Molecular Genetics of Mammalian Retrovirus Replication

Judith G. Levin, PhD, Head, Section on Viral Gene Regulation
Tiyun Wu, PhD, Staff Scientist
Kamil Hercik, PhD, Postdoctoral Fellow
Jiyang Jiang, MD, PhD, Postdoctoral Fellow
Mithun Mitra, PhD, Postdoctoral Fellow¹
Klara Post, MS, Senior Research Assistant
Amber L. Hertz, BS, Postbaccalaureate Fellow²
Pilar H. Saladores, BS, Postbaccalaureate Fellow³
Jianhui Guo, MD, PhD, Special Volunteer

The goal our research is to define the molecular mechanisms responsible for the replication of HIV and related retroviruses and to investigate the role of host proteins that block virus infection. Our studies are critical for developing new strategies to combat the AIDS epidemic, which continues to pose a global threat to human health. 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 structures. This activity is critical for highly efficient and specific viral DNA synthesis. We are also studying the mechanism of antiviral activity of two host cytidine deaminases, APOBEC3G (A3G) and APOBEC3A (A3A). In other studies, we are working to understand 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

Wu, Post, Levin; in collaboration with Gorelick, Musier-Forsyth, Rein

HIV-1 NC is a small basic protein with two zinc fingers, each containing the invariant CCHC zinc-coordinating residues. Its nucleic acid chaperone function consists of two independent activities: (1) nucleic acid aggregation (N-terminal basic residues) and (2) weak helix destabilizing activity (zinc fingers) (Levin et al., Prog Nucleic Acids Res Mol Biol 2005;80:217). NC plays 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, which is (−) 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. Our recent work on minus-strand transfer demonstrated that, instead of the overall structure, the local structure of the nucleic acid substrates at the nucleation site for annealing is a major determinant of NC’s chaperone function.

Current studies focus on a comparison of the nucleic acid chaperone activities of HIV-1 Gag and its proteolytic cleavage products, including NC (also known as NC7) with its immediate precursors, NC9 and NC15. We use the minus-strand transfer assay as the read-out for chaperone function, as the assay system represents the most stringent biochemical test for the chaperone activity. We performed the initial assays with Gag and the GagW-M mutant, which, unlike Gag, has weak dimerization activity. We found for the first time that both Gag and GagW-M stimulate minus-strand transfer and block the dead-end self-priming reaction (GagW-M stimulates slightly better than Gag), although not as efficiently as NC. The results showed that the two Gag proteins have sufficient helix destabilizing activity to unfold the highly stable TAR structures in acceptor RNA and (−) SSDNA. The data also indicate that our assay does not require Gag dimerization. Initial characterization of the chaperone activities of the NC precursors indicated that the order of activity in minus-strand transfer reactions is NC>NC9>NC15. We are now characterizing other Gag-related proteins.

In other work, we are investigating initiation of HIV-1 plus-strand DNA synthesis by the RNA polypurine tract (PPT) primer, along with the roles of HIV-1 NC and RNase H activity in ensuring the specificity of the synthesis. During the synthesis of minus-strand DNA, the RNase H activity of RT degrades viral genomic RNA sequences after their transcription into DNA. The degradation results in the release of several RNA fragments that have the potential to prime plus-strand DNA synthesis. We call such activity “mispriming.” If mispriming were to occur, viral DNA would not have the correct ends and would not be competent for integration. We have compared priming by the authentic PPT primer with the activities of other purine-rich, non–PPT RNAs, which have sequences from the region near the 3′ PPT. The PPT is the most efficient primer; the other primers show a range of activities (e.g., from 50 to less than 1 percent of PPT activity). Significant RNase H cleavage occurs only in the non–PPT reactions, suggesting that RNase H plays a major role in blocking mispriming. Interestingly, the addition of NC further reduces priming by all non–PPT primers to essentially baseline level but does not affect the PPT. Modest inhibition of mispriming by NC also occurs in the absence of RNase H. Collaborative experiments show that the biochemical data correlate with the biophysical properties of the PPT and non–PPT duplexes, as determined by circular dichroism spectroscopy, temperature-dependent UV absorption, and fluorescence anisotropy. These assays indicate that the PPT duplex is distinct from the non–PPT hybrids and help explain the inability of NC to affect PPT priming. Zinc coordination by NC is also required for inhibition of mispriming, implying that NC functions by destabilizing the non–PPT duplexes, leading to dissociation of the RNA strand. Collectively, our findings demonstrated a previously unrecognized role for NC nucleic acid chaperone activity, which, together with RNase H cleavage, dramatically increases the fidelity of plus-strand initiation.

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;35:3974-3987.

