Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev response element decoy consisting of the 13-nucleotide minimal Rev-binding domain

Abstract

Intracellular immunization is an anti-viral gene therapy strategy based on the introduction of DNA templates into cells to stably express genetic elements which inhibit viral gene expression and replication. We have recently developed an intracellular immunization strategy for human immunodeficiency virus (HIV) infection that uses RNA decoys. RNA decoys are short RNA oligonucleotides corresponding to the HIV trans activation response element (TAR) or Rev response element (RRE) sequences, which function by inhibiting the binding of the HIV regulatory proteins Tat and Rev to the authentic HIV RNA TAR and RRE regions, respectively. In this report we describe the characterization of potent RRE decoys containing the minimal 13-nucleotide primary Rev binding domain of the RRE. Using an improved tRNA cassette to express high levels of RRE transcripts in CEM cells, we found that this new generation of minimal RRE decoys were more potent inhibitors of HIV in isolated cell lines than previously described TAR or RRE decoys. CEM cells expressing RRE decoys exhibited diminished Rev function in cotransfection assays, confirming the specificity of inhibition of HIV by RRE decoys and indicating that the 13-nucleotide minimal Rev binding domain defined by using in vitro binding studies also binds Rev in vivo. Significant differences in the degree of HIV inhibition between individual CEM cell lines transduced with RRE decoy vectors which were not due to sequence alterations in the tRNA-RRE DNA template, differences in RRE decoy expression level, or endogenous variations in the resistance of CEM clonal cell lines to HIV were observed. In order to evaluate the efficacy of RRE decoys in a more realistic fashion than by comparison of individual clonal cell lines, polyclonal populations of transduced CEM cells were infected with HIV. By using a novel flow cytometric method for quantitating intracellular p24 expression, one version of the RRE decoys tested in this study was found to be capable of durably protecting polyclonal populations of CEM cells from HIV.

Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes

“…1C) was constructed as previously described (9). The antisense-TAR sequences were derived from the HIV-1 MN isolate (20) and were expressed as a tRNA i Met -antisense-TAR fusion transcript in a double-copy vector design (1,27,28,50). The antisense-Tat/Rev gene used to generate the antisense-Tat/Rev retroviral vector GC-(anti-Tat/Rev)SN DC (Fig.…”

Isolation of a nuclease-resistant decoy RNA that selectively blocks autoantibody binding to insulin receptors on human lymphocytes.

“…Unfortunately, isolation of small peptides that bind avidly to target proteins has been difficult, probably because short peptides cannot adopt stably folded structures very readily (2). By contrast, short RNA molecules can form fairly stable structures through basepairing interaction (3), and RNA decoys have been reported that avidly bind and block a variety of proteins (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Expression of TAR and RRE decoy RNAs has been shown to render CD4 + T cells resistant to HIV replication by inhibiting the functions of the RNAbinding proteins tat and rev (8)(9)(10)(11)(12).…”

“…By contrast, short RNA molecules can form fairly stable structures through basepairing interaction (3), and RNA decoys have been reported that avidly bind and block a variety of proteins (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Expression of TAR and RRE decoy RNAs has been shown to render CD4 + T cells resistant to HIV replication by inhibiting the functions of the RNAbinding proteins tat and rev (8)(9)(10)(11)(12). Furthermore, by using in vitro selection techniques, "designer" decoy RNAs have been isolated from large pools of random RNA molecules that bind with high affinity and specificity to several proteins, including a few that do not naturally interact with nucleic acids (13,(15)(16)(17)(18)(19)(20)(21)(22).…”

Intracellular expression of RNA transcripts complementary to the human immunodeficiency virus type 1 gag gene inhibits viral replication in human CD4+ lymphocytes

“…Gene therapy approaches (intracellular immunization) were proposed in 1988 (3) as an alternative treatment modality for human immunodeficiency virus type 1 (HIV-1) infection. Several therapeutic genes, including transdominant mutant proteins (2,4,33,34), RNA decoys (13,32), ribozymes (21,37), and antisense sequences (7,11,28,30), have been evaluated preclinically (9). Multiple therapeutic genes (a transdominant Rev gene and RNA decoys) have been evaluated previously for their potential to inhibit HIV-1 replication in vitro (4,5,8).…”