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MOLECULAR GENETICS OF MAMMALIAN RETROVIRUS
REPLICATION
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Judith G. Levin, PhD, Head, Section on Viral Gene Regulation Jianhui Guo, MD, PhD, Staff Scientist |
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Our goal is to define the molecular mechanisms involved in the replication of HIV and related retroviruses. Our studies are critical for developing new strategies to combat the AIDS epidemic. 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. As a nucleic acid chaperone, NC can facilitate nucleic acid conformational rearrangements that lead to formation of the most thermodynamically stable structure. Such activity is essential for viral DNA synthesis. In other studies, our efforts are directed toward understanding the function of the viral capsid protein (CA) in HIV-1 assembly and early postentry 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. NC function in virus replication is dependent on its dynamic interaction with nucleic acids 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 annealing of (-) strong-stop DNA to acceptor RNA, i.e., the strand transfer reaction. Mutational analysis has demonstrated that both NC zinc fingers are required for this function. In our recent work, we have focused on the influence of nucleic acid structure on 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 gain a better understanding of NC-mediated inhibition of self-priming, we have collaborated 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. The results show that, when NC binds to TAR DNA alone, only a modest shift occurs toward less folded conformations. In the presence of acceptor RNA, however, NC binding to TAR DNA results in a shift of the majority of molecules to the unfolded state. Interestingly, the FRET data are correlated with biochemical observations, indicating that self-priming is blocked only in the presence of acceptor RNA and NC, i.e., conditions that favor optimal minus-strand transfer. Taken together, the results support the possibility that NC-mediated annealing of nucleic acids is a concerted process involving a destabilization step that occurs in synchrony with hybridization. In other work on nucleic acid chaperone activity, we are investigating the structural and thermodynamic requirements for NC interaction with strand transfer nucleic acid intermediates. We have constructed a series of synthetic (-) strong-stop DNA and acceptor RNA truncation mutants and are studying them 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. As might be expected, truncations that disrupt the TAR DNA structure in (-) strong-stop DNA completely eliminate DNA self-priming. However, reducing or eliminating self-priming does not necessarily result in an increase in strand transfer efficiency; the structure of the acceptor RNA is also important. In fact, we have demonstrated that NC can mediate efficient strand transfer only when both (-) strong-stop DNA and acceptor RNA are moderately structured. Thus, little or no strand transfer occurs when the acceptor RNA is highly stable. The data demonstrate that a delicate thermodynamic balance between (-) strong-stop DNA and acceptor RNA must be maintained for efficient minus-strand transfer. Studies are now in progress to determine the roles of NC and RNase H activity in facilitating the removal of short terminal RNA fragments that result from RNase H-catalyzed degradation of genomic RNA and that are initially annealed to (-) strong-stop DNA. The fragments must be removed to allow acceptor RNA to anneal to (-) strong-stop DNA so that elongation of minus-strand DNA can proceed. Preliminary results suggest that NC is efficient in facilitating fragment removal, even in the absence of RNase H. Hong M, Harbron EJ, O'Connor DB, Guo J, Barbara PF, Levin JG, Musier-Forsyth K. Nucleic acid conformational changes essential for HIV-1 nucleocapsid protein-mediated inhibition of self-priming in minus-strand transfer. J Mol Biol 2003; 325:1-10. Nucleic acid and protein requirements for initiation of HIV-1 reverse transcription Iwatani, Levin; in collaboration with Gorelick, Musier-Forsyth We have been investigating the initiation step in HIV-1 reverse transcription, an event primed by a host tRNA, tRNA3Lys, which 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 the short DNA product known as (-) strong-stop DNA. In earlier work, we demonstrated that, in the absence of NC, the RNA template must contain a minimum of 24 bases downstream of the PBS when RNA primers (tRNA3Lys or an 18-nt RNA complementary to the PBS [R18]), but not a DNA primer, are used. Surprisingly, when NC is present, the additional 24 bases downstream of the PBS are dispensable for synthesis primed by tRNA but not for synthesis primed by the R18 primer. We proposed 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. Mutational analysis supports the possibility that NC facilitates an interaction between the 3' arm of the anticodon stem and part of the variable loop of tRNA3Lys and nt 143-149 in viral RNA. Collectively, the findings provide evidence that the nucleic acid chaperone activity of NC modulates the conformational stability of the initiation complex.
