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My laboratory investigations have been driven by an abiding interest in understanding the consequences of genetic rearrangement in evolution and disease, and in using viruses to elucidate fundamental mechanisms in biology. Starting with bacteriophages and moving to the retroviruses, my use of the tools of genetics, molecular biology, biochemistry, and biophysics has spanned more than half a century—from the time when DNA structure was just discovered to the present day of big data and epigenetics. Both riding and contributing to the successive waves of technology, my laboratory has elucidated fundamental mechanisms in DNA replication, repair, and recombination. We have made substantial contributions in the area of retroviral oncogenesis, delineated mechanisms that control retroviral gene expression, and elucidated critical details of the structure and function of the retroviral enzymes—reverse transcriptase, protease, and integrase—and have had the satisfaction of knowing that the fundamental knowledge gained from these studies contributed important groundwork for the eventual development of antiviral drugs to treat AIDS. While pursuing laboratory research as a principal investigator, I have also been a science administrator—moving from laboratory head to department chair and, finally, to institute director. In addition, I have undertaken a number of community service, science-related “extracurricular” activities during this time. Filling all of these roles, while being a wife and mother, has required family love and support, creative management, and, above all, personal flexibility—with not too much long-term planning. I hope that this description of my journey, with various roles, obstacles, and successes, will be both interesting and informative, especially to young female scientists.

Retroviral integrases are reported to form alternate dimer assemblies like the core-core dimer and reaching dimer. The core-core dimer is stabilized predominantly by an extensive interface between two catalytic core domains. The reaching dimer is stabilized by N-terminal domains that reach to form intermolecular interfaces with the other subunit's core and C-terminal domains (CTD), as well as CTD-CTD interactions. In this study, molecular dynamics (MD), Brownian dynamics (BD) simulations, and free energy analyses, were performed to elucidate determinants for the stability of the reaching dimer forms of full-length Avian Sarcoma Virus (ASV) and Human Immunodeficiency Virus (HIV) IN, and to examine the role of the C-tails (the last ~16-18 residues at the C-termini) in their structural dynamics. The dynamics of an HIV reaching dimer derived from small angle X-ray scattering and protein crosslinking data, was compared with the dynamics of a core-core dimer model derived from combining the crystal structures of two-domain fragments. The results showed that the core domains in the ASV reaching dimer express free dynamics, whereas those in the HIV reaching dimer are highly stable. BD simulations suggest a higher rate of association for the HIV core-core dimer than the reaching dimer. The predicted stability of these dimers was therefore ranked in the following order: ASV reaching dimer HIV reaching dimer composite core-core dimer. Analyses of MD trajectories have suggested residues that are critical for intermolecular contacts in each reaching dimer. Tests of these predictions and insights gained from these analyses could reveal a potential pathway for the association and dissociation of full-length IN multimers.

Collaborations between the Wlodawer and Skalka laboratories have covered a period of almost 30 years. During that time our groups have co-authored 18 publications, including several much cited journal articles, book chapters, and scholarly reviews. It has therefore been most rewarding for us to share enthusiasm, insights, and expertise with our Frederick colleagues over the years, and also to enjoy lasting friendships.

The retroviral integrases are virally encoded, specialized recombinases that catalyze the insertion of viral DNA into the host cell's DNA, a process that is essential for virus propagation. We have learned a great deal since the existence of an integrated form of retroviral DNA (the provirus) was first proposed by Howard Temin in 1964. Initial studies focused on the genetics and biochemistry of avian and murine virus DNA integration, but the pace of discovery increased substantially with advances in technology, and an influx of investigators focused on the human immunodeficiency virus. We begin with a brief account of the scientific landscape in which some of the earliest discoveries were made, and summarize research that led to our current understanding of the biochemistry of integration. A more detailed account of recent analyses of integrase structure follows, as they have provided valuable insights into enzyme function and raised important new questions.

