The goal of the research performed in the Section on Viral Gene Regulation is to define the molecular mechanisms involved in the replication of HIV and related retroviruses. The research is critical for developing new strategies to combat the AIDS epidemic, which has spread to all parts of the world and poses a serious threat to human health and security. We have developed reconstituted model systems to investigate HIV-1 reverse transcription and the role of the viral nucleocapsid protein (NC), which promotes highly efficient and specific viral DNA synthesis. Other studies, performed in cell culture, are directed toward elucidating the function of the viral capsid protein (CA) in virus assembly and early postentry events.

Role of Nucleocapsid Protein in HIV-1 Strand Transfer
Guo, Wu,* Levin in collaboration with Henderson,a Gorelick,a Musier-Forsythb
During reverse transcription, two strand transfer events are required for synthesis of full-length plus- and minus-strand DNA copies of the viral RNA genome. The process is efficient only when NC is present. HIV-1 NC has two zinc fingers, each containing invariant CCHC zinc-coordinating residues and other residues that form three short loops; only the sequence of the second loop is identical in both zinc fingers. A major question concerns the functional significance of the two zinc fingers in HIV-1 NC. Recently, we reported the first direct evidence that zinc coordination is required for the nucleic acid chaperone activity of NC. The chaperone activity catalyzes transient unfolding of the complex secondary structures in RNA and DNA strand transfer intermediates. We have now asked whether CCHH or CCCC, which are the zinc binding motifs found in cellular zinc finger proteins, can be substituted for CCHC and whether the amino acid context surrounding the CCHC motifs can be changed by exchanging or duplicating the zinc fingers. Our results demonstrate that, for optimal strand transfer, the CCHC motifs cannot be replaced by CCHH or CCCC and that the amino acid context in the loop regions must be preserved. We also find that the N-terminal zinc finger is a more crucial determinant of NC nucleic acid chaperone activity than the C-terminal finger. Interestingly, a comparison of our in vitro results with earlier in vivo replication data raises the possibility that NC may adopt multiple conformations that are responsible for different NC functions during virus replication. In addition to these studies, we are using Fluorescence Resonance Energy Transfer (FRET) to investigate how NC facilitates nucleic acid rearrangement during the minus-strand transfer step.

Nucleic Acid and Protein Requirements for Initiation of HIV-1 Reverse Transcription
Iwatani, Levin in collaboration with Musier-Forsythb
We have recently begun an investigation of the initiation step in HIV-1 reverse transcription. The event is primed by cellular tRNALys3, which is annealed to the 18-nucleotide priming binding site (PBS) near the 5' terminus of the viral RNA genome; extension of the primer leads to synthesis of (-) strong-stop DNA. As a first approach, we have compared the priming activities of tRNALys3 and RNA or DNA oligonucleotides with the sequence of the 3' 18 nucleotides (nt) of the tRNA. Surprisingly, with both the tRNA and 18-nt RNA primers, at least 24 nt downstream of the PBS must be included in the template RNA for optimal synthesis of (-) strong-stop DNA, whereas these sequences are entirely dispensable when a DNA primer is used. The priming activities of 18-nt chimeric primers, which contain nine RNA bases at the 5' end followed by nine DNA bases at the 3' end or vice versa, are dictated by the nature of the 9-nt sequence at the 3' end of the oligonucleotide. We hypothesize that differences in the helical structures of the RNA and DNA primers are responsible for the observed differences in activity and might affect interactions with the template RNA. Experiments to determine the effect of HIV-1 NC on primer-template requirements are currently in progress.

Function of HIV-1 Capsid Protein in Virus Assembly and Early Postentry Events
Tang, Agresta,* Levin in collaboration with Murakami,c Freed, c Campbelld
Studies on HIV-1 CA have focused on a group of conserved, hydrophobic residues in the N-terminal portion of the protein. We have now made five additional alanine substitution mutants. All these mutants have a distinct phenotype: the resultant virions are noninfectious, exhibit aberrant core morphology, and are unable to initiate viral DNA synthesis in the infected cell, although the particles contain a functional reverse transcriptase enzyme. Taken together, the results reveal the close connection between proper virion core morphology and the ability to undergo reverse transcription in vivo. Studies on the protein composition and stability of the mutant viral cores are now under way.