To analyze further the effect of NC on initiation, we tested by band-shift assay the affinity of reverse transcriptase (RT) for the viral RNA-tRNA complex in the presence or absence of NC. In the absence of dNTPs, NC does not affect RT binding to complexes constituted with either wild-type or mutant templates (substitution of the complementary bases in nt 143-149). In contrast, in a functional assay with a +1 extension of the tRNA primer, NC stimulates incorporation with the wild-type but not mutant templates. The results provide further evidence for a specific NC-dependent interaction outside the PBS region. In additional experiments conducted with low concentrations of dNTPs (5 microM), we find that NC significantly stimulates tRNA-primed (-) strong-stop DNA synthesis, even with a template with more than 24 downstream bases. Taken together, our data suggest that NC-facilitated interactions may also be important in vivo, where dNTP concentrations are thought to be relatively low and viral RNA is genome-size. Interestingly, studies with NC zinc finger mutants demonstrate that zinc coordination is not required for NC stimulation of (-) strong-stop DNA synthesis regardless of dNTP concentration. In our current work, we are performing enzymatic mapping studies to probe conformational differences in wild-type and mutant viral RNA-tRNA complexes in the presence and absence of NC. Guo J, Wu T, Kane BF, Johnson DG, Henderson LE, Gorelick RJ, Levin JG. Subtle alterations of the native zinc finger structures have dramatic effects on the nucleic acid chaperone activity of human immunodeficiency virus type 1 nucleocapsid protein. J Virol 2002;76:4370-4378. Iwatani Y, Rosen AE, Guo J, Musier-Forsyth K, Levin JG. Efficient initiation of HIV-1 reverse transcription in vitro: Functional analysis of HIV-2 reverse transcriptase activities Post, Guo, Levin; in collaboration with Hizi, Le Grice, Powell HIV-2 infection is a significant public health problem in West Africa and parts of Asia, yet there have been relatively few studies of HIV-2 replication as compared with the vast literature on HIV-1. Both viruses share similar genome organization, protein composition, and mode of transmission but differ in time of onset of AIDS and geographic pattern of infection. In addition, drugs that inhibit HIV-1 replication, e.g., nevirapine, 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 reverse transcriptase (RT), a potential target for antiviral therapy, using assays that model specific steps in reverse transcription. We conducted parallel studies with HIV-1 RT.
Post K, Guo J, Howard KJ, Powell MD, Miller JT, Hizi A, Le Grice SFJ, Levin JG. Human immuno- deficiency virus type 2 reverse transcriptase activity in model systems that mimic steps in reverse transcription. J Virol 2003;77:7623-7634. Function of HIV-1 capsid protein in virus assembly and early postentry events Tang, Levin; in collaboration with Freed Our laboratory has 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. Structural studies of CA showed that a group of conserved hydrophobic residues (including Trp23 and Phe40) 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. In our initial study, we used genetic, molecular, and ultrasound approaches and reported the unusual phenotype associated with single alanine substitution mutations in the residues. Mutant virions are not infectious and lack a cone-shaped core. Moreover, despite having a functional RT enzyme, the mutants are blocked in viral DNA synthesis in infected cells. The findings suggest that the mutations result in a defect in an early step preceding reverse transcription, which is correlated with a defect in assembly of viral cores. We have also initiated studies to determine whether substitutions other than alanine result in a different phenotype; for this purpose, we constructed a series of vertical mutations in residues Trp23 and Phe40. We made a total of 13 additional substitutions at position 23 and two at position 40 (F40W and F40Y) as well as a double mutation, W23F/F40W. We tested the mutants in a single-cycle infectivity assay and observed that only mutant W23F exhibited infectivity, albeit at a very low level. W23F has a phenotype that appears to be intermediate between wild-type virus and the original W23A mutant. Interestingly, the W23F mutant (unlike W23A) is able to replicate during long-term culture in MT-4 cells, although we observed delayed replication kinetics. Our current efforts focus on isolating and characterizing second-site suppressors of the W23F mutation, which has the potential to yield important information about CA structure. Tang S, Murakami T, Agresta BE, Campbell S, Freed EO, Levin JG. Human immunodeficiency virus type 1 N-terminal capsid mutants that exhibit aberrant core morphology and are blocked in initiation of reverse transcription in infected cells. J Virol 2001;75:9357-9366. COLLABORATORS Eric O. Freed, PhD, HIV Drug Resistance Program, NCI, Frederick MD (Until July 2003, Section Chief, Laboratory of Molecular Microbiology, NIAID, Bethesda MD) Robert J. Gorelick, PhD, AIDS Vaccine Program, SAIC Frederick, Inc., NCI, Frederick MD Amnon Hizi, PhD, Sackler School of Medicine, Tel Aviv University, Israel Stuart F.J. Le Grice, PhD, HIV Drug Resistance Program, Resistance Mechanisms Laboratory, NCI, Frederick MD Karin Musier-Forsyth, PhD, University of Minnesota, Minneapolis MN Michael D. Powell, PhD, Morehouse School of Medicine, Atlanta GA aArrived in 2003 bDeparted in 2003 |
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