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Molecular Genetics of Mammalian Retrovirus Replication
- Judith G. Levin, PhD, Head, Section on Viral Gene Regulation
- Tiyun Wu, PhD, Staff Scientist
- Mithun Mitra, PhD, Postdoctoral Fellow
- Klara Post, MS, Senior Research Assistant
- Dustin Singer, BA, Postbaccalaureate Fellow
The goal of the research performed in the Section on Viral Gene Regulation 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. The studies are critical for developing new strategies to combat the AIDS epidemic, which continues to be a global threat to human health. To this end, 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), a nucleic acid chaperone that remodels nucleic acid structures so that the most thermodynamically stable conformations are formed. This activity is critical for highly efficient and specific viral DNA synthesis. We are also investigating the mechanism of antiviral activity of human APOBEC3 (A3) proteins, which play a role in the cellular innate immune response to viral pathogens, including HIV-1. In other studies, our efforts have been directed toward 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 (sometimes referred to as NCp7) is a small basic protein with two zinc fingers (ZFs), each containing the invariant CCHC zinc-coordinating residues. Its nucleic acid chaperone function depends on three properties: (i) ability to aggregate nucleic acids, which is important for annealing (N-terminal basic residues); (ii) moderate helix-destabilizing activity (ZFs); and (iii) rapid on-off binding kinetics. NC plays a critical role in almost every step of viral DNA synthesis, including minus-strand transfer. In this case, the first product of reverse transcription, (-) strong-stop DNA, is annealed to the RNA sequence at the 3′ end of the genome (acceptor RNA), in a reaction mediated by base-pairing of the complementary repeat regions in the nucleic acid substrates. This is followed by reverse transcriptase (RT)–catalyzed elongation of minus-strand DNA. Our recent studies focused on a comparison of the nucleic acid chaperone activities of HIV-1 NC with HIV-1 Gag and Gag's proteolytic cleavage products. The sequence of Gag in the N to C direction is: matrix (MA)→capsid (CA)→spacer peptide 1 (SP1)→NC→SP2→p6. For this work, we use a reconstituted minus-strand transfer assay system, an especially sensitive read-out for chaperone function.
Our initial studies on Gag showed that it facilitates minus-strand transfer, indicating for the first time that it has both annealing and helix-destabilizing activities (Wu et al., Virology 2010;405:556). In fact, at low concentrations, Gag is a more efficient chaperone than NC. However, high concentrations of Gag severely inhibit the DNA extension step and block RT movement along the single-stranded (ss) nucleic acid template. We refer to this phenomenon as the “roadblock” mechanism for inhibition of HIV-1 reverse transcription. (This mechanism was originally proposed for the activity of A3G, a host restriction factor, as described below.)
In subsequent work, we have found that ZF mutations in the NC domain of Gag do not have a major effect on minus-strand transfer, in contrast to the very strict requirement for the native ZFs exhibited by mature NC. This discrepancy could reflect known differences in the conformations of mature NC and the NC domain in Gag. When Gag is free in solution, the NC domain is in close proximity to MA. It is possible that the positively charged residues in MA increase the annealing efficiency of the repeat sequences in acceptor RNA and (-) strong-stop DNA, via an aggregation-driven pathway, resulting in significant extension in the absence of ZF function. We also examined the effect of WT Gag on minus-strand initiation, i.e., annealing of tRNALys3 to viral RNA followed by RT–catalyzed elongation of the tRNA primer to synthesize (-) strong-stop DNA. Increasing concentrations of Gag inhibit activity in the assay. Given that Gag is known to facilitate the annealing reaction with high efficiency in vivo and in vitro, the data strongly suggest that Gag causes a major roadblock to tRNA primer extension. Thus, extension must occur during or following maturation, when mature NC rather than Gag is present in the virus. Gag ZF mutants have little or no activity, indicating a requirement for helix-destabilizing activity in the initiation assay.