Cellular identity in both normal and disease processes is determined by programmed epigenetic activation or silencing of specific gene subsets. Here, we have used human cells harboring epigenetically silent GFP-reporter genes to perform a genome-wide siRNA knockdown screen for the identification of cellular factors that are required to maintain epigenetic gene silencing. This unbiased screen interrogated 21,121 genes, and we identified and validated a set of 128 protein factors. This set showed enrichment for functional categories, and protein-protein interactions. Among this set were known epigenetic silencing factors, factors with no previously identified role in epigenetic gene silencing, as well as unstudied factors. The set included non-nuclear factors, for example, components of the integrin-adhesome. A key finding was that the E1 and E2 enzymes of the small ubiquitin-like modifier (SUMO) pathway (SAE1, SAE2/UBA2, UBC9/UBE2I) are essential for maintenance of epigenetic silencing. This work provides the first genome-wide functional view of human factors that mediate epigenetic gene silencing. The screen output identifies novel epigenetic factors, networks, and mechanisms, and provides a set of candidate targets for epigenetic therapy and cellular reprogramming.

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the "reaching dimer" previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.

Integrated retroviral DNA is subject to epigenetic transcriptional silencing at different frequencies. This process is mediated by repressive DNA methylation and histone modifications on viral chromatin. However, the detailed mechanisms by which retroviral silencing is initiated and maintained are not well understood. Using a model system in which avian sarcoma virus (ASV) DNA is epigenetically repressed in mammalian cells, we previously found that a cellular scaffolding protein, Daxx, acts as an antiretroviral factor that promotes epigenetic repression through recruitment of histone deacetylases (HDACs). Here we show that human Daxx protein levels are increased in response to retroviral infection and that Daxx acts at the time of infection to initiate epigenetic repression. Consistent with a rapid and active antiviral epigenetic response, we found that repressive histone marks and long terminal repeat (LTR) DNA methylation could be detected within 12 h to 3 days postinfection, respectively. Daxx was also found to be required for long-term ASV silencing maintenance and full viral DNA methylation, and it was physically associated with both viral DNA and DNA methyltransferases (DNMTs). These findings support a model in which incoming retroviral protein-DNA complexes are detected by Daxx, and the integrated provirus is rapidly chromatinized and repressed by DNA methylation and histone modification as part of an antiviral response. These results uncover a possible direct and active antiviral mechanism by which DNMTs can be recruited to retroviral DNA.

We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.

Oligonucleotide assays have been invaluable for elucidation of the molecular mechanisms of retroviral integrases. A suite of rapid and sensitive fluorescence assays to measure the DNA binding, processing, and joining activities of integrase (IN) is described here. The assays are especially useful for characterizing the major activities of the enzyme, and for handling large numbers of samples efficiently. They can greatly facilitate further biochemical and structural analyses for HIV-1 and other IN proteins. The assays can also be adapted for moderate-high throughput testing of various inhibitory compounds.

Background Retroviral integration depends on the interaction between intasomes, host chromatin and cellular targeting cofactors as LEDGF/p75 or BET proteins. Previous studies indicated that the retroviral integrase, by itself, may play a role in the local integration site selection within nucleosomal target DNA. We focused our study on this local association by analyzing the intrinsic properties of various retroviral intasomes to functionally accommodate different chromatin structures in the lack of other cofactors. Results Using in vitro conditions allowing the efficient catalysis of full site integration without these cofactors, we show that distinct retroviral integrases are not equally affected by chromatin compactness. Indeed, while PFV and MLV integration reactions are favored into dense and stable nucleosomes, HIV-1 and ASV concerted integration reactions are preferred into poorly dense chromatin regions of our nucleosomal acceptor templates. Predicted nucleosome occupancy around integration sites identified in infected cells suggests the presence of a nucleosome at the MLV and HIV-1 integration sites surrounded by differently dense chromatin. Further analyses of the relationships between the in vitro integration site selectivity and the structure of the inserted DNA indicate that structural constraints within intasomes could account for their ability to accommodate nucleosomal DNA and could dictate their capability to bind nucleosomes functionally in these specific chromatin contexts. Conclusions Thus, both intasome architecture and compactness of the chromatin surrounding the targeted nucleosome appear important determinants of the retroviral integration site selectivity. This supports a mechanism involving a global targeting of the intasomes toward suitable chromatin regions followed by a local integration site selection modulated by the intrinsic structural constraints of the intasomes governing the target DNA bending and dictating their sensitivity toward suitable specific nucleosomal structures and density. Electronic supplementary material The online version of this article (doi:10.1186/s12977-015-0145-9) contains supplementary material, which is available to authorized users.

