RNA aptamers directed to the HIV-1 GAG polyprotein as inhibitors of replication and as tools to dissect replication mechanisms
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The Gag polyprotein orchestrates the assembly of HIV-1 and is the only viral protein required for the formation of non-infectious virus-like particles in vitro. Gag is synthesized in the cytoplasm of HIV infected cells and has four major domains - matrix (MA), capsid (CA), nucleocapsid (NC) and p6. My thesis focuses on the development of RNA aptamers raised against the Gag polyprotein of HIV-1 as inhibitors of viral replication. Aptamers are highly structured nucleic acids that recognize their targets with high affinity. We raised RNA aptamers against a version of HIV-1 Gag that lacks the N-terminal myristate and the C-terminal p6 (DP6). We showed that the aptamers recognize purified MA or NC proteins in vitro..;In the first part of my thesis, we have investigated the potential of anti-Gag aptamers to inhibit HIV-1 replication. Upon co-transfection with the pNL4-3.Luc (VSV-G) molecular clone into 293T cells, we observed that several of the aptamers led to a 2-20 fold inhibition of virus production. This inhibition was also reflected in the intracellular Gag levels, which were, in turn, proportional to viral mRNA levels. We hypothesized that the ability of the aptamers to influence viral mRNA levels is mediated by perturbation of specific Gag-viral RNA interactions. One of the well-characterized interactions between the Gag protein and the viral mRNA involves the NC domain of Gag and the Psi-packaging signal (Psi) in the viral mRNA. We observed that the NC-specific aptamers specifically competed with the Psi of HIV-1 for binding to DP6 in vitro. Therefore, we tested the ability of two NC-specific inhibitory aptamers to affect the production of viruses engineered with partial deletions of their Psi and observed that these aptamers were no longer able to inhibit Gag synthesis and virus production. Our results reveal an essential interaction between the Gag protein and the viral mRNA, well before encapsidation, that is essential for viral mRNA stability.;The second part of my thesis focuses on the interactions of the HIV-1 NC protein with the psi-RNAs from different retroviruses. In vitro , the anti-HIV-1 Gag aptamers did not bind to the Gag proteins from SIVmac or RSV. Consistent with this result, upon cotransfecting the aptamers with a molecular clone of SIV or HIV, the aptamers inhibited the production of HIV but not SIV. The interaction of the Psi-region of a retrovirus with its cognate NC protein is believed to be highly specific and cross-packaging between retroviruses is known to occur only at low efficiencies. To understand the nature of interaction between the NC-specific aptamers and the HIV-1 NC protein, we performed in vitro competition assays with purified NC protein and used the psi-RNA transcripts from different retroviruses as competitors. While HIV-1-psi, when used in excess as unlabeled RNA, could robustly compete with the radiolabeled aptamers for binding to NC, the control RNAs like yeast tRNA, could not. Interestingly, psi-RNAs from HIV-2, SIVmac and MuLV also strongly competed with the aptamers for binding to NC. While HIV-1 NC has been shown to recognize the psi-regions of HIV-2 and SIVmac, it is not known to interact with the psi of MuLV. Our results suggest a novel possibility that the psi-RNA regions of diverse retroviruses contain conserved features at the level of secondary or tertiary structure, which are recognized by a binding pocket on NC. Further, discrimination between the genomic RNAs by HIV-1 NC might require a second signal unique to the virus.;In summary, we have characterized the interactions of aptamers raised against a near-full-length Gag polyprotein, both in vitro and in vivo and demonstrate that they mediate significant inhibition of virus production. Hence, we believe that the anti-Gag aptamers might have immense potential not only as anti-HIV-1 gene-therapeutic agents but also as potential tools to study interactions of the Gag protein with various host and viral factors.
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