Judith G. Levin, PhD, Head, Section on Viral Gene Regulation
Tiyun Wu, PhD, Staff Scientist
Yasumasa Iwatani, PhD, Research Fellow 1
Suchitra Derebail, PhD, Postdoctoral Fellow 2
Kamil Hercik, PhD, Postdoctoral Fellow 3
Klara Post, MS, Senior Research Assistant
Elizabeth Cramer, BS, Postbaccalaureate Fellow 3
Victoria Yang, BS, Postbaccalaureate Fellow 2
Jianhui Guo, MD, PhD, Special Volunteer
The goal of our research 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, which continues to spread to all parts of the world and poses a serious threat to human health. 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 is a nucleic acid chaperone; that is, NC facilitates nucleic acid conformational rearrangements that lead to formation of the most thermodynamically stable structure. This activity is critical for highly efficient and specific viral DNA synthesis. Recently, we initiated a new project on APOBEC3G, which is a cellular cytidine deaminase that blocks HIV-1 reverse transcription and replication in the absence of a viral protein known as Vif. In other studies, we focus on understanding the function of the viral capsid protein (CA) 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
Wu, Guo, Levin; in collaboration with Gorelick, 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 depends on NC's dynamic interaction with nucleic acids. In addition, NC plays a critical role in the two-strand transfer steps that occur during viral DNA synthesis. We have shown that during minus-strand transfer, NC nucleic acid chaperone activity destabilizes the highly structured complementary TAR stem-loop (TAR DNA) at the 3′ end of (-) strong-stop DNA [(-) SSDNA] and inhibits TAR-induced self-priming, a dead-end reaction that competes with annealing of (-) SSDNA to acceptor RNA. Both NC zinc fingers are required for this function. In recent work, we focused on the influence of nucleic acid structure on NC nucleic acid chaperone activity, with emphasis on the importance of local structure and NC-catalyzed removal of the 5′ terminal RNA fragments generated during RNase H degradation of genomic RNA.
To investigate the structural and thermodynamic requirements for NC interaction with strand-transfer nucleic acid intermediates, we constructed a series of synthetic (-) SSDNA and acceptor RNA truncation mutants. We studied these constructs with an in vitro assay of minus-strand transfer and self-priming, enzymatic structure probing, and analysis of secondary structure using mFold. Our results demonstrate that NC-mediated strand transfer is efficient only when (-) SSDNA and acceptor RNA are both moderately structured. Thus, we conclude that a delicate thermodynamic balance between (-) SSDNA and acceptor RNA must be maintained for efficient minus-strand transfer. Our conclusion is consistent with the fact that NC is a weak destabilizer of nucleic acid structure.
In continuing studies of acceptor RNAs, we unexpectedly found that a particular RNA (RNA 70) with a high delta G value has more strand transfer activity than a smaller RNA (RNA 50) with a lower delta G value, using the same (-) SSDNA in the assays of both RNAs. This result, in conjunction with mFold analysis of the RNA structures, led us to postulate that NC nucleic acid chaperone activity ultimately depends on the stability of local structure at the nucleation site for annealing rather than on the overall stability of the RNA. To test this hypothesis, we made mutations in the local structure at the potential nucleation sites found at the 5′ end of each RNA. In accord with our prediction, we observed that destabilizing changes in local structure dramatically increased strand transfer activity while stabilizing changes led to a striking reduction in the rate and extent of strand transfer and increased dependence on NC. Annealing assays yielded similar results. However, strand transfer appeared to be less efficient than annealing. We explained the apparent discrepancy by showing that Mg2+, which is not present in the annealing buffer but must be added for DNA synthesis, competes with NC for binding to the negatively charged phosphodiester backbone of the nucleic acids. We have also shown that mutations in the 6-nt TAR loop reduce both strand transfer and annealing. The result indicates that NC promotes loop-loop interactions between TAR RNA and TAR DNA. Moreover, the data demonstrate that NC interactions with bases in the loop and the stem both contribute to efficient strand transfer.
We performed other experiments to assess the roles of RNase H and NC in the removal of short 5′-terminal RNA fragments that are initially annealed to the 3′ end of (-) SSDNA. We modeled fragment removal in the context of minus-strand transfer by heat-annealing a 5′-terminal RNA oligonucleotide to a longer synthetic (-) SSDNA and then adding acceptor RNA, reverse transcriptase (RT), and NC. Our results demonstrate that, under these conditions, the efficiency of minus-strand transfer catalyzed by either RNase H-minus or wild-type RTs is very similar. Thus, NC nucleic acid chaperone activity alone can facilitate terminal fragment removal without a requirement for secondary RNase H cleavage (as previously thought). Interestingly, coordination of zinc by the CCHC motifs in NC is required for terminal fragment removal.
