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 two broad areas of interest: reverse transcription 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. 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 has two zinc fingers, each containing the invariant CCHC zinc-coordinating motifs. (A) Using a mutational approach, we have provided the first direct evidence that zinc coordination by the CCHC motifs is required for efficient strand transfer. The zinc fingers are also required for NC-mediated inhibition of dead-end self-priming reactions, which compete with minus-strand transfer and are induced by the complementary TAR secondary structure at the 3? end of (-) strong-stop DNA. 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. Biochemical data were correlated with enzymatic mapping and mFold analysis. We found that eliminating self-priming by disrupting the TAR DNA structure does not necessarily result in increased strand transfer efficiency: acceptor RNA structure is also critical. Thus, 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. (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) Other studies are being performed on the initiation step in HIV-1 reverse transcription. This event is primed by a host lysyl-tRNA, which is annealed to the 18-nt primer binding site near the 5? terminus of the viral RNA genome; extension of the primer leads to synthesis of (-) strong-stop DNA. Mutational analysis and band-shift experiments with wild-type and mutant template RNAs support the possibility that NC promotes an interaction between the 3? arm of the anticodon stem and part of the variable loop of the tRNA primer and nt 143-149 in viral RNA. Zinc coordination is not required to facilitate this interaction. (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 and lack a cone-shaped core. Moreover, despite having a functional RT protein, the mutants are blocked in viral DNA synthesis in cells, indicating a defect in an early postentry event preceding reverse transcription. Additionally, mutant cores have a marked deficiency in RT protein and enzymatic activity and a substantial increase in the retention 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 demonstrate the intimate connection between infectivity, proper core morphology, and the ability to undergo reverse transcription. (ii) We have also performed studies to determine whether substitutions other than alanine result in a different phenotype. A total of 11 substitutions were made at position 23 (Trp), but only one mutant, W23F, was infectious, albeit to a very small extent. W23F, unlike the original mutant W23A, was able to replicate during long-term culture in MT-4 cells, but with delayed replication kinetics. With continued passage, we eventually isolated two second-site suppressor mutants, which replicate like wild-type virus in MT-4 cells. We have also found that the original W23A mutant has trans-dominant inhibitory activity i.e., W23A can reduce the infectivity of wild-type virus produced by cells cotransfected with a mixture of wild-type and mutant particles. Interestingly, the trans-dominant inhibitory activity of W23F is approximately five-fold less than the corresponding activity of W23A. These observations indicate that the virions resulting from the cotransfection contain a co-assembled population of wild-type and mutant CA molecules. Moreover, the results imply that collaboration of many N-terminal CA domains is required for assembly of a virus particle, in accord with models for CA multimerization.

Project Start
Project End
Budget Start
Budget End
Support Year
32
Fiscal Year
2004
Total Cost
Indirect Cost
Name
U.S. National Inst/Child Hlth/Human Dev
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Tang, Shixing; Ablan, Sherimay; Dueck, Megan et al. (2007) A second-site suppressor significantly improves the defective phenotype imposed by mutation of an aromatic residue in the N-terminal domain of the HIV-1 capsid protein. Virology 359:105-15
Wu, Tiyun; Heilman-Miller, Susan L; Levin, Judith G (2007) Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein. Nucleic Acids Res 35:3974-87
Iwatani, Yasumasa; Chan, Denise S B; Wang, F et al. (2007) Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G. Nucleic Acids Res 35:7096-108
Iwatani, Yasumasa; Takeuchi, Hiroaki; Strebel, Klaus et al. (2006) Biochemical activities of highly purified, catalytically active human APOBEC3G: correlation with antiviral effect. J Virol 80:5992-6002
Opi, Sandrine; Takeuchi, Hiroaki; Kao, Sandra et al. (2006) Monomeric APOBEC3G is catalytically active and has antiviral activity. J Virol 80:4673-82
Miles, Lesa R; Agresta, Beth E; Khan, Mahfuz B et al. (2005) Effect of polypurine tract (PPT) mutations on human immunodeficiency virus type 1 replication: a virus with a completely randomized PPT retains low infectivity. J Virol 79:6859-67
Levin, Judith G; Guo, Jianhui; Rouzina, Ioulia et al. (2005) Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism. Prog Nucleic Acid Res Mol Biol 80:217-86
Heilman-Miller, Susan L; Wu, Tiyun; Levin, Judith G (2004) Alteration of nucleic acid structure and stability modulates the efficiency of minus-strand transfer mediated by the HIV-1 nucleocapsid protein. J Biol Chem 279:44154-65
Post, Klara; Guo, Jianhui; Howard, Kathryn J et al. (2003) Human immunodeficiency virus type 2 reverse transcriptase activity in model systems that mimic steps in reverse transcription. J Virol 77:7623-34
Imamichi, Tomozumi; Murphy, Michael A; Adelsberger, Joseph W et al. (2003) Actinomycin D induces high-level resistance to thymidine analogs in replication of human immunodeficiency virus type 1 by interfering with host cell thymidine kinase expression. J Virol 77:1011-20

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