|
Potential use of retrovirus vectors against HIV-1: construction of ribozymes to interfere with HIV-1 replication |
|
Principal Investigator : Akhil C Banerjea Project Associates/Assistants Ph D Students The main focus of the work is to understand the mechanisms for the rapid spread of HIV-1 in India that includes the host, virological and immunological factors. The identification of various factors will provide us tool to interfere effectively with the spread of the virus. Many host genes, in particular the chemokines (chemokines, chemokine receptors, etc) are involved in transmission, tropism and pathogenesis of the virus. We wish to identify the genetic variants that might affect the progression of the disease AIDS. Nucleic acid molecules (ribozymes & 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 and some model viral genes (hepatitis X and reovirus S1 gene). Construction of a di-ribozyme (S1-553-984-Rz) Hammer-head ribozyme
(Rz-984) against the reovirus S1 gene was cloned downstream of the
ribozyme-553 in direct tandem and then cloned into a T-Tailed vector. The
recombinant clones were subjected to sequencing that confirmed the
authenticity of the clone. This clone directed the formation of correct size
ribozyme transcript once linearized at the 3’-end and subjected to in vitro
transcription. The di-ribozyme mediated cleavage was more effective than the
two mono-ribozymes individually. These ribozymes when introduced into a
mammalian cell (now under CMV promoter) and subsequently challenged with
reovirus showed remarkable protection against the CPE caused by the virus. We
have identified reovirus S1 gene as the major determinant for cytopathic
effects caused by the virus. Ribozyme and DNA-enzymes against CXCR-4 genes We reported earlier the
construction of hammer-head ribozyme and a 10-23 catalytic motif containing
DNA-enzyme against the CXCR-4 gene. CXCR-4 is the most important HIV-1
coreceptor present on the surface of T lymphocytes. These molecules showed
sequence-specific cleavage activities and could functionally interfere in a
cell membrane fusion assay. These constructs have been given to Dr Samit
Chatterjee, NCCS, Pune, so that it could be placed into a retrovirus vector
with the ultimate aim to introduce them in a stem cell. Host factors that modulate HIV progression Chemokines and chemokine
receptors play a vital role in the life cycle of HIV-1. We were first in
identifying the HIV-1 protective mutation (The D32 mutation in the CCR5 gene)
in Asia (Gene 207, 141-147, 1998). This mutation is very rare in India (1 out
of 600 screened). The cis- acting regions controlling the expression of the
CCR5 gene is known to be very polymorphic, some of these mutations are known
to affect progression of HIV-1. We have screened few normal individuals and we
found 59353 T to C and 59402 G to A mutation, with majority of the individuals
(6 out of 8) having both the mutations. The a-chemokine SDF-1 gene mutation (G
to A) that is known to modulate the progression of HIV is quite common in
India (40%, 32/80) with ~ 8% being homozygous. We reported a remarkable double
transition (GG to AA) in monkeys. We also carried out the genetic analysis of
the CCR5 promoter region in monkeys and novel mutations with unique
deletions/substitutions were observed. We established that the CCR5 promoter
of humans have acquired additional sequences (compared to monkeys) to achieve
higher level of complex regulation. RANTES is a powerful b-chemokine that is
known to inhibit HIV-1 replication. Recently many mutations have been
identified (mainly at -28 and -403 regions from start site) in humans that can
affect the progression of HIV. We analyzed about ~400 bases of the RANTES
promoter region in humans and monkeys and observed several polymorphic regions
in monkeys and interestingly, they also showed unique polymorphisms at -28 and
-403 region. Mutations unique among different species of monkeys were also
reported. Our data provides important clues as to why the course of HIV-1,
HIV-2 and SIV infection and disease pattern is so different in different
species of monkeys. Construction of hammer-head ribozymes and DNA-enzymes
against HIV-1 gag, Tat & Tat/Rev Two mono-ribozymes or
DNA-enzymes were placed in tandem to make multitarget ribozymes or
DNA-enzymes. We identified two potent HIV-1 gag-DNA enzymes from a
combinatorial library. The multitarget ribozymes or DNA-enzymes when incubated
with their target RNA, specific cleavage products could be observed. HIV-1 gag
and Tat/Rev ribozymes have been given to Dr Samit Chatterjee, NCCS, Pune for
gene transfer studies as described earlier. Ribozymes & DNA-enzymes against X gene of
hepatitis B virus Two mono-ribozymes and
two DNA-enzymes against the X gene were placed in tandem through recombinant
techniques. In vitro synthesized full length X RNA was cleaved in a sequence
specific manner into multiple fragments that retained the cleavage specificity
of the mono-ribozymes or mono-DNA-enzymes. RNAse protection assay was used to
quantitate the levels of target RNA. Publications Original peer-reviewed articles 1. Shahi S, Shanmugasundaram GK and Banerjea AC
(2001) Ribozymes that cleave reovirus genome segment S1 also protect cells
from pathogenesis caused by reovirus infection. Proc Natl Acad Sci USA (in
press). 2. Goila R, Felix K and Banerjea AC (2001)
MIP-1a promoter polymorphism in humans and monkeys: identification of two
polymorphic regions characterized by insertion of unique sequences in monkeys.
AIDS (in press). 3. Goila R and Banerjea AC (2001) Inhibition of
hepatitis B virus X gene expression by novel DNA enzymes. Biochem J
353:701-708. 4. Sriram B and Banerjea AC (2000) In
vitro-selected RNA cleaving DNA-enzymes from a combinatorial library are
potent inhibitors of HIV-1 gene expression. Biochem J 352:667-673. 5. *Ramamoorti N and Banerjea, AC (2000) Novel
SDF-1 gene mutations in Simians: presence of GG to AA transition in the 3’
untranslated region. AIDS 14:1279-1281 (in press last year, since published). 6. *Basu S, Sriram B, Goila R and Banerjea AC
(2000) Targeted cleavage of HIV-1 coreceptor-CXCR-4 by RNA-cleaving
DNA-enzyme: inhibition of coreceptor function. Antiviral Res 46:125-134 (*in
press last year, since published). 7. Shanmugasundaram GK, Ramamoorti N and
Banerjea AC (2000) Novel HIV-1 coreceptor-CCR5 promoter mutations in Simians:
identification of two highly polymorphic regions with extensive deletions.
AIDS 14:2201-2202. 8. Ramamoorti N, Goila R and Banerjea AC (2000)
Extensive polymorphism in regulated upon activation: normal T cell
expressed/secreted promoter of bonnet, baboon and rhesus monkeys. AIDS
14:2401-2403. Reviews/Proceedings 1. Shanmugasundaram GK, Ramamoorti N, Shoeb F,
Sunderashan G, Saraswati V, Husain S, Arumugam N, Felix K, Kumarvelu J and
Banerjea AC (2000). Review: Influence of host factor genes in AIDS - Analyses
of HIV-1 resistant genes in India. Ind J Microbiol (in press). 2. Banerjea AC (2000) Role of host factors in
progression of HIV-1. In: Proceedings of the Sixth Round Table Conference,
Ranbaxy Science Foundation, New Delhi. (Eds: Gupta S and Sood OP) Yogesh
Prakashan, New Delhi, 163-167. |