The goal of our research is to define the molecular mechanisms involved in the replication of HIV and related retroviruses. These studies are critical for developing new strategies to combat the AIDS epidemic, which has continued to spread to all parts of the world and is a serious threat to the health and well-being of both children and adults. To address these issues, we have developed reconstituted model systems to investigate the individual steps in HIV-1 reverse transcription, a major target of HIV therapy. Much of our work focuses on the viral nucleocapsid protein (NC), which promotes highly efficient and specific viral DNA synthesis. NC is a nucleic acid chaperone, which means that it can facilitate nucleic acid conformational rearrangements that lead to formation of the most thermodynamically stable structure. This activity is essential for viral DNA synthesis. In other studies, our efforts are directed toward understanding the function of the viral capsid protein in HIV-1 assembly and early post-entry events during the course of virus replication in vivo.

Role of Nucleocapsid Protein in HIV-1 Strand Transfer
Guo, Heilman-Miller, Levin; in collaboration with Gorelick, Henderson, Musier-Forsyth
HIV-1 NC is a small basic protein with two zinc fingers, each containing the invariant CCHC zinc-coordinating residues. The remaining amino acids within each finger are not identical. NC function in virus replication depends on its dynamic interaction with nucleic acids, and the protein plays a critical role in the two strand-transfer steps that occur during viral DNA synthesis. We have shown that, during minus-strand transfer, the nucleic acid chaperone activity of NC destabilizes the highly structured complementary TAR stem-loop (TAR DNA) at the 3' end of (-) strong-stop DNA and inhibits TAR-induced self-priming, a dead-end reaction that competes with strand transfer. In our recent work, we have focused on the functional significance of the two zinc fingers for NC nucleic acid chaperone activity and on measurement of NC-induced conformational changes in (-) strong-stop DNA, which form the basis for inhibition of self-priming.

To address the issue of zinc finger function, we have used a mutational approach. Initially, we investigated the effects of an NC mutation that eliminates zinc coordination by changing the two CCHC motifs to SSHS. Our results provided the first direct evidence that zinc coordination is required for inhibition of self-priming and efficient minus-strand transfer. More recent findings demonstrate that, for optimal minus-strand transfer, CCHH or CCCC (zinc-binding motifs found in cellular proteins) cannot replace the CCHC motifs and that the amino acid context within each finger must be preserved. Context changes also reduce the ability of NC to facilitate tRNA primer removal, a step that is required for plus-strand transfer. In addition, we have found that the N-terminal zinc finger is a more critical determinant of NC nucleic acid chaperone activity than the C-terminal finger. Interestingly, our in vitro results correlate with earlier in vivo replication data.

To gain a better understanding of NC-mediated inhibition of self-priming, we are collaborating with Karin Musier-Forsyth and colleagues who have developed a fluorescence resonance energy transfer (FRET) assay that makes it possible to monitor conformational changes directly in TAR DNA when NC is present. The results show that when NC binds to TAR DNA alone, there is only a modest shift toward less-folded states. In the presence of acceptor RNA, NC binding to TAR DNA results in a shift of the majority of molecules to the unfolded state. These data agree with biochemical observations we made by using a modified strand transfer system containing preformed (-) strong-stop DNA and acceptor RNA. Thus, in the absence of acceptor RNA, NC has little effect on self-priming, whereas when acceptor RNA is present, NC stimulates strand transfer, and self-priming is dramatically reduced. Work is now under way to define the structural requirements for NC interaction with the strand transfer nucleic acid intermediates. A series of synthetic (-) strong-stop DNA and acceptor RNA truncation mutants have been constructed and are under investigation by in vitro assay of minus-strand transfer and self-priming, enzymatic structure probing, and analysis of secondary structure using RNA and DNA structure prediction algorithms.

Nucleic Acid and Protein Requirements for Initiation of HIV-1 Reverse Transcription
Iwatani, Levin; in collaboration with Musier-Forsyth
We have begun an investigation of the initiation step in HIV-1 reverse transcription. The event is primed by a host tRNA, tRNA3Lys, that is annealed to the 18-nt priming binding site (PBS) near the 5' terminus of the viral RNA genome; extension of the primer leads to synthesis of a short DNA product known as (-) strong-stop DNA. Earlier studies showed that cis-acting genomic RNA sequences upstream of the PBS are important for efficient (-) strong-stop DNA synthesis, but a possible role of sequences downstream of the PBS has not yet been established. As a first approach, we constructed a series of 14 RNA templates with varying lengths of downstream sequences. (-) Strong-stop DNA synthesis catalyzed by RT was first measured under conditions wherein the template was heat-annealed to each of three primers: tRNA3Lys; and 18-nt RNA (R18) or DNA (D18) oligonucleotides, each bearing the sequence of the 3' 18 nt of the tRNA. Interestingly, a minimum of 24 bases downstream of the PBS was required when the RNA primers, but not the D18 primer, were used. To investigate this phenomenon further, we also tested 18-nt DNA-RNA chimeric primers in our assay; 5'-R9D9 and 5'-R17D1 behaved like the D18 and R18 primers, respectively, while 5'-D9R9 exhibited “RNA-like” primer activity. Thermal stability (as determined by melting studies) and circular dichroism spectra of 18-nt primer:PBS duplexes were correlated with the biochemical data. The results point to the importance of helical conformation and stability as determinants of priming activity. Surprisingly, when NC was present, the additional 24 bases downstream of the PBS were dispensable for synthesis primed by tRNA but not for synthesis primed by the R18 primer.