HIV-1 integrase, which catalyzes the joining of viral DNA to the host cell DNA, has attracted considerable attention as a target for the design and screening of novel anti-HIV drugs as it is essential for virus replication and the establishment of persistent infection. Progress in the identification of different classes of compounds that block integrase activity has been summarized recently in several excellent reviews. Here, we present a brief overview of integrase inhibition, highlighting some of the unusual properties of this protein and important considerations in searching for potential new inhibitors and their evaluation.

The cellular protein Daxx was identified as an interactor with avian sarcoma virus (ASV) integrase (IN) in a yeast two-hybrid screen. After infection, Daxx-IN interactions were detected by coimmunoprecipitation. An association between Daxx and viral DNA, likely mediated by IN, was also detected by chromatin immunoprecipitation. Daxx was not required for early events in ASV replication, including integration, as Daxx-null cells were transduced as efficiently as Daxx-expressing cells. However, viral reporter gene expression from ASV-based vectors was substantially higher in the Daxx-null cells than in Daxx-complemented cells. Consistent with this observation, histone deacetylases (HDACs) were found to associate with viral DNA in Daxx-complemented cells but not in Daxx-null cells. Furthermore, Daxx protein was induced in an interferon-like manner upon ASV infection. We conclude that Daxx interacts with an IN-viral DNA complex early after infection and may mediate the repression of viral gene expression via the recruitment of HDACs. Our findings provide a novel example of cellular immunity against viral replication in which viral transcription is repressed via the recruitment of antiviral proteins to the viral DNA.

The study of retroviruses over the last century has revealed a wide variety of disease-producing mechanisms, as well as apparently harmless interactions with animal hosts. Despite their potential pathogenic properties, the intrinsic features of retroviruses have been harnessed to create gene transfer vectors that may be useful for the treatment of disease. Retroviruses, as all viruses, have evolved to infect specific cells within the host, and such specificities are relevant to both pathogenesis and retrovirus-based vector design. The majority of cells of an animal host are not progressing rapidly through the cell cycle, and such a cellular environment appears to be suboptimal for replication of all retroviruses. Retrovirus-based vectors can therefore be restricted in many important target cells, such as post-mitotic differentiated cells or stem cells that may divide only infrequently. Despite intense interest, our understanding of how cell cycle status influences retroviral infection is still quite limited. In this review, we focus on the importance of the cell cycle as it relates to the early steps in retroviral replication. Retroviruses have been categorized based on their abilities to complete these early steps in non-cycling cells. However, all retroviruses are subject to a variety of cell cycle restrictions. Here, we discuss such restrictions, and how they may block retroviral replication, be tolerated, or overcome.

The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 avian sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected. However, RNA polymerase II transcription units (protein-encoding genes) appear to be favored targets of ASV integration. The integration frequency within genes is similar to that previously described for murine leukemia virus but distinct from the higher frequency observed with human immunodeficiency virus type 1. We found no evidence for preferred ASV integration sites over the length of genes and immediate flanking regions. Microarray analysis of uninfected HeLa cells revealed that the expression levels of ASV target genes were similar to the median level for all genes represented in the array. Although expressed genes were targets for integration, we found no preference for integration into highly expressed genes. Our results provide a more detailed description of the chromosomal features that may influence ASV integration and support the idea that distinct, virus-specific mechanisms mediate integration site selection. Such differences may be relevant to viral pathogenesis and provide utility in retroviral vector design.

