RNAi has shown great potential to investigate gene functions and as a potential new class of drugs to suppress disease-causing genes. The beauty of the system that makes it a powerful tool lies in sequence specificity towards particular gene, its quick effect, easiest and most cost effective. RNAi based potential therapeutics would offer advantage over traditional drug molecules as 1) RNAi can target any gene so eventually every protein, but traditional drugs majorly target only certain classes of proteins e.g. G-protein-coupled receptors and ion channels etc. 2) RNAi can block the production of disease-causing proteins before they are made, therefore have greater potential in disease control and intervention. 3) Most importantly, the junction of two protein producing genes as well as regulatory elements could be an important target site, which is only possible by siRNA. 4) As the HIV genome integrates into host genome and persists thus there is great potential to inactivate only HIV genome by using siRNA based approaches.
Unfortunately, HIV may escape the effect of siRNA by mutations in the siRNA target sequences. These escape mutations can decrease or altogether abolish the siRNA inhibition effect. We have collected 107 such HIV escape mutant sequences targeted by 41 siRNAs. Substitution mutations are observed in majority of sequences and clustered around the middle region of the target sequence i.e. 8 to 11 positions. It seems that escape substitution mutations are preferred in the middle region than flanking regions of the target sequences. Simultaneously, deletion mutations were scattered through all the positions in the target sequences.
The mutation generated at target site or nearby results either (A) lack of 100% complementarities between target and siRNA
[26],
[27] or (B) target site forms a secondary structure and thus not accessible for siRNAs that abolish the siRNA effect
[28]. To overcome escape mutants, design of siRNAs targeting highly conserved genome regions that are essential for virus life cycle are preferred. Another promising approach is co-expressing multiple shRNAs that simultaneously target different regions of the viral genome
[29],
[30],
[31] or inhibition of HIV-1 replication with RNAi against cellular co-factors
[32].
We have checked the conservation of target sequences of each siRNA among 1496 HIV reference sequences. Analysis showed that for only 1% siRNA were conserved in over 90% target sequences. It could be attributed to high variation among HIV reference sequences. For 13% siRNA target sequences conservation was between 80–90% while 45% siRNA has less than 50% target conservation.
In the database, most of the siRNA efficacy was observed in 293T and Hela cell lines. One potential problem could be the interpretation of these results when comparing them to the natural infection in primary cells. We have analyzed a few siRNA experiments carried out in both cell lines as well as in the primary cells (peripheral blood mononuclear cells: PBMCs
[33],
[34], peripheral blood lymphocytes: PBLs
[10],
[35] and CD4+ T cells etc.
[36],
[37]).
Chang et al. reported the efficient inhibition of HIV infection (>90%) using siRNAs targeting highly conserved pol and vpu sequences against different strains of HIV-1 in 293T cells and marked inhibition effect in primary PBMCs
[35]. Sang-Kyung Lee et al. showed that three anti-HIV shRNAs targeting rev, gag, and vif reduced the p24 level by more than 90%, demonstrating their ability to protect primary CD4
+ T cells, which are the major targets of HIV-1 infection
in vivo, from homologous virus as they did for HeLa-CD4 cells
[37]. Jean-Marc Jacque et al. also described inhibition of early and late steps of HIV-1 replication in Magi cells and primary PBLs by six siRNAs targeted to LTR, vif and nef regions of the HIV-1 genome
[10]. These studies suggest that that siRNA perform equally well in both cell lines and primary cells.
RNAi has shown great potential of providing a new class of antiviral therapeutic molecules. Sequence-specific degradation of viral RNA by siRNAs/shRNA gives promising results for the treatment of HIV infection. Various studies have shown selective inhibition of viral genes/proteins that are crucial for HIV-1 replication through transiently expressed synthetic siRNA or continuous expression of vectors containing shRNAs expression cassettes. However, there is no single platform having complete information about siRNA used against HIV. This results in delay in the literature mining, especially in the presence related articles describing siRNA targeting the host genes. So as to speedup research, we have developed HIVsirDB database having comprehensive information about the siRNA/shRNA used in the past targeting every region of the HIV genome.
Users can explore information about the siRNAs/shRNAs sequences, target HIV genome region, efficacies and the experimental conditions prior their experiments in user friendly manner using the search and browsing facility. siRNA analysis tools will help the users to map siRNA on their target sequences and to know the siRNA sequence conservation among 1496 reference strains. Simultaneously, HivsirMut will provide effect of escape mutations and nucleotide mismatch between siRNA and target on the potency. This information would help in picking up the best and effective siRNAs and target genome region for further research.
HIVsirDB, the freely available open source database, would be very useful to experimentalists in deciding the highly potent siRNAs targeting the most susceptible target site in HIV genome and its experimental procedure to suppress HIV infection. The online submission facility will be helpful for updating this database.
Limitations and future prospects
Our major limitation in developing the database is that the information about siRNAs/shRNAs targeting HIV are too scattered. Extensive literature search is required to further expand the database as numbers of articles were on siRNA/shRNA targeting the host organism.
In future, HIVsirBD would be expanded by including the comprehensive information about siRNAs targeting host genes and their effect on viral entry and propagation. Data of the multiple siRNA targeting different genome regions simultaneously would also be integrated. This database will be updated manually as soon as enough data will be available.
Availability and requirements
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