Anti-HIV-1 therapy using ribozymes has focused mainly on either targeting the viral RNA or mRNA of cellular genes that may have a role to play in the viral life cycle. In order to achieve a near complete knockdown of viral gene expression, one needs to take into account several factors that may influence the cleavage efficiency of these antiviral agents. The most important considerations are target site accessibility and colocalization of the ribozyme and target. Although a number of guiding parameters are available to aid the selection of efficient target sites, most of these parameters are established in vitro and then tested in cells. Most approaches to ribozyme design begin with choosing an appropriate NU(A, C, or U) target site in the message of interest. These approaches do not take into account factors that can alter the efficiency of the ribozymes in an intracellular environment, such as local structural features in the target mRNA and RNA–protein interactions. By using an in vivo or intracellular library approach these concerns are minimized. Moreover, by modifying the selection process, one can enrich for ribozymes that achieve the desired phenotype without necessarily requiring complete target knockdown.
In this report, we have exploited the potent killing effect of the TK-gancyclovir axis in an HIV-1 proviral DNA setting. The hammerhead ribozymes were incorporated in the U16 snoRNA stem to localize them to the nucleolus, an intracellular compartment that we found to be effective for ribozyme targeting of singly spliced and unspliced HIV-1.10
We reasoned that this approach would result in a large number of ineffective ribozymes being eliminated in the first round of selection. Indeed, we saw almost ~80% of the cells detached from the surface during the first round of selection. On the other hand, cells harboring a protective ribozyme would inhibit expression of TK and survive. The ribozyme molecules from these cells could then be rescued by reverse transcriptase-PCR of total RNA using primers specific to the U16 stem. After three rounds of selection with this approach, random sampling of the colonies revealed two sequences that had been enriched during the process. We also randomly tested other library ribozymes along with these two for their activity against HIV-1. We were surprised to find one that routinely resulted in a higher p24 output as compared to the U16-transfected control. When we tested this ribozyme in cell survival assays using HIV-1 pNL-TK we found that the ribozyme promoted cell survival at a level comparable to that observed with RzC36. We are currently investigating the mode of inhibition and the potential target site for this ribozyme sequence. Another ribozyme sequence that we had obtained in four different clones did not show any inhibition of HIV-1 p24 levels. However, further analysis showed that the ribozyme had a marked complementarity with the TK open reading frame and could promote cell survival when cotransfected with pNL-TK in the presence of gancyclovir (Supplementary Figure S1
). This could explain why the ribozyme had been selectively enriched during screening but was unable to show inhibition of viral gene expression with HIV-1 pNL4-3, because HIV-1 pNL4-3 lacks the target site for RzC43. However, the target site is not a canonical NUH target and is, in fact, a GUG. Earlier studies have presented conflicting observations regarding cleavage of GUG targets. Koizumi et al
found that a target site with this sequence could not be cleaved, whereas Sheldon and Symons24
observed considerable cleavage of a GUG substrate in a unimolecular ribozyme substrate reaction. Perriman et al.25
have demonstrated that a GUG triplet could be induced to cleave by modifying the helix II. Indeed, there were minor differences among the catalytic motifs used in these studies. In our work, we have used a minimal helix II reported by Persson et al.26
It is quite possible that the selection process we employed transcends the guidelines of cleavage established in vitro
using perfectly matched Watson–Crick base pairing, and takes into account interactions between the bulges and mismatches in helix I and III with the helix II. Using in vitro
selection procedures, Kore et al
. were able to isolate hammerhead ribozymes with a mutation at position 7 in the catalytic motif,27
which is capable of cleaving GAC triplets, another anomaly to the established NUX rule.
With the goal of determining effective target sites in the HIV-1 genome, we decided to proceed with the investigation of RzC36 which, in addition to surviving gancyclovir, showed potent inhibition of HIV-1 gene expression. RzC36 did not share perfect complementarity with any region of the HIV-1 genome. However, a cDNA array analysis of cells cotransfected with RzC36 and HIV-1 pNL4-3 showed a complete reversal of the cDNA array pattern observed with HIV-1 pNL4-3 cotransfection with the control U16 backbone vector. These results suggested that RzC36 was targeting viral RNA, which could explain the complete reversal of virus-mediated changes observed when this ribozyme was cotransfected with HIV-1 pNL4-3.
In order to identify potential target sites for RzC36, we relaxed the rules of target site determination to a minimum of 6 nt of base pairing in the hybridizing arms of the ribozyme with the target sequence. Using this approach we were able to identify two potential target sites in HIV-1 NL4-3 in the U5 region and in pol. The U5 site is present in all forms of viral transcripts, whereas the site in pol at position 4850 would not be present in the partially or fully spliced forms of the viral RNAs. Both sites turned out to be very good ribozyme sites in HIV-1, resulting in ~75 and 80% inhibition of p24 levels in transient transfection assays with pNL4-3, respectively. Moreover, these ribozymes were protective in HIV-1 challenge assays when they were stably expressed in CEM cells, thereby suggesting that RzC36 could have been selected for its ability to target both sites. This is not surprising, given that a significant level of knockdown of TK expression would be required for the cells to survive the gancyclovir treatment.
In summary, the use of nucleolar-localizing hammerhead ribozymes has proven to provide potent inhibition of HIV-1 replication in cell culture.10
The results presented in this study provide further support for our model that HIV-1 traffics through the nucleolus, and also provides a rationale for the using nucleolar-localizing ribozymes to inhibit HIV-1 infection.