We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PK CS ) and the damage-signaling kinases (ATM and ATR) have been implicated in this process. However, some studies have suggested that NHEJ proteins may not be required for the completion of lentiviral DNA integration. Here we provide additional evidence that NHEJ proteins are required for stable transduction by human immunodeficiency type 1 (HIV-1)-based vectors. Our analyses with two different reporters show that the number of stably transduced DNA-PK CS -deficient scid fibroblasts was reduced by 80 to 90% compared to the number of control cells. Furthermore, transduction efficiency can be restored to wild-type levels in scid cells that are complemented with a functional DNA-PK CS gene. The efficiency of stable transduction by an HIV-1-based vector is also reduced upon infection of Xrcc4 and ligase IV-deficient cells, implying a role for these components of the NHEJ repair pathway. Finally, we show that cells deficient in ligase IV are killed by infection with an integrase-competent but not an integrase-deficient HIV-1 vector. Results presented in this study lend further support to a general role for the NHEJ DNA repair pathway in completion of the retroviral DNA integration process.

Caffeine is an efficient inhibitor of cellular DNA repair, likely through its effects on ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases. Here, we show that caffeine treatment causes a dose-dependent reduction in the total amount of HIV-1 and avian sarcoma virus retroviral vector DNA that is joined to host DNA in the population of infected cells and also in the number of transduced cells. These changes were observed at caffeine concentrations that had little or no effect on overall cell growth, synthesis, and nuclear import of the viral DNA, or the activities of the viral integrasein vitro. Substantial reductions in the amount of host-viral-joined DNA in the infected population, and in the number of transductants, were also observed in the presence of a dominant-negative form of the ATR protein, ATRkd. After infection, a significant fraction of these cells undergoes cell death. In contrast, retroviral transduction is not impeded in ATM-deficient cells, and addition of caffeine leads to the same reduction that was observed in ATM-proficient cells. These results suggest that activity of the ATR kinase, but not the ATM kinase, is required for successful completion of the viral DNA integration process and/or survival of transduced cells. Components of the cellular DNA damage repair response may represent potential targets for antiretroviral drug development.

The integrase-catalyzed insertion of the retroviral genome into the host chromosome involves two reactionsin vivo: 1) the binding and endonucleolytic removal of the terminal dinucleotides of the viral DNA termini and 2) the recombination of the ends with the host DNA. Kukolj and Skalka (Kukolj, G., and Skalka, A. M. (1995) Genes Dev. 9, 2556–2567) have previously shown that tethering of the termini enhances the endonucleolytic activities of integrase. We have used 5′-5′ phosphoramidites to design reverse-polarity tethers that allowed us to examine the reactivity of two viral long terminal repeat-derived sequences when concurrently bound to integrase and, additionally, developed presteady-state assays to analyze the initial exponential phase of the reaction, which is a measure of the amount of productive nucleoprotein complexes formed during preincubation of integrase and DNA. Furthermore, the reverse-polarity tether circumvents the integrase-catalyzed splicing reaction (Bao, K., Skalka, A. M., and Wong, I. (2002) J. Biol. Chem. 277, 12089–12098) that obscures accurate analysis of the reactivities of synapsed DNA substrates. Consequently, we were able to establish a lower limit of 0.2 s−1 for the rate constant of the processing reaction. The analysis showed the physiologically relevant U3/U5 pair of viral ends to be the preferred substrate for integrase with the U3/U3 combination favored over the U5/U5 pair.

Integrase (IN) catalyzes the insertion of retroviral DNA into chromosomal DNA of a host cell and is one of three virus-encoded enzymes that are required for replication. A library of monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1) IN was raised and characterized in our laboratory. Among them, monoclonal antibody (mAb) 33 and mAb32 compete for binding to the C-terminal domain of the HIV-1 IN protein. Here, we show that mAb33 is a strong inhibitor of IN catalytic activity, whereas mAb32 is only weakly inhibitory. Furthermore, as the Fab fragment of mAb32 had no effect on IN activity, inhibition by this mAb may result solely from its bivalency. In contrast, Fab33 did inhibit IN catalytic activity, although bivalent binding by mAb33 may enhance the inhibition. Interaction with Fab33 also prevented DNA binding to the isolated C-terminal domain of IN. Results from size-exclusion chromatography, gel electrophoresis, and matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses revealed that multiple Fab33 small middle dotIN C-terminal domain complexes exist in solution. Studies using heteronuclear NMR showed a steep decrease in (1)H-(15)N cross-peak intensity for 8 residues in the isolated C-terminal domain upon binding of Fab33, indicating that these residues become immobilized in the complex. Among them, Ala(239) and Ile(251) are buried in the interior of the domain, whereas the remaining residues (Phe(223), Arg(224), Tyr(226), Lys(244), Ile(267), and Ile(268)) form a contiguous, solvent-accessible patch on the surface of the protein likely including the epitope of Fab33. Molecular modeling of Fab33 followed by computer-assisted docking with the IN C-terminal domain suggested a structure for the antibody-antigen complex that is consistent with our experimental data and suggested a potential target for anti-AIDS drug design.

