ABSTRACT
DNAzymes are easier to prepare and less sensitive to chemical and enzymatic degradation than ribozymes; however, a DNA enzyme expression system has not yet been developed. In this study, we exploited the mechanism of HIV-1 reverse transcription (RT) in a DNA enzyme expression system. We constructed HIV-1 RT-dependent lentiviral DNAzyme expression vectors including the HIV-1 primer binding site, the DNA enzyme, and either a native tRNA (Lys-3), tR(M)DtR(L), or one of two truncated tRNAs (Lys-3), tR(M)DΔARMtR(L) or tR(M)D3'-endtR(L). Lentiviral vector-mediated DNAzyme expression showed high levels of inhibition of HIV-1 replication in SupT1 cells. We also demonstrated the usefulness of this approach in a long-term assay, in which we found that the DNAzymes prevented escape from inhibition of HIV. These results suggest that HIV-1 RT-dependent lentiviral vector-derived DNAzymes prevent the emergence of escape mutations.
Subject(s)
Antiviral Agents/metabolism , DNA, Catalytic/metabolism , HIV-1/genetics , Reverse Transcription , Virus Replication , Antiviral Agents/chemistry , Cell Line , DNA, Catalytic/genetics , Gene Expression , Genetic Vectors/chemistry , HIV-1/metabolism , HIV-1/physiology , Humans , Lentivirus/genetics , Mutation , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , RNA, Transfer/genetics , RNA, Viral/metabolism , env Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
The RNA interference (RNAi) phenomenon is a recently discovered process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of mRNA containing the same sequence. We designed mammalian expression vectors that direct the synthesis of small interfering RNA (siRNA)-like transcripts and examined them for their siRNA-mediated gene interference targeting the env gene (NL4-3:7490-7508, E7490). We constructed siRNA expression vectors for two different strands (sense and antisense; tandem promoter) and for siRNA expressed from the short hairpin RNA (shRNA). The inhibition efficacy on HIV-1 replication differed between these two vectors. Notably, the shRNA vector pU6-env-shRNA inhibited p24 production more effectively than the tandem promoter expression vector pU6-env-siRNA. Furthermore, we examined the ability of lentiviral vectors expressing shRNA to suppress HIV-1 expression in HIV-1-infected SupT1 cells. The env-shRNA (E 7490) almost completely suppressed HIV-1 expression in infected cells for up to 15 days.
Subject(s)
Anti-HIV Agents/pharmacology , Gene Expression Regulation, Viral/drug effects , Gene Silencing/drug effects , HIV-1/drug effects , HIV-1/genetics , RNA, Small Interfering/pharmacology , Animals , COS Cells , Cell Line , Cells, Cultured , Chlorocebus aethiops , Genetic Vectors/pharmacology , Humans , Microbial Sensitivity Tests , Structure-Activity Relationship , Virus Replication/drug effectsABSTRACT
We designed a vector to produce single-stranded DNA (ssDNA). We used HIV-1 reverse transcription for the purpose of constructing a DNAzyme expression vector against the HIV-1 env V3 loop. Initiation of HIV-1 reverse transcription requires the formation of a complex containing the viral RNA, tRNALys and reverse transcriptase. The expression vector contains the HIV-1 primer binding site (PBS) and tRNALys at the 3' end of its RNA transcript, thus enabling to an ssDNA would be synthesized by HIV-1 reverse transcriptase. We have demonstrated that the DNAzyme expressed by the lentiviral vectors suppressed HIV-1 replication in SupT1 cells.
Subject(s)
DNA, Catalytic/metabolism , Genetic Therapy/methods , Genetic Vectors , HIV-1/genetics , Lentivirus/genetics , Cell Line , DNA, Catalytic/biosynthesis , DNA, Single-Stranded/biosynthesis , HIV Infections/therapy , Humans , RNA, Viral/metabolism , Reverse Transcription , Virus ReplicationABSTRACT
Since phenoxazine is an essential structure of actinomycin D, which exerts a strong anticancer effect, we examined the anticancer effect of 2-aminophenoxazine-3-one (Phx-3) on mouse malignant melanoma B16 cells in vitro and in vivo. Phx-3 inhibited proliferation of the B16 cells in a dose-dependent manner in vitro. We furthermore studied the in vivo effects of Phx-3 on mouse malignant melanoma B16 cells transplanted in female C57BL/6Cr Slc mice. Treatment with Phx-3 (0.5 mg/kg) completely suppressed the growth of mouse malignant melanoma B16 cells transplanted in mice as compared with the control group. Phx-3 was found to exert few adverse effects, in terms of bodyweight loss, changes in serum levels of blood biochemical parameters such as aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN) and creatinine, dysfunction of the liver and the kidney examined by pathological methods, piloerection and wasting, when mice were treated with a dose of 0.5 mg/kg. These results suggest that Phx-3 may be used to treat patients affected by malignant melanoma in future.
Subject(s)
Cell Proliferation/drug effects , Melanoma, Experimental/prevention & control , Oxazines/pharmacology , Alanine Transaminase/blood , Animals , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/therapeutic use , Aspartate Aminotransferases/blood , Blood Urea Nitrogen , Cell Line, Tumor , Creatinine/blood , Female , Melanoma, Experimental/blood , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Oxazines/therapeutic use , Time FactorsABSTRACT
The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (> or = 90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490-7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.
Subject(s)
Anti-HIV Agents/pharmacology , Gene Expression Regulation, Viral/drug effects , Gene Silencing , HIV-1/genetics , RNA, Small Interfering/pharmacology , Transduction, Genetic , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Gene Silencing/drug effects , Genes, env/drug effects , Genes, env/genetics , Genetic Therapy/methods , Genetic Vectors/genetics , HIV-1/drug effects , Humans , Microbial Sensitivity TestsABSTRACT
The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of a mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that dsRNA specifically suppresses the expression of HIV-1 genes. To study dsRNA-mediated gene interference in HIV-1-infected cells, we have designed six long dsRNAs containing the HIV-1 gag and env genes. HIV-1 replication was totally suppressed in a sequence-specific manner by the dsRNAs in HIV-1-infected cells. Especially, E2 dsRNA containing the major CD4-binding domain sequence of gp120, as the target of the HIV-1 env gene, dramatically inhibited the expression of the HIV-1 p24 antigen in PBMCs for a relatively long time. The dsRNA interference method seems to be a promising new strategy for anti-HIV-1 gene therapeutics.
