Inhibition of HIV-1 Replication by an HIV-1 Dependent Ribozyme Expression Vector with the Cre/loxP (ON/OFF) System

Abstract

Antiviral strategies to inhibit HIV-1 replication have included the generation of gene products that provide the intracellular inhibition of an essential viral protein or RNA. When used in conjunction with the HIV-1 long terminal repeat (LTR), an inducible promoter dependent on the virus-encoded trans-activator (tat), relatively high background activity is still observed in the absence of tat (Caruso & Klatzmann, 1992; Dinges et al., 1995). In order to circumvent this problem, we used the Cre/loxP (ON/OFF) recombination system as a tool for our investigation. In the present study, we constructed a loxP-cassette vector with the ribozyme (Rz) expression portion under the control of the tRNAi(Met) promoter between two loxP sequences (plox-Rz-U5). We also constructed an HIV-1 LTR promoter-driven Cre recombinase gene (pLTR-Cre). These vectors were triple-transfected into HeLa CD4 cells with the HIV-1 pseudotype viral expression vector. Basal activity was not detectable before HIV-1 infection. The LTR-dependent Cre protein product in HIV-1 infected HeLa CD4 cells expressed the ribozyme by inducing loxP homologous recombination, which strongly inhibited the HIV-1 gene expression. These results demonstrate the potential of an anti-ribozyme with the Cre/loxP system for controlling HIV-1 infection via gene therapy.

Suppression of Human Immunodeficiency Virus Type 1 (HIV-1) Replication by an HIV-1-dependent Double Locked Vector with the Cre/loxP System

“…However, the pLTR-Cre showed slight expression of the luciferase gene in HIV-1 uninfected HeLa CD4 + cells. [18] Recently, Miyake et al reported the use of the LTR promoter including the region from the 5 U3 to the middle of gag. [20] Hence, we also constructed an HIV-1-specific Cre expression vector that includes the LTR-gag-p17 promoter (from the 5 LTR U3 to the end of gag-p17, 1.2 kb, Figure 1).…”

“…After digestions of the PCR amplified LTR product with SpeI and EcoRI, and the pGEM-Cre vector with MfeI and BamHI, each fragment was ligated into the SpeI/BamHI sites of the plasmid containing the Cre gene (pBS185), to yield pLTR-gag-p17-Cre. In order to add the XhoI cloning site to the plox-Rz-U5 [18] fragment, plox-Rz-U5 was first digested by NotI and SpeI. This fragment was then cloned into the NotI and SpeI sites in pVAX-1 (Invitrogen) to yield pVAX-LRU, which contained the needed XhoI site.…”

“…[18] First, to characterize the luciferase gene expression under the control of the CreloxP system, we compared the luciferase activities of an HIV-1 minimal LTRdriven luciferase expression plasmid (pLTR-luc) and a new HIV-1 LTR-gagp17 driven Cre expression plasmid, pLTR-gag-p17-Cre and ploxP-luc, in the absence or presence of pNL4-3 ( Figure 2). When HeLa CD4 + cells were transfected with the minimal pLTR-luc expression plasmid, the luciferase activity was observed in the absence of pNL4-3 (lane 3).…”

“…[13] Previously, we demonstrated the function of an HIV-1-dependent ribozyme expression vector, with which the site-specific excision of loxP sequences [14][15][16] can be achieved by using the HIV-1 long terminal repeat (minimal LTR)-Cre-loxP system (ON/OFF) as a molecular switch in an acute HIV-1 infection. [17,18] It is also demonstrated that the retroviral vector incorporated ribozyme inhibit HIV-induced apoptotic cell death. [19] However, our expression system also exhibited lower, non-specific expression of the anti-HIV-1 ribozyme in the absence of tat.…”

Suppression of HIV-1 Replication by a Combination of Endonucleolytic Ribozymes (RNase P and tRNase ZL)

“…The RT-PCR reactions were used to establish the level of uncleaved HIV-1 mRNA (product I and II). [28] The uncleaved HIV-1 mRNA was amplified by the vifF1and tat-F1 primers, and the vif-R and tat-R primers ( Figure 5A). The level of product I and II were expected to decrease after cleavage of the HIV-1 mRNA.…”