Retroviral infection induces integrase-dependent apoptosis in DNA-PK-deficient murine scid lymphocytes. Furthermore, the efficiency of stable transduction of reporter genes is reduced in adherent cell lines that are deficient in cellular DNA-repair proteins known to mediate nonhomologous end joining (NHEJ), such as DNA-PK and XRCC4 (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Here we report that wortmannin, an irreversible inhibitor of phosphatidylinositol 3-kinase (PI-3K)-related PKs, including the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) and ATM, sensitizes normal murine lymphocytes to retrovirus-mediated cell killing. We also show that the efficiency of stable transduction of reporter genes in human (HeLa) cells, mediated by either an avian sarcoma virus or a human immune deficiency virus type 1 vector, is reduced in the presence of wortmannin. The dose dependence of such reduction correlates with that for inhibition of PI-3K-related protein kinase activity in these cells. Results from wortmannin treatment of a panel of cell lines confirms that formation and/or survival of transductants is dependent on components of the NHEJ pathway. However, stable transduction is virtually abolished by wortmannin treatment of cells that lack ATM. These results suggest that ATM activity is required for the residual transduction observed in the NHEJ-deficient cells. Our studies support the hypothesis that DNA repair proteins of the NHEJ pathway and, in their absence, ATM are required to avoid integrase-mediated killing [corrected] and allow stable retroviral DNA transduction. The studies also suggest that cells can be sensitized to such killing and stable retroviral DNA integration blocked by drugs that inhibit cellular DNA repair pathways.

Model oligodeoxyribonucleotide substrates representing viral DNA integration intermediates with a gap and a two-nucleotide 5' overhang were used to examine late steps in human immunodeficiency virus, type 1 (HIV-1) retroviral integrase (IN)-catalyzed DNA integration in vitro. HIV-1 or avian myeloblastosis virus reverse transcriptase (RT) were capable of quantitatively filling in the gap to create a nicked substrate but did not remove the 5' overhang. HIV-1 IN also failed to remove the 5' overhang with the gapped substrate. However, with a nicked substrate formed by RT, HIV-1 IN removed the overhang and covalently closed the nick in a disintegration-like reaction. The efficiency of this closure reaction was very low. Such closure was not stimulated by the addition of HMG-(I/Y), suggesting that this protein only acts during the early processing and joining reactions. Addition of Flap endonuclease-1, a nuclease known to remove 5' overhangs, abolished the closure reaction catalyzed by IN. A series of base pair inversions, introduced into the HIV-1 U5 long terminal repeat sequence adjacent to and/or including the conserved CA dinucleotide, produced no or only a small decrease in the HIV-1 IN-dependent strand closure reaction. These same mutations caused a significant decrease in the efficiency of concerted DNA integration by a modified donor DNA in vitro, suggesting that recognition of the ends of the long terminal repeat sequence is required only in the early steps of DNA integration. Finally, a combination of HIV-1 RT, Flap endonuclease-1, and DNA ligase is capable of quantitatively forming covalently closed DNA with these model substrates. These results support the hypothesis that cellular enzyme(s) may catalyze the late steps of retroviral DNA integration.

