Potential use of retrovirus vectors against HIV-1: construction of ribozymes to interfere with HIV-1 replication

Project Associates / Assistants
G K Shanmugasundaram

Ph D Students
Ritu Goila
Hoshang Unwalla
Samitabh Chakraborti

Host genes, besides viral genes, play a major role in spread and pathogenesis of HIV /AIDS. We wish to identify those genes and selectively interfere with their functions. This goal will be achieved by using nucleic acid based approaches to interfere with the replication of HIV-1. We wish to place these interfering genes in retrovirus vectors and obtain stable gene expression in the host cells that are exploited by HIV, mainly T lymphocytes and macrophages.

HIV-1 uses the HIV-1 coreceptors, the chemokine receptors to gain entry and initiate infection in host cells. Chemokine and chemokine recptors are known to influence transmission, tropism and pathogenesis of virus. Mutations in promoter elements that govern the levels of chemokine and chemokine receptors are now known to modulate the expression of these genes. Several such mutations in CCR5, SDF-1, MCP-1, and MIP-1a and RANTES have been recognized. Besides, certain interleukins (IL4 and IL10) are also known to modulate the expression of chemokine receptors. Ribozymes and DNA-enzymes are novel therapeutic molecules that we wish to exploit to interfere with some of the HIV-1 genes (TAT and Rev) and host genes (CCR5 and CXCR4) that play important roles in HIV-1 replication. Model genes like the X gene of hepatitis B virus and reovirus genes will be exploited to design effective nucleic acid based anti-viral approaches.

DNA-enzymes against HIV-1 TAR element

Since TAR element is very important for HIV-1 transcription and replication, we constructed several DNA-enzymes against the HIV-1 TAR region. HIV-1 TAR DNA containing the entire TAR element was amplified by PCR using specific primers. They were initially cloned into pGEM and then into pcDNA3 expression vector. The recombinant clones were linearized at their 3’ends and transcripts were generated by using a transcription kit (Riboprobe, Promega). Labeled transcripts of the expected sizes were generated. Plasmid pGEM-TAR1 yielded a transcript that contained 77 nucleotides at its 5’end but pCDNA-TAR1 plasmid gave a transcript that was close to the authentic size of TAR RNA. Several DNA-enzymes containing either 10-23 or 8-17 catalytic motif were synthesized and its activity was tested on the above two TAR RNAs. Two DNA-enzymes, 470 and 475 (number reflects its position in the infectious clone pNL4-3), were identified that cleaved pCDNA-TAR1 transcript in a sequence specific and catalytic manner. Mutant DNA-enzyme-475 that contained a point mutation (G to C) in the catalytic motif, failed to cleave the target RNA completely. Since this mutant DNA-enzyme failed to cleave the target RNA completely, it served as an important control for all intracellular studies that are aimed at assessing the anti-viral potential of DNA-enzymes. We also obtained a single Dz-8-17 that cleaved the target RNA specifically. Cleavage reactions were also carried out on TAR RNAs that were synthesized from pGEM vectors. Significantly more number of DNA-enzymes were identified along with DNA-enzyme-470 and 475 in this case also. This experiment suggests that additional sequences at 5’-end (derived from vector) of TAR RNA had rendered it susceptible to more cleavages. These two DNA-enzymes (470 and 475) were introduced into HeLa cells using lipofectin and challenged with HIV-1 encoding DNA – pNL4-3. These DNAs were cotransfected in 1:1(1mg for 1 X106 cells) ratio and amount of virus production was measured by p24 antigen assay. Approximately 80% reduction in the amounts of p24 antigen was detected by DNA-enzymes 470 and 475. Mutant Dz-475 also showed about 40% reduction when compared to control (cells that received only pNL4-3 DNA). This is very likely due to the antisense component of the DNA-enzyme. We also measured the levels of one of the target RNAs, the TAT RNA by RT-PCR from DNA-enzyme transfected cells. HIV-1 TAT specific RNA was down regulated to about 10 fold with either Dz-470 or 475. Under similar conditions, Mutant DNA-enzyme-475 (disabled) showed about 4-fold reduction. Using these two approaches, we could conclude that about 40% specific reduction in virus yields could be attributed to the catalytic nature of these DNA-enzymes. The remarkable finding of this work was that the target sites in RNA that were cleaved by DNA-enzymes were directed towards the single stranded bulge region of TAR (required for base pairing with DNA-enzymes). Regions that were completely base paired, showed no cleavage. Thus, DNA-enzymes provide a powerful tool to predict the secondary structure of a target RNA.

DNA-enzyme against HIV-2 TAR element

HIV-2 TAR element was also amplified by PCR using specific primers. It was cloned into pGEM and pCDNA3 vectors. The latter vector gave a transcript with multiple pause sites. In contrast, the pGEM derived vector gave a single well defined transcripts. Thus insertion of unrelated sequences between the IST element and LTR promoter rendered the IST promoter completely inactive. Earlier multiple short transcripts were shown to be directed by the IST promoter element located down stream of the LTR promoter. This inducer of short transcripts promoter (IST) seems to be far more active in case of HIV-2 than HIV-1. This is an original observation that may explain why HIV-2 infection takes a significantly longer time to cause HIV/AIDS.

Publications

Original peer-reviewed articles

1.     Chakraborti S and Banerjea AC (2003) Inhibition of HIV-1 gene expression by novel DNA-enzymes targeted to cleave HIV-1 TAR RNA: potential effectiveness against all HIV-1 isolates. Mol Ther (in press).

2.     Chakraborti S and Banerjea AC (2003) Identification of cleavage sites in the HIV-1 TAR RNA by 10-23 and 8-17 catalytic motif containing DNA-enzymes. Biomacromol (in press).

3.     Shahi S and Banerjea AC (2002) Multitarget ribozyme against the S1 genome segment of reovirus possesses novel cleavage activities and is more efficacious than its constituent mono-ribozymes. Antiviral Res 55:129-140.

4.     Gaur K, Gupta PK, Banerjea AC and Singh Y (2002) Effect of nasal immunization with protective antigen of Bacillus anthracis on protective immune response against anthrax toxin. Vaccine 20:2836-2839.

5.     *Azim T, Bogaerts J, Yirrell DL, Banerjea AC, Sarkar MS, Ahmed G, Amin NM, Rahman AS and Husain AM (2002) Injecting drug users in Bangladesh: prevalence of syphilis, hepatitis, HIV and HIV subtypes. AIDS 16:121-123 (*in press last year, since published).