We are now focusing on the nucleic acid chaperone activity of the immediate NC (i.e., NCp7) precursors: NCp9 (NC + SP2) and NCp15 (NC + SP2 + p6). Assays of tRNALys3–primed (-) strong-stop DNA synthesis show that, at low concentrations of protein, the rank order of activity is NCp9>NCp15>NC. Surprisingly, activity is dramatically reduced when the concentration of NCp9 is increased. Moreover, this phenomenon is also seen with high concentrations of NCp15, albeit to a lesser extent. Similarly, although NCp9 is more active than NC or NCp15 in minus-strand transfer reactions, at high concentrations of NCp9, synthesis of the transfer product reaches a plateau over time and then declines as incubation proceeds. Results of sedimentation assays suggest that this unusual behavior reflects NCp9’s strong nucleic acid aggregation activity, which is enhanced by its highly basic C-terminal SP2 domain. Interestingly, the SP2 peptide by itself can stimulate the annealing step in minus-strand transfer, but not the overall reaction [annealing and (-) strong-stop DNA extension]. NCp15 has much lower minus-strand transfer activity than NCp9 or NCp7, which we hypothesized is attributable to NCp15’s acidic C-terminal p6 domain. Indeed, mutants with Ala substitutions in the acidic residues have improved chaperone activity and considerably higher binding affinity to ssDNA (i.e., lower Kd values). Taken together, the results are in good agreement with published data on the biological activity of virions containing NCp9 or NCp15 instead of mature NC: (i) HIV-1 containing NCp15 is not infectious; (ii) although virus containing NCp9 is initially infectious, continued passage in culture results in reversion to WT. Thus, our data help explain why fully processed NCp7 has evolved as the critical cofactor in HIV-1 reverse transcription and is optimal for viral fitness.
In other work, we investigated the requirements for an obligatory RNA removal reaction preceding minus-strand transfer (1,2). During synthesis of (-) strong-stop DNA, the RNase H activity of RT degrades the viral RNA template. As RT reaches the end of the template, short 5′ RNA fragments remain annealed to the DNA because RNase H cleavage of blunt-ended substrates is inefficient. However, the fragments must be removed so that minus-strand transfer can proceed. Using our strand transfer assay, we showed that removal of a 20-nt RNA fragment pre-annealed to the 3′ end of (-) strong-stop DNA requires both NC and RNase H activity. Results obtained from novel gel mobility shift and FRET assays, which each directly measures RNA fragment release from a duplex in the absence of DNA synthesis, demonstrated that the architectural integrity of NC’s ZF domains is absolutely required for this reaction. This suggests that NC’s helix-destabilizing activity (associated with the ZFs) facilitates strand exchange through the displacement of short terminal RNAs by the longer 3′ acceptor RNA, which forms a more stable duplex with (-) strong-stop DNA. Our findings are in excellent agreement with our earlier studies of the tRNA primer removal step in plus-strand transfer (Wu et al., J Virol 1999;73:4794) and the ability of NC to block mispriming during initiation of plus-strand DNA synthesis (Post et al., Nucleic Acids Res 2009;37:1755). Most importantly, we can now conclude that HIV-1 has evolved a single mechanism for RNA removal reactions that are critical for successful reverse transcription.
Molecular analysis of human APOBEC proteins
An interest in host proteins that might affect HIV-1 reverse transcription led us to investigate the activities of human APOBEC3 (A3) proteins, a family of seven cytidine deaminases that convert dC residues to dU in ssDNA. The proteins function as cellular restriction factors that play an important role in the innate immune response to viral pathogens. Our initial studies focused on A3G, which blocks HIV-1 reverse transcription and replication in the absence of the viral protein known as Vif. We and others found that A3G can inhibit reverse transcription by deaminase-dependent and deaminase-independent mechanisms. Based on A3G’s biophysical properties, i.e., its slow dissociation from bound nucleic acid and its ability to oligomerize, we proposed that deaminase-independent inhibition results from an A3G−induced roadblock to RT−catalyzed DNA elongation (Iwatani et al., Nucleic Acids Res 2007;35:7096). A deamination mechanism requires rapid binding and release of ssDNA, while a roadblock mechanism requires slow binding. To determine whether these binding modes could be detected experimentally, we collaborated with Mark Williams and colleagues, who performed force-induced melting (“DNA stretching”) experiments with purified A3G (3). The data show that A3G initially binds to ssDNA with rapid on-off rates. However, A3G subsequently converts via oligomerization to a slowly dissociating mode, which would inhibit reverse transcription in the absence of deamination. Interestingly, an A3G mutant that cannot oligomerize does not exhibit slow ssDNA binding kinetics. Collectively, the findings provide a molecular basis for the dual mechanism used by A3G to block HIV-1 infection.
We have also been studying the human A3A protein, which inhibits a wide range of viruses (including HIV-1) and endogenous retroelements such as LINE-1. Surprisingly, A3A has an opposing biological activity. It can also edit genomic DNA, which may play a role in carcinogenesis. To understand the molecular mechanisms that underlie the biological activity of A3A, it was important to have highly purified A3A and to determine its structure. We have been collaborating with members of a structural biology group led by Angela Gronenborn, who solved the A3A solution structure at high resolution using NMR spectroscopy (4). The overall structure of A3A is similar to structures described for A3C and for the C-terminal domain of A3G (A3G−CD2), despite some differences, mainly in the loop regions. This analysis defined the surface for A3A interaction with short ssDNA substrates (equal to or larger than 15 nt) and identified the positions of the catalytic residues as well as the residues required for substrate binding.