Monoclonal antibodies (mAbs) have been used extensively in the biochemical analysis of proteins. Molecular identification of a specific epitope can enhance our understanding of the relationship between the structure and function of a protein. We recently developed a protein footprint technique for mapping mAb epitopes that employs limited proteolysis followed by peptide analysis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Here we describe the rational for the technique and illustrate its use in mapping the epitopes of two mAbs that bind to the C-terminal domain of human immunodeficiency virus type-1 integrase. The results provide a plausible explanation for the fact that one mAb inhibits enzyme activity while the second does not. © 2001 John Wiley & Sons, Inc. Biopolymers (Pept Sci) 55: 308–318, 2000

In the presence of a divalent metal cofactor (Mg2+ or Mn2+), retroviral-encoded integrase (IN) catalyzes two distinct reactions: site-specific cleavage of two nucleotides from both 3' ends of viral DNA, and sequence-independent joining of the recessed viral ends to staggered phosphates in a target DNA. Here we investigate human immunodeficiency virus type 1 (HIV-1) IN-DNA interactions using surface plasmon resonance. The results show that IN forms tight complexes both with duplex oligonucleotides that represent the viral DNA ends and with duplex oligonucleotides with an unrelated sequence that represent a target DNA substrate. The IN-DNA complexes are stable in 4.0 M NaCl, or 50% (v/v) methanol, but they are not resistant to low concentrations of SDS, indicating that their stability is highly dependent on structural features of the protein. Divalent metal cofactors exert two distinct effects on the IN-DNA interaction. Mn2+ inhibits IN binding to a model target DNA with the apparent Kd increasing approximately 3-fold in the presence of this cation. On the other hand, Mn2+ (or Mg2+) stimulates the binding of IN to a model viral DNA end, decreasing the apparent Kd of this IN-viral DNA complex approximately 6-fold. Such metal-mediated stimulation of the binding of IN to the viral DNA is totally abolished by substitution of the subterminal conserved CA/GT bp with a GT/CA bp, and is greatly diminished when the viral DNA end is recessed or "pre-processed." IN binds to a viral duplex oligonucleotide whose end was extended with nonviral sequences with kinetics similar to the nonviral model target DNA. This suggests that IN can distinguish the integrated DNA product from the viral donor DNA in the presence of divalent metal ion. Thus, our results show that preferential recognition of viral DNA by HIV-1 IN is achieved only in the presence of metal cofactor, and requires a free, wild-type viral DNA end.

Retroviral DNA integration is catalyzed by the viral protein integrase. Here, it is shown that DNA-dependent protein kinase (DNA-PK), a host cell protein, also participates in the reaction. DNA-PK-deficient murine scid cells infected with three different retroviruses showed a substantial reduction in retroviral DNA integration and died by apoptosis. Scid cell killing was not observed after infection with an integrase-defective virus, suggesting that abortive integration is the trigger for death in these DNA repair–deficient cells. These results suggest that the initial events in retroviral integration are detected as DNA damage by the host cell and that completion of the integration process requires the DNA-PK–mediated repair pathway.

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration in vitro with specially designed mini-donor HIV-1 DNA, a supercoiled plasmid acceptor, purified bacterium-derived HIV-1 integrase (IN), and host HMG protein family members. This system is comparable to one previously described for avian sarcoma virus (ASV) (A. Aiyar et al., J. Virol. 70:3571–3580, 1996) that was stimulated by the presence of HMG-1. Sequence analyses of individual HIV-1 integrants showed loss of 2 bp from the ends of the donor DNA and almost exclusive 5-bp duplications of the acceptor DNA at the site of integration. All of the integrants sequenced were inserted into different sites in the acceptor. These are the features associated with integration of viral DNA in vivo. We have used the ASV and HIV-1 reconstituted systems to compare the mechanism of concerted DNA integration and examine the role of different HMG proteins in the reaction. Of the three HMG proteins examined, HMG-1, HMG-2, and HMG-I(Y), the products formed in the presence of HMG-I(Y) for both systems most closely match those observed in vivo. Further analysis of HMG-I(Y) mutants demonstrates that the stimulation of integration requires an HMG-I(Y) domain involved in DNA binding. While complexes containing HMG-I(Y), ASV IN, and donor DNA can be detected in gel shift experiments, coprecipitation experiments failed to demonstrate stable interactions between HMG-I(Y) and ASV IN or between HMG-I(Y) and HIV-1 IN.

