/Nxf1 pathway. Previous work from many laboratories have shown that these export pathways are separate and use different cellular factors (15
If cotargeting were the dominant reason for the observed antisense inhibition, the efficiency would also have been expected to remain the same when RevM10-Tap was used to replace Rev in the export of the HIV-1 target and antisense RNA. Instead, the inhibition was significantly reduced, demonstrating that trafficking through the Rev pathway is a major determinant of efficient antisense inhibition. However, nuclear cotargeting may still contribute to antisense inhibition, since the CTE-driven antisense more efficiently targeted the RRE-virus that was forced to use the Tap/Nxf1 pathway. In addition, trafficking of both RRE and CTE-driven antisense RNAs and targets on the Tap/Nxf1 pathway also led to nearly identical inhibition profiles.
Our results clearly demonstrate that nuclear retention of HIV-1 target RNA did not contribute to the antisense inhibition that was achieved with the RRE-driven antisense RNA in the presence of Rev. Even when p24 levels were efficiently reduced, the GagPol RNA was still exported to the cytoplasm and present in polyribosomal complexes. In addition, our experiments with a proviral HIV-1 clone that did not give rise to virus particles demonstrated that the inhibition did not occur at the level of particle assembly or release. Thus, the antisense effects are manifested at the cytoplasmic level, after the association of the mRNA with the translation machinery, but before particle assembly.
We also showed that the reduction in p24 was not due to a general effect on protein synthesis, since Nef protein levels were unaffected by antisense expression. This specificity of inhibition for the HIV-1 target was somewhat surprising, since in mammalian cells, long double-stranded RNAs often activate protein kinase R and 2′5′-oligoadenylate synthetase, which leads to the interferon response and a general downregulation of translation and RNA degradation (29
). This suggests that trafficking of the antisense RNA through the Rev/RRE pathway somehow allows the interferon response to be bypassed. An analogous bypass mechanism has been suggested for inhibition by some siRNAs (12
Although in most cases, the appearance of an RNA in polyribosome complexes leads to production of protein, a reduction in the rate of initiation concomitant with a decrease in the rate of elongation can give rise to significantly reduced levels of protein (“ribosome stalling”) that could explain the reduced GagPol protein levels seen (68
). Another possibility is that protein is produced but rapidly degraded. Both of these mechanisms have been proposed to function in miRNA-mediated translation inhibition (51
). Alternatively, the high-molecular-weight complexes could represent complexes that are EDTA sensitive and appear to be polyribosomes but are actually recently described pseudo-polyribosomal complexes (70
). Further experiments will be needed to distinguish between these possibilities.
Independent of the detailed mechanism utilized, our results clearly point to a novel, previously unknown, mechanism for antisense inhibition. One model would be that Rev and the RRE allow the target/antisense RNA complex to be exported to the cytoplasm, where the antisense RNA functions to inhibit protein production. Intriguingly, there have been several reports that some of the more complex retroviruses produce natural antisense transcripts (5
). The best characterized of these RNAs in HIV-1 appears to initiate from multiple transcription start sites 5′ of the 3′ LTR and extend into the pol
region, where a novel polyadenylation site has recently been described (37
). Although additional studies are needed to validate the presence of antisense RNA in HIV-1-infected cells, the evidence for the existence of an antisense transcript in the human T-cell leukemia retrovirus is much more compelling (7
). In light of our finding that antisense transcripts can be potent inhibitors of gene expression in the HIV-1 system, further studies on the role of these natural transcripts in the regulation of HIV-1 and other retroviruses seem warranted.
Although we do not know whether a similar mechanism of antisense inhibition normally operates in the host cell to function in gene regulation, recent evidence indicates that expression of natural antisense RNA to normal gene transcripts may be a common occurrence (9
). Although most mRNAs probably do not traffic down the Crm1 pathway used by Rev and the RRE, this pathway has been reported to be utilized by some mRNAs (35
). Thus, it is possible that mechanisms similar to the one we have described are utilized to regulate expression of cellular mRNAs.
In the case of cellular, as well as viral mRNA, it has been shown that regions of double-stranded RNA, resulting from the presence of inverted sequences in the RNA or association with antisense RNA, are subject to deamination by ADAR, leading to multiple inosines in the RNA (1
). Such RNAs are normally retained in the nucleus through interaction with nuclear matrix proteins and eventually degraded (36
). However, a previous study using the Xenopus
oocyte export model showed that edited RNA was exported to the cytoplasm if the RNA contained an RRE and Rev was provided in trans
). In addition, a previous study by Lu et al. using a lentivirus antisense vector reported that HIV-1 RNA recovered from cells expressing antisense RNA showed multiple mutations in the antisense target region of the HIV-1 genome that were consistent with ADAR activity (42
). These results suggest that at least some of the genomic RNA, which was complexed with antisense RNA and edited as a result of formation of dsRNA, was eventually exported to the cytoplasm and packaged. Therefore, it seems possible that editing activity plays a role in antisense inhibition. It also follows that naturally occurring antisense could help provide retroviruses with an additional pathway for sequence diversification.
However, to date, reverse transcription-PCR sequencing of cytoplasmic RNA in cells transfected with proviral clones and plasmids expressing antisense RNA has failed to detect any mutations indicating ADAR editing (data not shown). Thus, we do not believe that editing is directly connected to the reduced protein levels we observe. Also, the target region is downstream of the GagPol ORF, and mutations in the target region, the 3′ untranslated region, will thus not affect the GagPol protein per se. However, editing of even a small amount of the RNA could potentially lead to the production of miRNA from the antisense RNA (52
). Inhibition by a miRNA-mediated mechanism would be consistent with the efficient inhibition we observe, and miRNA often exerts its effect at the translation level (55
Irrespective of the mechanism utilized for antisense inhibition, our results are of importance for future development in the gene therapy field. The data suggest that it will be advantageous to ensure that any long antisense RNA designed to combat HIV-1 contains the RRE to allow trafficking along the Rev/RRE pathway, since this will likely significantly increase the efficiency of antisense inhibition. Our data also show that an RRE-driven antisense RNA, in combination with Rev, is able to efficiently inhibit a target that utilizes the CTE pathway. This raises the possibility that Rev/RRE trafficking of antisense RNA could also be exploited to make antisense RNA inhibition more efficient for non-HIV-1 applications.