Reverse transcription is the process by which a retrovirus such as HIV-1 converts its genetic material (single-stranded RNA) into a double-stranded DNA copy that is integrated into host chromosomal DNA. This process is complex and is catalyzed by the virion-associated enzyme, reverse transcriptase (RT). However, another viral protein, the nucleocapsid protein (NC), is also required to ensure efficient and specific viral DNA synthesis. We study the mechanistic basis for NC activity. HIV-1 NC is a small, basic nucleic acid binding protein with two zinc fingers, each containing the invariant CCHC zinc-coordinating motifs. It is a nucleic acid chaperone, i.e., it has the ability to catalyze conformational rearrangements that lead to the most thermodynamically stable nucleic acid structures. This property is critical for promoting the two strand transfer events that are needed for synthesis of full-length plus- and minus-strand viral DNA. In minus-strand transfer, the initial product of reverse transcription, (-) strong stop DNA, is translocated to the 3-prime end of viral RNA (termed acceptor RNA) in a reaction facilitated by base-pairing of the complementary repeat regions, which are present at the ends of the RNA and DNA partners. (A) In recent work on NC, we have focused on the effect of changes in nucleic acid structure and thermostability on NC-mediated minus-strand transfer. We found that strand transfer is efficient only when (-) strong-stop DNA and acceptor RNA are moderately structured and a delicate thermodynamic balance between these two reactants is maintained. This finding is consistent with the fact that NC is a weak destabilizer of secondary structure. Using mutational analysis, we have now obtained evidence demonstrating that NC nucleic acid chaperone activity is ultimately dependent on the stability of acceptor RNA local structure at the nucleation site for annealing, rather than on the overall stability of the structure. In addition, we have made a novel finding showing that when NC-facilitated destabilization of acceptor RNA is required, annealing appears to be more efficient than strand transfer (i.e., annealing plus elongation). We explain this apparent discrepancy by showing that Mg2+, which is not present in assays for annealing alone, but must be added for RT-catalyzed elongation, successfully competes with NC for binding to the negatively charged phosphodiester backbone of the nucleic acids. These studies underscore the importance of having optimal ionic conditions for studies of NC chaperone activity. (B) We have recently initiated a new project on human APOBEC3G (A3G), a cellular cytidine deaminase with two zinc finger domains, which blocks HIV-1 reverse transcription and replication in the absence of the viral protein known as Vif. The antiviral effect has been shown to be largely deaminase-dependent, but there is also a deaminase-independent component. One focus of our A3G studies has been to elucidate the mechanism for A3G inhibition of reverse transcription. We have succeeded in purifying catalytically active A3G, allowing us to provide a comprehensive molecular analysis of its deaminase and nucleic acid binding activities. We have demonstrated that A3G deaminates cytosines in single-stranded DNA only, whereas it binds efficiently to single-stranded DNA and RNA. Moreover, we have also shown that A3G and NC do not interfere with each others binding to RNA. This suggested that inhibition of reverse transcription is likely to be unrelated to an effect on NC chaperone function. To test this hypothesis, we investigated the interplay between A3G, NC, and RT in reconstituted reactions representing individual steps in the reverse transcription pathway. For the first time, we report that A3G does not affect the kinetics of NC-mediated annealing or the RNase H activity of RT. In sharp contrast, A3G significantly inhibits all RT-catalyzed elongation reactions with or without NC, without a requirement for A3Gs catalytic activity. Collaborative studies involving single-molecule DNA stretching analyses and fluorescence anisotropy have been performed. The data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC, and RT. (C) Our laboratory has also been investigating the role of the HIV-1 capsid protein (CA) in early postentry events, a stage in the infectious process that is still not completely understood. Initial efforts focused on the unusual phenotype associated with single alanine substitution mutations in conserved N-terminal hydrophobic residues of HIV-1 CA. Our findings illustrated the intimate connection between infectivity, proper core morphology, structural integrity of the CA protein, and the ability to undergo reverse transcription. (i) More recently, we have performed a study to provide new information on the plasticity of CA, i.e., its ability to tolerate changes in hydrophobic residues crucial for CA structure that do not totally abrogate biological activity. Our approach was to make mutant constructs that might retain the ability to replicate and thereby present an opportunity to isolate second-site suppressors. Of a total of 13 single substitutions, only one mutant, W23F, was found to exhibit infectivity in a single-cycle assay, albeit at a very low level. W23F was able to replicate during long-term culture in MT-4 cells, but with delayed replication kinetics. With continued passage, we could eventually isolate virions with a second-site suppressor mutation, W23F/V26I, which partially restored the wild-type phenotype. A structural model that accommodates the spatial changes induced by the W23F and V26I mutations shows that hydrophobic interactions between Phe23 and Ile26 are possible and can explain the suppressor phenotype. (iii) In current work, we are investigating the effect of point mutations in the linker region that connects the N- and C-terminal domains of CA. We are also interested in the effect of mutations in two lysine residues (one, N-terminal and the other, C-terminal) that on the basis of cross-linking studies are thought to be important for interactions between the two CA domains. Thus far, the mutant phenotypes appear to be less severe than those resulting from changes in the conserved hydrophobic residues.
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