Selection of transgenic embryos prior to embryo transfer is a means to increase the efficiency of transgenic livestock production. Among transgenic reporters, cytoplasmic expression of green fluorescent protein (GFP) has features that make it ideal for transgenic embryo selection. The primary objective of this study was to assess cytoplasmic expression of a specially designed GFP reporter as a tool for transgenic bovine embryo selection. A second objective was to evaluate this reporter for studying transgenic mosaicism related to timing of integration of pronuclear microinjected DNA. Transgenic embryos produced by pronuclear injection showed a discrete pattern of GFP expression with clusters at 25, 50, and 100% of blastomeres expressing GFP. This pattern of mosaicism is interpreted to indicate that the integration of microinjected DNA occurred, not only at the pronuclear stage, but also in the subsequent cell divisions. Among the GFP-positive transgenic embryos, only in 21% did all the blastomeres show the green fluorescence. Using the fraction of positive blastomeres within an embryo, the timing of integration of microinjected DNA was estimated. The frequency of nonmosaic embryos expressing GFP is consistent with published germline transmission success rates of transgenic cattle derived from pronuclear microinjected embryos. These results indicate the possible application of GFP as a marker of transgenic embryos and graphically illustrate underlying complexities in DNA integration in embryos subjected to pronuclear microinjection.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) undergoes a reversible metal-induced conformational change that activates the enzyme (Asante-Appiah, E., and Skalka, A. M. (1997) J. Biol. Chem. 272, 16196-16205). In this report, key structural features that mediate this conformational change have been identified by site-directed mutagenesis, limited proteolysis, and mass spectrometry studies. The results reveal two separable metal-induced effects. One depends on residues in the N-terminal domain (amino acids 1-50) and a C-terminal tail (amino acids 274-288) and is detected by increased resistance of the full-length protein to proteolytic digestion. This effect appears to depend on metal binding at an undefined location distinct from the known sites in the N-terminal and catalytic core domains. The second conformational change depends on metal binding at the active site in the catalytic core domain. Substitution of acidic residues Asp64 or Glu152 in the catalytic core D,D(35)E motif or truncation of the Src homology 3 (SH3)-like domain in the C-terminal region of the enzyme abolishes this metal-induced change. Comparison of tryptic digests of an HIV-1 IN derivative competent for metal-induced conformational change and a conformation-defective D64N derivative identified specific regions in HIV-1 IN that are affected by this second change. A region in the N terminus that spans Lys14, an extended loop and the adjacent region in the core domain (including lysines 136, 156, and 160 and Arg173), and residues at the C terminus beyond the SH3-like domain all become less accessible to proteolysis in the conformation-competent protein. In contrast, a region that encompasses Lys258 in the putative DNA binding groove of the SH3-like domain becomes more sensitive to proteolysis in the presence of Mn2+. The results are consistent with a model in which the binding of the metal ion by residues of the D,D(35)E motif elicits specific changes in all three domains of HIV-1 IN, inducing the restructuring of the enzyme for catalytic competence.

A sequence of 21 amino acids (aa) in the C-terminal region of the 286-aa avian sarcoma virus (ASV) integrase (IN) protein has been shown previously to mediate nuclear localization of both IN and β-galactosidase (βGal) protein fused to it. This karyophilic sequence includes a high proportion of prolines and residues with basic side chains. In this report, site-directed mutagenesis was used to introduce single aa substitutions of several of these residues. Indirect immunofluorescence showed that IN-βGal fusion constructs with Ala substitutions for sequence constituents K206, P215, K225 or R227 had lost the exclusive nuclear localization capability of the wild-type fusion. A fusion protein with the conservative substitution K206R retained the nuclear localization capacity. The site-specific substitutions that reduced karyophilic activity had no effect on the processing or joining activities of IN in vitro. However, the introduction of three of the four Ala codon substitutions into viral DNA clones caused a significant delay in viral replication following transfection of cycling chicken embryo fibroblasts. These results are consistent with a possible role for ASV IN in nuclear targeting.