Molecular analysis of the functional activities of APOBEC proteins

Hercik, Mitra, Wu, Iwatani,⁴ Levin; in collaboration with Gronenborn, Musier-Forsyth, Rouzina, Strebel, Williams

A3G is a cellular cytidine deaminase with two zinc-finger domains. It acts as a potent inhibitor of HIV-1 reverse transcription and replication in the absence of the viral protein Vif. Most studies on human A3G have been performed primarily in cell-based systems or with unfractionated enzyme from viral lysates, but our interest lies in the host factors that can influence reverse transcription. We succeeded in producing highly purified, catalytically active protein expressed in a baculovirus system, permitting us for the first time to provide a detailed molecular analysis of A3G’s deaminase and nucleic acid–binding activities.

Interestingly, in the course of our work, we found that A3G does not interfere with NC binding to single-stranded RNA (and vice versa), thereby suggesting that inhibition of reverse transcription by A3G is unlikely to be related to an effect on NC chaperon function. To test such a hypothesis and 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. We were thus able 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—consistent with in vivo studies showing that deamination alone does not fully account for antiviral activity. The interpretation of our results is based on collaborative studies of the nucleic acid–binding properties of NC, A3G, and RT as measured by single-molecule DNA stretching and fluorescence anisotropy. The biophysical data suggest that A3G competes effectively with RT for binding to a single-stranded nucleic acid template but that it is not effective in 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.

We have initiated studies on another human APOBEC protein, A3A, a cytidine deaminase with only one zinc-finger domain. A3A does not affect HIV replication but does inhibit retrotransposition of non–LTR (long-terminal repeat) elements representing 17 percent (LINE-1) and 11 percent (Alu) of the human genome, respectively. We expressed this protein in soluble form in E. coli and are now in the process of obtaining large amounts of purified protein for biochemical and structural analysis. Initial results showed that the partially purified protein has deaminase activity with a target sequence specificity that differs from that of A3G. Future work will focus on further characterization of A3A’s functional activities.

Iwatani Y, Chan DS-B, Wang F, Maynard KS, Sugiura W, Gronenborn A, Rouzina I, Williams MC, Musier-Forsyth K, Levin JG. Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G. Nucleic Acids Res 2007;35:7096-7108.
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;80:4673-4682.

Function of HIV-1 capsid protein in virus assembly and early post-entry events

Jiang, Derebail,⁴ 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 infection 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, lack a cone-shaped core, and, despite their functional RT enzyme, are blocked in the initiation of viral DNA synthesis in infected cells. Mutant viral cores display a marked deficiency in RT and have substantially increased amounts of CA, suggesting a defect in core disassembly. Taken together, our findings demonstrated the intimate connection among infectivity, proper core morphology, structural integrity of the CA protein, and ability to undergo reverse transcription.

More recently, we performed a study to provide new information on the plasticity of CA, i.e., its ability to tolerate changes in residues crucial for CA structure without total abrogation of biological activity. We constructed and tested mutants to determine whether they might retain the ability to replicate and thus offer an opportunity to isolate second-site suppressors. One mutant, W23F, exhibited 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. 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 were eventually able to isolate virions with a second-site suppressor mutation (W23F/V26I). The suppressor mutation 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. Our novel findings demonstrated that, despite the limits imposed on assembly of CA structure, HIV-1 is able to adapt partially to severe structural distortions in a major viral protein.

The past year saw a major advance with a report of the three-dimensional structure of the hexameric arrays of full-length HIV-1 CA at 9 Å resolution. Nonetheless, there is still little information on the nature of the short linker peptide (residues 146–150; SPTSI) that connects the two domains. In current work, we have made alanine-scanning mutations in all of the residues in the linker region as well as in the two residues (Y145 and L151) that flank the linker. All mutants except P147A produce virus particles; we have therefore substituted P147L in this series. Interestingly, of the five linker mutants, only two (S146A and T148A) have substantial infectivity in a single-cycle assay; Y145A and L151A display almost no infectivity. The mutants all have a normal protein profile, but, in the electronmicroscope, mutants with low infectivity exhibit aberrant core structures. Experiments to examine the properties of isolated cores are now in progress. In addition, we have made mutations in two interacting lysine residues, N-terminal (K70) and C-terminal (K182). Substitution of alanine or aspartic acid resulted in loss of infectivity, but a conservative change to arginine resulted in a substantial level of infectivity. Transmission electronmicroscopy showed that only mutants K70R and K182R have conical cores. Further characterization of these mutants is under way.

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;359:105-115.

¹ Joined the laboratory in 2008.
² Joined the laboratory in 2008.
³ Left the laboratory in 2008.
⁴ Yasumasa Iwatani, PhD, former Research Fellow
⁵ 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 and Cancer Virus 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
Alan Rein, PhD, HIV Drug Resistance Program, NCI at Frederick, Frederick, MD
Ioulia Rouzina, PhD, University of Minnesota, Minneapolis, MN
Klaus Strebel, PhD, Laboratory of Molecular Microbiology, NIAID, Bethesda, MD
Shixing Tang, MD, PhD, Center for Biologics Evaluation and Research, FDA, Bethesda, MD
Mark C. Williams, PhD, Northeastern University, Boston, MA

For further information, contact jlevin@mail.nih.gov or visit http://svgr.nichd.nih.gov.