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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
Kamil Hercik, PhD, Postdoctoral Fellow
Jiyang Jiang, MD, PhD, Postdoctoral Fellow
Mithun Mitra, PhD, Postdoctoral Fellow
Klara Post, MS, Senior Research Assistant
Gabriel Nam, BA, Postbaccalaureate Fellow
Dustin Singer, BA, Postbaccalauareate 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 developed reconstituted model systems to investigate the individual steps in HIV-1 reverse transcription, a major target of HIV therapy. Much of our work is focused on the viral nucleocapsid protein (NC), a nucleic acid chaperone that remodels nucleic acid structures so that the most thermodynamically stable conformations are formed, an activity critical for highly efficient and specific viral DNA synthesis. We are also investigating the mechanism of antiviral activity of human APOBEC3 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 that occurs during 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 its 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.

We discovered that Gag facilitates minus-strand transfer, indicating that it has both annealing and helix-destabilizing activities (Wu et al. Virology 2010;405:556). At low concentrations, Gag is a more efficient chaperone than NC. However, high concentrations of Gag severely inhibit the DNA extension step, consistent with "nucleic acid–driven multimerization" of Gag (facilitated by CA domain interactions) and the known slow dissociation of Gag from bound nucleic acid, which in turn block RT movement along the single-stranded nucleic acid template. We refer to this phenomenon as the "roadblock" mechanism for inhibition of HIV-1 reverse transcription. Taken together, our results illustrate how the presence of NC in the multidomain Gag protein modulates the nature of its nucleic acid chaperone activity and emphasize that an effective nucleic acid chaperone for reverse transcription must exhibit rapid on-off nucleic-acid binding kinetics. The data also help explain why NC, rather than the Gag precursor, has evolved as the critical cofactor in viral DNA synthesis.

More recently, we found that ZF mutations in the NC domain of Gag—either mutation of CCHC to CCCC in one or both ZFs or deletion of one or both ZFs—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, thereby resulting in significant extension in the absence of ZF function. We also examined the effect of Gag on minus-strand initiation, i.e., annealing of tRNA^Lys3 to viral RNA followed by RT–catalyzed elongation of the tRNA primer to synthesize (-) strong-stop DNA. In this case too, 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 also 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) in assays for minus-strand initiation and strand transfer.

In other work, we continued to study the RNA removal steps that occur in the course of reverse transcription and to investigate an obligatory reaction preceding minus-strand transfer. 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 that RNase H cleavage of blunt-ended substrates is inefficient. However, these fragments must be removed so that minus-strand transfer can proceed. We hypothesized that fragment removal is facilitated by NC destabilization of the short duplex and/or by RNase H cleavage (2). To test this prediction, we heat-annealed a 20-nt RNA, whose sequence is complementary to the 3′ end of (-) strong-stop DNA, to the DNA. Additional components were added (e.g., acceptor RNA, NC, RT, dNTPs etc.) and, following incubation, we determined the percent transfer product synthesized. Given that strand transfer cannot occur unless the RNA is removed, this assay uses formation of the transfer product as the readout for RNA removal. The results showed that minus-strand transfer occurred to the same extent in the absence or presence of the 20-nt RNA, indicating that the small RNA was removed with high efficiency. In the context of reverse transcription (the context in which RNA removal occurs), both NC and RNase H activity as well as NC's ZF function (ability to coordinate zinc and helix destabilizing activity) were required for maximal activity. We also measured the efficiency of fragment removal directly, i.e., in the absence of reverse transcription, using novel gel-shift mobility and FRET (fluorescence resonance energy transfer) assays with radioactively labeled RNA or RNA with a fluorescent label (FAM). NC was able to facilitate RNA removal in each case and, as in the RT assay, the reaction was dependent on NC's ZF function. Our findings are in excellent agreement with our earlier studies on the tRNA primer removal step in plus-strand transfer (Wu et al. J Virol 1999;73:4794) and on 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 uses a common strategy for all RNA removal reactions that occur during reverse transcription.

Molecular analysis of human APOBEC proteins

Our interest in host proteins that might affect HIV-1 reverse transcription led us to investigate the activities of human A3 proteins, a family of seven cytidine deaminases that convert dC residues to dU in single-stranded DNA. The proteins function as cellular restriction factors that play an important role in the innate immune response to viral pathogens including HIV-1. Our initial studies focused on A3G, which blocks HIV-1 reverse transcription and replication in the absence of the viral protein known as Vif. In current work, we have been studying the human A3A protein, which degrades foreign DNA, blocks HIV-1 replication in myeloid cells, and inhibits retrotransposition of LINE-1 elements, a class of mobile genetic elements present in the human genome. The elements may be detrimental to the human genome, given that they can insert into coding regions of functional genes and cause severe and often fatal genetic diseases. To test the role of A3A deaminase activity in the inhibition of LINE-1 retrotransposition, we performed cell-based assays with A3A mutants, which were designed by using structure-guided mutagenesis. In general, mutants defective in deamination were also found to be poor inhibitors of retrotransposition. However, the absence of deaminase activity did not always abolish anti-LINE-1 activity, suggesting that, in addition to A3A's catalytic activity, other factors are involved.