Analysis of HIV-1 polypurine tract mutations and effect on virus replication
Agresta, 4 Tang, 5 Levin; in collaboration with Powell
In all retroviruses, the polypurine tract (PPT), which is derived from genomic RNA by specific RNase H cleavage, serves as the primer for plus-strand DNA synthesis. The HIV-1 PPT contains 15 bases. To investigate the effect of changes in the PPT sequence on HIV-1 replication, we introduced mutations into the viral clone NL4-3KFSΔnef. Previously, we tested priming activity with most of these mutant PPT sequences in an in vitro assay for initiation of plus-strand DNA synthesis. More recently, we assayed each mutant for virion production and infectivity in a single-cycle assay. All the PPT mutations lowered infectivity; however, mutation of the 5′ end appeared to have less of an effect on infectivity than mutation of′the 3¿ end of the PPT sequence. Curiously, a mutant in which the entire PPT sequence was randomized (PPTSUB) retained 12 percent of wild-type infectivity. Supernatant fluids from infected cells contained virus particles, as evidenced by the presence of p24 antigen. Two-Long Terminal Repeat (2-LTR) circle junction analysis following PPTSUB infection revealed that the mutant could form a high percentage of normal junctions. Quantification of the 2-LTR circles using Real Time PCR demonstrated that the number of 2-LTR circles from cells infected with the PPTSUB mutant was 3.5 orders of magnitude greater than that of 2-LTR circles from cells infected with wild-type virus. To determine if the progeny virions from a PPTSUB infection could undergo further rounds of replication, we inserted the PPTSUB mutation into a replication-competent virus. Our results showed that the mutant virus was able to replicate. In addition, the infectivity of the progeny virions increased with each passage and resulted in rapid reversion to a wild-type PPT sequence. Collectively, these experiments confirm our earlier findin′ that the 3¿ end of the PPT is important for plus-strand priming, indicating that a virus completely lacking the PPT sequence can replicate at a low level.
Molecular analysis of the functional activities of APOBEC3G (APO3G)
Iwatani, Levin; in collaboration with Gronenborn, Strebel
Given our strong interest in host factors that can influence reverse transcription, we undertook a molecular analysis of APO3G, which is a cellular cytidine deaminase with two zinc finger domains that acts as a potent inhibitor of HIV-1 reverse transcription and replication in the absence of the viral protein Vif. Until now, studies on human APO3G have been performed primarily in cell-based systems and with unfractionated enzyme from viral lysates. We have succeeded in producing highly purified, catalytically active protein expressed in a baculovirus system, allowing us, for the first time, to provide a detailed molecular analysis of APO3G's deaminase and nucleic acid-binding activities. Using the purified enzyme, we demonstrated that APO3G deaminates dCs in single-stranded (ss)DNA, but not in ssRNA, double-stranded (ds)DNA, dsRNA, or a DNA/RNA hybrid. However, electrophoretic mobility shift assays (EMSAs) showed that APO3G binds efficiently to ssDNA or ssRNA with a Kd similar to that for NC binding, less efficiently to a DNA/RNA hybrid, and very poorly to dsDNA or dsRNA. The data demonstrate that the substrate specificities for nucleic acid binding and deamination differ. We have also shown that although NC and APO3G are both nucleic acid-binding proteins, they 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. In the case of an RNA stem-loop (SL-3) that is part of the HIV-1 packaging signal, we found that NC actually promotes APO3G binding. This result presumably reflects NC chaperone activity, which transiently destabilizes the SL-3 RNA. In turn, destabilization promotes increased binding of APO3G, which has a preference for binding to single-stranded nucleic acids. Interestingly, when bound to an 18-nt ssDNA, APO3G forms a dimer, although recent studies performed in infected cells indicate that multimerization is not required for the antiviral effect.