In recent work, we performed biochemical as well as structure-guided mutagenesis studies designed to further characterize A3A’s deaminase and nucleic acid−binding properties and to determine their impact on biological function (5). We have found that A3A binds to and deaminates ssDNA in a length-dependent manner. Interestingly, although A3A also binds to ssRNA, NMR analysis demonstrates that the RNA and DNA binding surfaces differ. Some of the major interactions observed with ssDNA are absent during ssRNA binding, and no deamination of ssRNA is detected in real-time NMR assays. The binding data suggest that A3A cannot deaminate ssRNA because the cytosine ring in an RNA substrate cannot be positioned accurately in the catalytic site. In other experiments, using catalytically active and inactive A3A mutants, we found that, although the determinants of deaminase and anti-LINE-1 restriction activity can overlap, they are not identical, indicating that deaminase and LINE-1 restriction activities are not linked. We also observed that, unlike A3G, A3A does not inhibit RT−catalyzed elongation of DNA by a roadblock mechanism. The result is consistent with our finding that A3A is monomeric and slightly acidic (theoretical pI value of 6.34) and binds to nucleic acids with low affinity. To rationalize A3A’s ability to deaminate genomic DNA (double-stranded [ds] DNA is not a substrate), we postulated that this might occur during cellular processes such as transcription, which require transient opening of the duplex. To investigate this possibility, we designed 40-nt dsDNA substrates containing unpaired regions at the center to mimic transcription bubbles of different sizes and we tested these constructs in a deaminase assay. The results clearly indicate that access of A3A to the reactive dC in the transcription bubble becomes more facile with increasing ss character. We also showed that the process could be counteracted by slowly dissociating ssDNA–binding proteins. Taken together, the studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions. Research on A3 proteins is continuing, and our current efforts focus on A3H haplotype II, which has potent anti-HIV activity in the absence of Vif.
Function of HIV-1 capsid protein in virus assembly and early postentry events
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. In recent studies, we showed that the interdomain linker region (residues 146-150) is a critical determinant of proper core assembly and stability. In the course of this work, we identified two novel mutants (P147L and S149A). While poorly infectious, the mutants display an attenuated phenotype and, surprisingly, their infectivity is rescued when env-minus virions are pseudotyped with the vesicular stomatitis envelope glycoprotein (VSV-G). Moreover, despite having unstable cores, the mutants (i) synthesize viral DNA in infected cells, although less efficiently than WT virus, (ii) assemble a significant number of viral cores with seemingly normal architecture, and (iii) assemble tubular structures in vitro that resemble WT CA assemblies. The results underscore the unusual plasticity of CA, which, despite the rigorous structural requirements that govern assembly and integrity of viral cores, permits some expression of biological activity even under less than optimal circumstances. In future work, we plan to examine in vitro CA assemblies by cryo-electron tomography to determine the nature of the defect in the cores of these unusual mutants.
- Hergott CB, Mitra M, Guo J, Wu T, Miller JT, Iwatani Y, Gorelick RJ, Levin JG. Zinc finger function of HIV-1 nucleocapsid protein is required for removal of 5'-terminal genomic RNA fragments: a paradigm for RNA removal reactions in HIV-1 reverse transcription. Virus Res 2013;171:346-355.
- Levin JG. Obituary. Virus Res 2013;171:356.
- Chaurasiya KR, McCauley MJ, Wang W, Qualley DF, Wu T, Kitamura S, Geertsema H, Chan DSB, Hertz A, Iwatani Y, Levin JG, Musier-Forsyth K, Rouzina I, Williams MC. Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid binding protein. Nat Chem 2013;Epub ahead of print.
- Byeon I-JL, Ahn J, Mitra M, Byeon C-H, Hercik K, Hritz J, Charlton LM, Levin JG, Gronenborn AM. NMR structure of human restriction factor APOBEC3A reveals substrate binding and enzyme specificity. Nat Commun 2013;4:1890.
- Mitra M, Hercik K, Byeon I-JL, Ahn J, Hill S, Hinchee-Rodriguez K, Singer D, Byeon C-H, Charlton LM, Nam G, Heidecker G, Gronenborn AM, Levin JG. Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties. Nucleic Acids Res 2013;Epub ahead of print.
- 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
- Yasumasa Iwatani, PhD, National Hospital Organization Nagoya Medical Center, Nagoya, Japan
- Karin Musier-Forsyth, PhD, Ohio State University, Columbus, OH
- Ioulia Rouzina, PhD, University of Minnesota, Minneapolis, MN
- Mark C. Williams, PhD, Northeastern University, Boston, MA