To obtain further insights into the molecular properties of A3A, we are using highly purified recombinant protein to perform more detailed studies of A3A's cytidine deaminase activity and nucleic acid binding affinity. A3A has only one zinc-coordinating domain, which must function in catalysis and substrate binding. It is a slightly acidic protein and binds to nucleic acids with low affinity, with an apparent Kd value in the micromolar range. In this respect, A3A differs dramatically from A3G, which has two zinc-coordinating domains, is a basic protein, and has high affinity for nucleic acids, with an apparent Kd value in the nanomolar range (Iwatani et al. J Virol 2006;80:5992). However, we found that, like A3G, A3A deamination efficiency is exquisitely sensitive to the sequence at the recognition site in the DNA substrate. In other work on this project, we are collaborating with members of a structural biology group led by Angela Gronenborn, who recently solved the solution structure of A3A by NMR spectroscopy. Her analysis defines the interface that is critical for interaction with the single-stranded DNA substrate and identifies the positions of the catalytic residues as well as the residues required for substrate binding. The availability of the new structural information is the basis for learning more about A3A's cellular and antiviral functions.

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

Our laboratory investigates 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. Our initial studies of certain N-terminal domain (NTD) mutants illuminated the intimate connection between infectivity, proper core assembly, structural integrity of the CA protein, and ability to undergo reverse transcription. We are now focusing on the interdomain linker region.

Early structural studies indicated that HIV-1 CA consists of two independently folded domains, an NTD (residues 1-145) and a C-terminal domain (CTD) (residues 151–231), which are connected by a short, flexible linker (residues 146–150). Despite advances in our knowledge of CA structure and the considerable sequence conservation of the linker residues, only limited information on their biological and molecular properties has been so far available. To investigate the role of this region in virus assembly and replication, we made alanine-scanning mutations in all linker residues (except for P147, which was changed to leucine) and in the two flanking residues (Y145 and L151) (1). We identified three classes of mutants: (i) S146A and T148A, which behave like the wild type (WT) and exhibit infectivity in a single-cycle replication assay; (ii) Y145A, I150A, and L151A, which are noninfectious, assemble unstable cores with aberrant morphology, and synthesize almost no viral DNA; and (iii) P147L and S149A, which display a poorly infectious, attenuated phenotype. Surprisingly, despite their poor replication capacity, infectivity of P147L and S149A env⁻ mutants can be rescued in an efficient and specific manner when these virions are pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G). Although it is known that VSV-G–mediated entry occurs via pH–dependent endocytosis, the reason why this mode of entry can result in rescue of infectivity is not clear. We suggested that the known faster fusion kinetics with VSV-G than with HIV-1 Env might result in bypass of the infectivity defect conferred by the P147L and S149A mutations. Several different assays indicated that P147L and S149A cores are unstable. Nevertheless, analysis by transmission electron microscopy (TEM) showed that these mutants were able to assemble conical cores (about 50% of the number seen in the WT population) and formed WT–like tubular assemblies from purified CA proteins in vitro. In contrast, Y145A and I150A CA proteins did not assemble into any recognizable structure. The distinctive nature of the P147L and S149A mutants was also apparent in other assays. For example, when viral DNA products were assayed in infected cells by qPCR, both mutants synthesized approximately 10-fold less DNA than WT, whereas viral DNA products were reduced about 10⁴-fold in cells infected with I150A.

Taken together, our findings demonstrate that the HIV-1 interdomain linker region is a critical determinant of proper core assembly and stability. Moreover, this study has important implications for understanding the molecular nature of HIV-1 assembly, as it underscores 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 an effort to identify potential ultrastructural differences between the WT and P147L/S149A mutant core structures and the relation to CA function, experiments exploiting higher-resolution EM techniques are now in progress.

Publications

Jiang J, Ablan S, Derebail S, Hercík K, Soheilian F, Thomas JA, Tang S, Hewlett I, Nagashima K, Gorelick RJ, Freed EO, Levin JG. The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability. Virology 2011;421:253-265.
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 2012;E-pub ahead of print.
Levin JG. Obituary. Virus Res 2012;E-pub ahead of print.

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
Indira Hewlett, PhD, Center for Biologics Evaluation and Research, FDA, Bethesda, MD
Yasumasa Iwatani, PhD, National Hospital Organization Nagoya Medical Center, Nagoya, Japan
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
Shixing Tang, MD, PhD, Center for Biologics Evaluation and Research, FDA, Bethesda, MD
Mark C. Williams, PhD, Northeastern University, Boston, MA

Contact

For more information, email levinju@mail.nih.gov or visit jlevinlab.nichd.nih.gov.

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