To identify the individual roles of the zinc finger domains, we expressed and purified three zinc finger mutant proteins: C100S, zinc finger one; C291S, zinc finger two; and the double mutant. EMSA data indicated that, although both zinc fingers bind to nucleic acids, zinc finger one contributes more to binding activity and encapsidation of APO3G into virions than does zinc 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. We have also undertaken a study to identify those residues in the zinc fingers that are important for APO3G's distinctive molecular properties. We focused initially on the P/A residue between the H and E residues in the zinc finger motif (HXE-X24-30-PCXXC), which is Pro in zinc finger one (normally Ala in all other human APO3 proteins) and Ala in finger two. Based on structural models, exchange of Pro/Ala should not cause significant structural perturbation. Indeed, the Pro/Ala substitutions do not alter nucleic acid binding in vitro or encapsidation of APO3G into virions. However, mutation of Pro to Ala in zinc finger one cannot confer catalytic activity while substitution of Ala to Pro in zinc finger two abolishes deaminase activity and reduces antiviral activity. A set of other Ala substitutions is under construction to determine whether deaminase and antiviral activities can be retained by replacing Ala with other residues. Taken together, the data imply that the structural context of the two zinc fingers is a crucial determinant of catalytic activity and that the motif in zinc finger one may not be critical for substrate recognition. Experiments to elucidate the mechanism(s) by which APO3G inhibits HIV-1 reverse transcription are in progress.
Function of HIV-1 capsid protein in virus assembly and early post-entry events
Derebail,2 Levin; in collaboration with Freed, Tang
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. In our initial study, we used genetic, molecular, and ultrastructural approaches to describe the unusual phenotype associated with single alanine substitution mutations in a group of N-terminal, conserved hydrophobic residues (including Trp23 and Phe40. We found that mutant virions are not infectious and lack a cone-shaped core. Moreover, despite the fact that they contain a functional RT enzyme, the mutants are blocked in the initiation of viral DNA synthesis in infected cells. In further work, we demonstrated that isolated mutant viral cores display a marked deficiency in RT, which is consistent with the inability of the mutants to synthesize viral DNA after entry. We also observed a substantial increase in the retention of CA, suggesting that mutant cores might be defective in core disassembly. Taken together, the findings demonstrate the intimate connection among infectivity, proper core morphology, structural integrity of the CA protein, and ability to undergo reverse transcription.
More recently, we initiated a study to provide new information on the plasticity of CA, that is, its ability to tolerate changes in residues crucial for CA structure without total abrogation of biological activity. Our approach called for making mutant constructs that might retain the ability to replicate and thereby present an opportunity to isolate second-site suppressors. We made a total of 13 additional substitutions 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 has a phenotype that is intermediate between wild-type virus and the original W23A mutant. For example, although W23A and W23F both exhibit transdominant inhibitory activity, W23F has one-fifth of the activity of W23A. In addition, the W23F mutant (unlike W23A) 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 the mutations, W23F/V26I, partially restores the wild-type phenotype, including production of particles with conical cores and normal CA content, and displays 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.
Although the structures of the N- and C-terminal domains were solved some time ago, there is little information on the short linker region (residues 146-150) that connects the two CA domains. In current work, we have made Ala scanning mutations in the three conserved residues in the linker region and found that, with the exception of P147A, the mutants all produce virus particles with a normal protein profile. Infectivity in a single-cycle assay is reduced, but not completely abolished. The results show that, unlike the situation with the hydrophobic residues that we previously studied, Ala substitutions in some of the linker residues are tolerated. We plan further analysis of these mutants, including the biochemical properties of isolated cores, the ability to synthesize viral DNA post-entry, and replication kinetics in several cell lines.
1 Appointed Research Fellow in 2006.
2 Departed the laboratory in 2006.
3 Joined the laboratory in 2006.
4 Beth Agresta, PhD, former Postdoctoral Fellow
5 Shixing Tang, MD, PhD, former Postdoctoral Fellow
COLLABORATORS
Eric O. Freed, PhD, HIV Drug Resistance Program, NCI, Frederick, MD
Robert J. Gorelick, PhD, AIDS Vaccine Program, SAIC Frederick, Inc., NCI, Frederick, MD
Angela M. Gronenborn, PhD, University of Pittsburgh Medical School, Pittsburgh, PA
Karin Musier-Forsyth, PhD, University of Minnesota, Minneapolis, MN
Michael D. Powell, PhD, Morehouse School of Medicine, Atlanta, GA
Klaus Strebel, PhD, Laboratory of Molecular Microbiology, NIAID, Bethesda, MD
Shixing Tang, MD, PhD, Laboratory of Molecular Virology, CBER, FDA, Bethesda, MD
For further information, contact levinju@mail.nih.gov.