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

Project Associates
N Arumugam
R Kaushik
G K Shanmugasundaram

Ph D Students
Shweta Shahi (till Jan 2001)
Ritu Goila
Hoshang Unwalla
Samitabh Chakrabarti

HIV-1 virulence, progression and pathogenesis is governed by multiple factors that include the host genes, the genetic make up of the virus and immunological factors, besides many others. We wish to exploit some of the genes that play a critical role in progression of HIV-1 by using ribozymes and DNA-enzymes that are designed to interfere with the gene expression in a sequence specific manner. These interfering genes will be placed in retrovirus-based vectors for gene transfer studies.

Variety of host genes, in particular the chemokines (chemokines, chemokine receptors, etc.) are involved in transmission, tropism and pathogenesis of the virus. Recently certain mutations in the interleukinn-10 promoter region have been identified that affect the progression of HIV-1 in humans. IL-4 is known to regulate the expression of CCR5 and CXCR4 and also the regulatory protein TAT. We wish to identify the genetic variants in chemokine/cytokine genes that might affect the progression of the HIV/AIDS. Nucleic acid molecules (ribozymes and RNA cleaving DNA-enzymes) that possess sequence specific cleavage activities are novel therapeutic molecules that have the potential to interfere with the expression of a target gene. We wish to exploit the use of these catalytic molecules to interfere with the expression of HIV structural and regulatory genes, host factor genes that are important for HIV infection and replication along with some model viral genes (hepatitis X and reovirus S1 gene).

A.        Construction of DNA-enzymes against HIV-1 and HIV-2 TAR element

A unique feature about all the messenger RNAs in HIV-1 is the presence of a unique stem-loop structure where HIV-1 regulatory protein TAT binds. Therefore, ribozymes or DNA-enzymes that are targeted to cleave at this region have potential to down regulate all the HIV-1 messenger RNAs and their encoded proteins. We, therefore cloned the substrate (HIV-1 TAR element) into an expression vector using specific primers using the infectious HIV-1 DNA-pNL4-3. A number of DNA-enzymes with a 10-23 catalytic motif were synthesized and tested in vitro for their sequence specific cleavage activities. Two DNA-enzymes that were targeted against the loop region of the TAR element possessed the cleavage activity and the rest that were targeted against the stem, possessed no activity. Thus these DNA-enzymes can be used to probe the secondary structure of RNA. HIV-2 TAR element has been cloned in an expression vector and ribozymes or DNA-enzymes will be designed. We have identified inducer of short transcripts (IST) promoter determinants in case of HIV-1 that governs the synthesis of multiple short transcripts in the absence of HIV-1 TAT protein.

B.    Functional analyses of the ribozymes and DNA-enzymes against HIV-1 genes

HIV-1 TAT is known to have multiple effects on the cell and contribute significantly towards pathogenesis. We reported earlier number of mono- and multi-ribozymes against HIV-1 TAT/ Rev region. Conditions for apoptosis were established in a T cell line (Molt-1) that was transfected with CMV driven HIV-1TAT plasmid DNA. Upon transfection, these cells showed apoptosis as visualized by a kit by observing peroxide stained cells under inverted microscope. Preliminary experiments indicated protective effects of ribozyme against HIV-1 TAT mediated apoptosis. This will be repeated with the DNA-enzyme also that have been constructed against HIV-1 TAT/Rev RNA.

C.    Host factors that modulate HIV progression

Chemokines and interleukins play a critical role in the life cycle of HIV. Chemokine receptors act as HIV-1 coreceptors whereas the chemokines can interact with their receptors and block HIV-1 entry. The role of various interleukins may be indirect in this setting. It is now known that certain promoter mutations in IL-10, RANTES and IL-4, may influence the progression of HIV-1. Chemokine stromal cell derived factor-1 (SDF-1) is a ligand for the chemokine receptor CXCR4 that T-tropic isolates of HIV-1 use to gain entry. Studying the regulation of this gene is, therefore, important. We earlier reported high frequency of transition mutation (G to A in the 3’-untranslated region) in normal Indian population. We extended this study with more number of individuals (n = 100) and the trend remained the same, i. e., about 40 % have this mutation with about 8% being homozygous. We identified at least 3 proteins that bind to this untranslated region of SDF-1 RNA in humans and monkeys by gel shift analyses and there was change in electrophoretic mobility in one of the binding proteins when compared with humans and monkeys. Wild-type humans as well as heterozygous of homozygous individuals did not show any change in the binding pattern. Certain IL-10 promoter mutations have recently been linked with progression of HIV. We wish to find out the status of the mutations in Indian population. We carried out genetic analyses of the IL-10 promoter mutation in monkeys and found three novel types of mutations (substitution of large sequences, deletion and unique insertions). The functional implications of these changes will be studied in future. We also reported the nature of polymorphism in the HIV-1 coreceptor-CXCR4 promoter in the monkeys and found out that one of the two highly polymorphic regions in the CCR5 promoter region is lacking in rabbits (NZB) when compared with monkeys.

Publications

Original peer-reviewed articles

1.     Azim T, Bogaerts J, Yirrell DL, Banerjea AC, Sarker 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.

2.     Ramamoorti N, Kumarvelu J, Shanmugasundaram GK, Rani K and Banerjea AC (2001) High frequency of G to A transition mutation in the stromal cell derived factor-1 gene in India, a chemokine that blocks HIV-1 (X4) infection: multiple proteins bind to 3’-untranslated region of SDF-1 RNA. Genes & Immunity 2:408-410.

3.     Kumarvelu J, Shanmugasundaram GK, Unwalla H, Ramamoorti N and Banerjea AC (2001) Genetic analyses of the promoter region of interleukin-10 gene in different species of monkeys: implications for HIV/AIDS progression. Genes & Immunity 2:404-407.

4.     Shanmugasundaram GK, Sundaresan G, Shoeb F, Arumugam N, Kumarvelu J, Unwalla H, Chakraborti S and Banerjea AC (2001) Genetic analyses of cis-acting sequences controlling expression of HIV-1 coreceptor-CCR5 in rabbits and CXCR4 gene in monkeys. J Hum Virol 4:188-194.

5.     Unwalla H and Banerjea AC (2001) Novel mono- & di-DNA-enzymes targeted to cleave TAT or TAT-Rev RNA inhibit HIV-1 gene expression. Antiviral Res 51:127-139.

6.     Unwalla H and Banerjea AC (2001) Inhibition of HIV-1 gene expression by novel macrophage-tropic-DNA-enzymes targeted to cleave HIV-1 TAT/Rev RNA. Biochem J 357:97-105.

7.     *Shahi S, Shanmugasundaram GK and Banerjea AC (2001) Ribozymes that cleave reovirus genome segment S1 also protects cells from pathogenesis caused by reovirus infection. Proc Natl Acad Sci USA 98:4101-4106 (*in press last year, since published).

8.     *Goila R, Felix K and Banerjea AC (2001) MIP1-a promoter polymorphism in humans and monkeys: identification of two polymorphic regions characterized by insertion of unique sequences in monkeys. AIDS 15:1066-1068 (*in press last year, since published).