MOLECULAR GENETICS OF MAMMLIAN RETROVIRUS REPLICATION
, Head, Section on Viral Gene Regulation
Tiyun Wu, PhD, Staff Scientist
Yasumasa Iwatani, PhD, Research Fellow1
Jiyang Jiang, PhD, Postdoctoral Fellow2
Kamil Hercik, PhD, Visiting Fellow
Klara Post, MS, Senior Research Assistant
Elizabeth Cramer, BS, Postbaccalaureate Fellow3
Pilar H. Saladores, BS, Postbaccalaureate Fellow2
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 facilitates nucleic acid conformational rearrangements leading to formation of the most thermodynamically stable structure. Such activity is critical for highly efficient and specific viral DNA synthesis. Recently, we initiated a new project on APOBEC3G, 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 direct our efforts to 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 reverse transcription
HIV-1 NC is a small basic protein bearing two zinc fingers, each containing the invariant CCHC zinc-coordinating residues. NC function in virus replication is dependent on NC’s dynamic interaction with nucleic acids, with the protein playing a critical role in the two-strand transfer steps that occur during viral DNA synthesis. In minus-strand transfer, the first product of reverse transcription, (−) strong-stop DNA [(−) SSDNA], is annealed to the RNA sequence at the 3¢ end of the genome (acceptor RNA) in a reaction facilitated by base-pairing of the complementary repeat regions in the nucleic acid substrates. This reaction is followed by reverse transcriptase– (RT) catalyzed elongation of minus-strand DNA. Current work focuses on understanding the influence of nucleic acid structure on NC nucleic acid chaperone activity, with an emphasis on the importance of local structure, and on the role of NC in fidelity of reverse transcription.
In previous studies on NC-facilitated HIV-1 minus-strand transfer, we found that the structure and thermostability of nucleic acid intermediates are major determinants for the transfer process. In fact, we concluded that a delicate thermodynamic balance between (−) SSDNA and acceptor RNA must be maintained for efficient minus-strand transfer. Thus, if either acceptor RNA or (−) SSDNA is highly stable, minus-strand transfer will not occur. This conclusion is consistent with the fact that NC is a weak destabilizer of nucleic acid structure.
In the course of our work, we made an unexpected observation: an acceptor RNA (RNA 70) with a high delta G value had more strand transfer activity than a smaller RNA (RNA 50) with a lower delta G value [we used the same (−) SSDNA in the assays]. Taken in conjunction with mFold analysis of the RNA structures, the result led us to postulate that NC nucleic acid chaperone activity is ultimately dependent on the thermostability of local structure at the nucleation site for annealing, not on the overall stability of the RNA. If true, of the two acceptors, RNA 70 likely has the weaker local structure at the nucleation site. To test such a hypothesis, we made mutations at the potential nucleation site at the 5¢ end of each RNA. As predicted, we observed that destabilizing changes in local structure dramatically increased strand transfer activity, whereas stabilizing changes led to a striking reduction in strand transfer and increased dependence on NC. Interestingly, our data also pointed to different consequences for annealing versus strand transfer when in vitro reactions were strongly or weakly dependent on NC. When NC had little or no effect, annealing and strand transfer occurred with similar efficiencies. However, when NC-catalyzed destabilization of acceptor structure was required, annealing appeared to be more efficient than strand transfer. We could explain 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. Collectively, our findings provide new insights into the mechanism of NC-dependent and -independent minus-strand transfer.
In other experiments, we are investigating initiation of HIV-1 plus-strand DNA synthesis by the RNA polypurine tract (PPT) primer and the roles of HIV-1 NC and RNase H activity in ensuring the specificity of this initiation process. Our interest in the process arises from the fact that many potential RNA primers are generated by RNase H degradation of viral RNA during an early step in reverse transcription. Our approach involves comparison of plus-strand DNA synthesis primed by the authentic PPT primer with the activity of other purine-rich RNA oligonucleotide sequences near the PPT whose activities are termed “mispriming.” The primers fall into three groups: (1) priming efficiency about 50 percent of that seen with the PPT; (2) modest priming efficiency at about 10 to 15 percent of PPT activity; and (3) very poor priming activity. Interestingly, when wild-type RT (RNase H+) is used, NC dramatically reduces DNA extension by all non–PPT primers. PPT priming is unaffected by RNase H or NC. NC alone can also inhibit mispriming, although the effect is greater when both NC and RNase H activities are present. Studies to address the mechanism by which NC reduces mispriming are in progress.
Levin JG, Guo J, Rouzina I, Musier-Forsyth K. Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism. Prog Nucleic Acids Res Mol Biol 2005;80:217-86.
Wu T, Heilman-Miller SL, Levin JG. 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 2007;35:3974-87.
Molecular analysis of the functional activities of APOBEC3G (A3G)
We are interested in host factors that can influence reverse transcription. A3G, a cellular cytidine deaminase with two zinc finger domains, 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 A3G have been performed primarily in cell-based systems and with unfractionated enzyme from viral lysates. We 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 A3G’s deaminase and nucleic acid binding activities. Using the purified enzyme, we demonstrated that A3G 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 A3G binds efficiently to ssDNA or ssRNA, less efficiently to a DNA/RNA hybrid, and very poorly to dsDNA or dsRNA. The data demonstrate that the requirements for nucleic acid binding and deamination differ. We also showed that, although NC and A3G 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, possibly 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 A3G binding. This result presumably reflects NC chaperone activity, which transiently destabilizes the SL-3 RNA. In turn, destabilization promotes increased binding of A3G, which has a preference for binding to single-stranded nucleic acids.
