Molecular Genetics Of Mammalian Retrovirus Replication

Child Health And Human Development

Investigators

Linked publications & trials

Abstract

The goal of this project is to define the molecular mechanisms involved in the replication of HIV and related retroviruses and to develop new strategies for AIDS therapy. Our research is currently focused on several broad areas of interest: reverse transcription; inhibition of virus replication by the host protein APOBEC3G; and virus assembly. During reverse transcription, there are two strand transfer events that are required for synthesis of full-length plus- and minus-strand DNA copies of the viral RNA genome. In minus-strand transfer, the initial product of reverse transcription, (-) strong stop DNA, is translocated to the 3? end of viral RNA in a reaction facilitated by base-pairing of the complementary R regions (which includes the stable TAR stem-loops) in the RNA and DNA molecules. This process is dependent on the viral nucleocapsid protein (NC), a nucleic acid chaperone with the ability to catalyze conformational rearrangements that lead to the most thermodynamically stable nucleic acid structures. HIV-1 NC is a small, basic nucleic binding protein with two zinc fingers, each containing the invariant CCHC zinc-coordinating motifs. (A) In recent work on NC, we have focused on two issues that affect minus-strand transfer. (i) To investigate the effect of changes in nucleic acid structure and thermostability on NC-mediated minus-strand transfer, we designed a series of (-) strong-stop DNA and acceptor RNA constructs. 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. More recently, using mutational analysis, we have 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 shown that NC interactions with bases in the TAR stem and in the 6-nt loop both contribute to efficient minus-strand transfer. (ii) Short 5? terminal RNA fragments generated during RNase H degradation of genomic RNA are initially annealed to the 3? end of (-) SSDNA. These fragments must be removed so that strand transfer can occur. By modeling this reaction in the context of minus-strand transfer, we have shown that fragment removal can be catalyzed by NC alone without a requirement for secondary RNase H activity (as previously thought). Coordination of zinc by the CCHC motifs is required. (B) We have recently initiated a new project on APOBEC3G (APO3G), 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. We have succeeded in purifying catalytically active APO3G, allowing us for the first time, to provide a comprehensive molecular analysis of its deaminase and nucleic acid binding activities. We have demonstrated that APO3G deaminates cytosines in single-stranded (ss)DNA only, whereas it binds efficiently to ssDNA and ssRNA. APO3G and NC (both nucleic acid binding proteins) do not interfere with each other?s binding to RNA. In fact, a high-molecular-weight complex containing both proteins is formed through an RNA bridging mechanism. Although both zinc finger domains have the ability to bind nucleic acids, the first zinc finger contributes more to binding and APO3G encapsidation into virions than finger two. In contrast, deamination is associated exclusively with the second zinc finger. Moreover, zinc finger two is more important than finger one for the antiviral effect, demonstrating a correlation between deaminase and antiviral activities. Experiments to elucidate the mechanism(s) by which APO3G inhibits HIV-1 reverse transcription are the focus of current efforts. (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) We have previously described the unusual phenotype associated with single alanine substitution mutations in conserved N-terminal hydrophobic residues. Mutant virions are not infectious, lack a cone-shaped core, and are blocked in viral DNA synthesis in cells. Additionally, mutant cores have a marked deficiency in RT protein and abnormally high amounts of CA. The high level of core-associated CA would interfere with proper disassembly and taken together with the RT defect, would also account for the failure of the mutants to synthesize viral DNA postentry. Our results illustrate 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 initiated a study to provide new information on the plasticity of CA, i.e., its ability to tolerate changes in residues crucial for CA structure without total abrogation of 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. A total of 13 additional substitutions were made at position 23, two at position 40, and a double mutation. Only one mutant, W23F, was found to exhibit infectivity in a single-cycle assay, albeit at a very low level. W23F is 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 two second-site mutations. In particular, one of these mutations, W23F/V26I, partially restores the wild-type phenotype, including production of particles with conical cores and normal CA content, as well as wild-type replication kinetics in MT-4 cells. 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. The mutant phenotypes appear to be less severe than those resulting from changes in the conserved hydrophobic residues.