Molecular Genetics Of Mammalian Retrovirus Replication

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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 for efficient and specific viral DNA synthesis. [unreadable] [unreadable] (A) We study the mechanistic basis for NCs 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. (i) 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. (ii) We have also shown that NC chaperone activity together with RNase cleavage block mispriming by non-polypurine tract RNAs during plus-strand DNA synthesis. These findings demonstrate a previously unrecognized role for NC in ensuring the fidelity of plus-strand initiation. [unreadable] [unreadable] (B) Our interest in host proteins that might affect HIV-1 reverse transcription has led us to investigate 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. (i) 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 shown, for example, that A3G and NC do not interfere with each other's 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 have reported 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 and without a requirement for A3G catalytic activity. Data from single-molecule DNA stretching analyses and fluorescence anisotropy 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. (ii) In current work, we have begun to study the APOBEC3A (A3A) protein, which has only one zinc finger domain and is a potent inhibitor of retrotransposition by Line-1 and Alu non-LTR elements. Efforts are underway to express and purify large enough amounts of protein for biochemical and structural analysis. [unreadable] [unreadable] (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. (i) Our initial findings illuminated the intimate connection between infectivity, proper core morphology, structural integrity of the CA protein, and the ability to undergo reverse transcription. (ii) 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. Mutants were constructed and tested to determine whether they might retain the ability to replicate and thereby present an opportunity to isolate second-site suppressors. When one of these mutants, W23F, was subjected to long term passage, a second-site suppressor mutation, W23F/V26I was isolated that partially restored the wild-type phenotype. A structural model that accommodates the spatial changes induced by the W23F and V26I mutations can explain the suppressor phenotype. These findings are novel and demonstrate that despite the limits imposed on assembly of CA structure, HIV-1 is able to partially adapt to severe structural distortions in a major viral protein. (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 studying the effect of mutations in two lysine residues (one, N-terminal and the other, C-terminal) that are thought to be important for interactions between the two CA domains. Although all of the mutants produce virus particles, most have little or no infectivity in a single-cycle assay. The lack of infectivity is correlated with the appearance of defective cores in the electron microscope. The results obtained thus far indicate that in general, residues involved in interdomain interactions cannot be mutated without loss of proper structure and function. Further characterization of these mutants is in progress.