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 A3G into virions than zinc finger two. In contrast, deamination is associated exclusively with the second zinc finger. Moreover, zinc finger two is more important than zinc finger one for the antiviral effect, demonstrating a correlation between deaminase and antiviral activities but also suggesting that the antiviral activity may have a small deaminase-independent component.
Our finding that A3G does not interfere with NC binding to ssRNA (and vice versa) (see above) raised the possibility that inhibition of reverse transcription by A3G is likely to be unrelated to an effect on NC chaperone function. To test this hypothesis and to probe the mechanism that might be involved, we took advantage of defined biochemical assay systems that we have developed over the years for studies of viral DNA synthesis. Using our highly purified A3G as well as purified NC and RT, we investigated the effect of A3G on a series of reconstituted reactions that occur during reverse transcription. The reconstituted reactions allowed us to perform an independent analysis of individual steps in the pathway, which is not possible with cell-based systems. We found that A3G inhibited all RT-catalyzed DNA elongation reactions, but not RNase H activity or NC’s ability to promote annealing. Inhibition of RT polymerase function was independent of A3G’s deaminase activity, which is consistent with in vivo studies showing that deamination alone does not fully account for antiviral activity. We base the interpretation of our results on collaborative studies of the nucleic acid binding properties of NC, A3G, and RT as measured by single-molecule DNA stretching and fluorescence anisotropy (FA). The biophysical data suggest that A3G competes very effectively with RT for binding to an ss nucleic acid template but is not effective at displacing NC. Thus, our results support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC, and RT.
Iwatani Y, Takeuchi H, Strebel K, Levin JG. Biochemical activities of highly purified, catalytically active human APOBEC3G: correlation with antiviral effect. J Virol 2006;80:5992-6002.
Opi S, Takeuchi H, Kao K, Khan MA, Miyagi E, Goila-Gaur R, Iwatani Y, Levin JG, Strebel K. Monomeric APOBEC3G is catalytically active and has antiviral activity. J Virol 2006;80:4673-82.
Function of HIV-1 capsid protein in virus assembly and early post-entry events
Our laboratory has been investigating the role of the the HIV-1 CA protein in early post-entry events, a stage in the infectious process that is still not completely understood. In our initial study using genetic, molecular, and ultrastructural approaches, we described 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 presence of 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 post-entry. We also observed a substantial increase in the retention of CA, suggesting that mutant cores might be defective in core disassembly. Taken together, our findings demonstrate the intimate connection among infectivity, proper core morphology, structural integrity of the CA protein, and the ability to undergo reverse transcription.
More recently, we performed 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. We developed mutant constructs that might retain the ability to replicate, thus providing an opportunity to isolate second-site suppressors. We made a total of 13 additional substitutions at position 23, 2 at position 40, and a double mutation. Only one mutant, W23F, exhibited infectivity in a single-cycle assay, albeit at a very low level. The phenotype of W23F 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 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, as well as 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.
Even though the structures of the N- and C-terminal domains were solved some time ago, little information exists on the short linker region (residues 146–150) that connects the two CA domains. In current work, we have made alanine-scanning mutations in several conserved residues in the linker region and, so far, have found that, with the exception of P147A, all the other mutants 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 mutations in the hydrophobic residues that we have previously studied, alanine substitutions in some of the linker residues are tolerated. We also made mutations in two lysine residues (one N-terminal, the other C-terminal) that can be cross-linked. Further analysis of the mutants, including the biochemical properties of isolated cores, the ability to synthesize viral DNA post-entry, replication kinetics in several cell lines, and characterization of virion architecture by electron microscopy, is in progress.
Tang S, Ablan S, Dueck M, Ayala-López W, Soto B, Caplan M, Nagashima K, Hewlett IK, Freed EO, Levin JG. 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 2007;359:105-15.
1 Left the laboratory in 2006.
2 Joined the laboratory in 2007.
3 Left the laboratory in 2007.
4 Suchitra Derebail, PhD, former Postdoctoral Fellow
COLLABORATORS
Eric O. Freed, PhD, HIV Drug Resistance Program, NCI at Frederick, Frederick, MD
Robert J. Gorelick, PhD, AIDS Vaccine Program, SAIC Frederick, Inc., NCI at Frederick, Frederick, MD
Angela M. Gronenborn, PhD, University of Pittsburgh Medical School, Pittsburgh, PA
Karin Musier-Forsyth, PhD, Ohio State University, Columbus, OH
Ioulia Rouzina, PhD, University of Minnesota, Minneapolis, MN
Klaus Strebel, PhD, Laboratory of Molecular Microbiology, NIAID, Bethesda, MD
Shixing Tang, MD, PhD, CBER, FDA, Bethesda, MD
Mark C. Williams, PhD, Northeastern University, Boston, MA
For further information, contact jlevin@mail.nih.gov.