Taken together, our findings suggest that, in the absence of NC, sequences downstream of the PBS are required to induce a conformation of the initiation complex that results in more efficient (-) strong-stop DNA when an RNA primer is used. We propose that NC abrogates this requirement by facilitating stable formation of extended interactions between the full-length tRNA and the RNA template, which are not possible with an 18-nt RNA.

Functional Analysis of HIV-2 Reverse Transcriptase Activities
Post, Guo, Levin; in collaboration with Powell, Le Grice, Hizi
The number of HIV-2 replication studies is relatively small as compared with the enormous literature on HIV-1 replication. Both viruses show similar genome organization, protein composition, and mode of transmission but differ in time of onset of AIDS and geographic pattern of infection (HIV-2 infection occurs primarily in West Africa and parts of Asia, where it is a significant public health issue). In addition, drugs that inhibit HIV-1 replication are not necessarily effective against HIV-2, posing a major problem for clinical treatment of HIV-2–infected individuals. We have conducted a systematic evaluation of the functional activities of HIV-2 RT, a potential target for antiviral therapy, by using assays that mimic specific steps in reverse transcription. We also performed parallel studies with HIV-1 RT.

In general, under standard assay conditions, the RNA- and DNA-dependent polymerase activities as well as the 5'-directed RNase H activities of the two enzymes are comparable (i.e., differences lie within an approximately two-fold range). In contrast, when the concentration of RT is reduced 100- or 1,000-fold, more striking differences are observed and the activity of HIV-2 RT is significantly reduced. Furthermore, under the conditions used for an assay that models tRNA primer removal, the ability of HIV-2 RT to catalyze secondary RNase H cleavage is also impaired. Moreover, initiation of plus-strand DNA synthesis is much less efficient with HIV-2 RT compared with HIV-1 RT unless the salt and magnesium ion concentrations are lowered to 5 mM and 3 mM, respectively. We have previously shown that plus-strand initiation depends on nucleic acid contacts with “primer grip” residues in the palm subdomain of the p66 RT subunit. We now propose that the reduced activity of HIV-2 RT in the plus-strand priming reaction may reflect architectural differences in the primer grip regions of the two enzymes. Taken together, our findings should be useful for development of specific high-throughput screening assays of potential HIV-2 inhibitory agents.

Function of HIV-1 Capsid Protein in Virus Assembly and Early Post-Entry Events
Tang, Levin; in collaboration with Freed
Our laboratory has been investigating the role of the HIV-1 capsid protein (CA) in early post-entry events, a stage in the infectious process that is still not completely understood. Structural studies of CA showed that a group of conserved hydrophobic residues faces the interior of the coiled coil–like structure within the N-terminal domain, and it was suggested that these residues might be important for CA structure and function. Recently, as a result of using genetic, molecular, and ultrastructural approaches, we reported the unusual phenotype associated with single alanine substitution mutations in these residues. Mutant virions are not infectious and lack a cone-shaped core. Moreover, despite their having a functional reverse transcriptase, the mutants are blocked in viral DNA synthesis in infected cells, indicating a defect in an early post-entry event preceding reverse transcription.

Current efforts focus on elucidating the mechanism by which these CA mutations disrupt virus infectivity. The work has uncovered several novel properties of the mutants. We have observed that the mutations block the incorporation of host cyclophilin A (a peptidyl-prolyl cis-trans isomerase required for virus replication) into virions. This finding was unexpected, given that the mutated residues are distant from the cyclophilin A binding loop in CA. The results indicate that the mutations induce conformational changes in CA that have global effects on CA structure and function. To investigate the possibility that the mutants might also be compromised in an early post-entry step, we modeled disassembly (i.e., viral uncoating) in vitro by generating viral cores following treatment of virus particles with mild detergent. In contrast to wild-type, less RT is associated with mutant cores. What is most striking, however, is the extraordinarily high retention of CA in mutant cores, indicating that mutant cores are unusually stable. Such stability would interfere with proper disassembly and, together with the reduced level of RT in mutant cores, would account for the failure of these mutants to synthesize viral DNA following virus entry into cells.

Our results reveal for the first time the crucial role of the N-terminal hydrophobic core in HIV1 replication. The critical importance of these residues for maintaining CA structure and function indicates that the hydrophobic motif represents a potential new target for antiviral drugs and other therapeutic approaches for combating the AIDS epidemic.