Efficacy and mode of action of hammerhead and hairpin ribozymes against various HIV-1 target sites.
Ribozymes have been proposed as gene therapy agents against HIV-1, although many fundamental questions about their mechanism of action remain unclear. Few studies have compared directly the potential of different modified ribozyme species against a particular target. Here we compare the relative abilities of hammerhead (HhU5) and hairpin (HpU5) ribozymes directed against a well-studied target RNA that has therapeutic potential, located in the untranslated 5' region (U5), to inhibit HIV-1 replication. The two types of ribozymes showed similar antiviral efficacy after being stably transfected into HUT78 cells and subsequently challenged with HIV-1(SF2), but the HhU5 ribozyme showed faster cleavage kinetics when tested in a cell-free system. In the second part of this study, we examined whether different ribozymes were able to inhibit the integration of proviral DNA in infected HUT78 cells. We found that cell pools stably expressing HpU5 could limit the appearance of integrated provirus, indicating that they could inhibit the infecting viral RNA before reverse transcription. A preintegration effect was also found for cell pools expressing a ribozyme targeting the nef gene (HhNef) or a ribozyme targeting the LTR (HhLTR). However, no discernible preintegration effects were seen for the HhU5 ribozyme or an active ribozyme directed against an RNA target site in the pol gene (HhPol). Thus, the results suggest that the mode of ribozyme action varied between sites and is not dependent solely on inhibiting the infecting viral RNA. Evidence for a preintegration effect is extremely encouraging and indicates that "resistant" cells have some chance to repopulate the immune system through such a selective advantage. We also studied the ability of the different ribozymes to down regulate viral RNA postintegration.
Hotchkiss
Graham
G
Clinical Research Centre, Department of Laboratory Medicine, Karolinska Institutet, 141 57 Stockholm, Sweden.
Maijgren-Steffensson
Catharina
C
Ahrlund-Richter
Lars
L
D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D015658:HIV Infections / Q000628:therapy; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011533:Proviruses / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016662:Virus Integration / Q000235:genetics / Q000502:physiology; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
D020121:5' Untranslated Regions; D016337:RNA, Catalytic; D012367:RNA, Viral; C418481:hairpin ribozyme; C427204:hammerhead ribozyme; D004247:DNA