Retroviruses utilize balanced splicing to express multiple proteins from a single primary transcript. A number of cis -acting signals help maintain this balance, including the branch point sequence (BPS), polypyrimidine tract (PPyT) and sequences within the downstream exon. In general, regulated splicing requires weak splicing signals and we have previously shown the same requirement for the simple retrovirus, avian sarcoma virus (ASV). Here we take advantage of the requirement for balanced splicing in retroviral replication to examine the sequence constraints of an intronic splicing element. Selection for replication competence makes it possible to amplify and identify functional sequences from a pool of all possible sequences. In this report we examine the role of pyrimidines within the PPyT. Our results provide in vivo confirmation that the functional strength of a PPyT is related to its length and uridine content and that the PPyT plays a role in the second step of the splicing reaction. We also show that the minimal distance between the 3'-splice site and the BPS in this system is 16 nt. With modification, the selection system described here can be used to examine the sequence constraints of other exonic or intronic splicing elements in vivo .

Retroviruses display a unique form of alternative splicing in which both spliced and unspliced RNAs accumulate in the cytoplasm. Simple retroviruses, such as avian sarcoma virus, do not encode regulatory proteins that affect splicing; this process is controlled solely through interactions between the viral RNA and the host cell splicing machinery. Previously, we described the selection and characterization of novel avian sarcoma virus mutants. These viruses were separated into two classes based upon analysis of splicing intermediates produced in infected cells and in a cell-free system. One class, which included mutants with altered polypyrimidine tract or branch point sequences, showed significant accumulation of intermediates, suggesting that splicing was regulated in step 2. The other class, which included mutants with deletions of exonic enhancer sequences, did not accumulate splicing intermediates, suggesting that splicing was regulated before step 1 of the splicing reaction. In this report, we show that a mutant blocked at step 1 fails to form a stable spliceosomal complex, whereas one blocked at step 2 shows a defect in its ability to transit through the last spliceosomal complex. Using UV cross-linking methods, we show that regulation at each step is associated with specific changes in the binding of cellular splicing factors. Regulation at step 1 is correlated with decreased cross-linking of the factor U2AF65, whereas regulation at step 2 is correlated with enhanced cross-linking of the factor SAP49. Because these mutations were isolated by selection for replication-competent viruses, we conclude that retroviral splicing may be regulated in vivo through altered binding of constitutive splicing factors.

The integrase protein of retroviruses catalyzes the insertion of the viral DNA into the genomes of the cells that they infect. Integrase is necessary and sufficient for this recombination reaction in vitro; however, the enzyme's activity appears to be modulated in vivo by viral and cellular components included in the nucleoprotein pre-integration complex. In addition to integrase, cis-acting sequences at the ends of the viral DNA are important for integration. Solution of the structures of the isolated N- and C-terminal domains of HIV-1 integrase by nuclear magnetic resonance (NMR) and the available crystal structures of the catalytic core domains from human immunodeficiency virus type-1 (HIV-1) and avian sarcoma virus (ASV) integrases are providing a structural basis for understanding some aspects of the integration reaction. The role of the evolutionarily conserved acidic amino acids in the D,D(35)E motif as metal-coordinating residues that are critical for catalysis, has been confirmed by the metal-integrase (core domain) complexes of ASV integrase. The central role that integrase plays in the life cycle of the virus makes it an attractive target for the design of drugs against retroviral diseases such as AIDS. To this end, several compounds have been screened for inhibitory effects against HIV-1 integrase. These include DNA intercalators, peptides, RNA ligands, and small organic compounds such as bis-catechols, flavones, and hydroxylated arylamides. Although the published inhibitors are not very potent, they serve as valuable leads for the development of the next generation of tight-binding analogues that are more specific to integrase. In addition, new approaches are being developed, exemplified by intracellular immunization studies with conformation-sensitive inhibitory monoclonal antibodies against HIV-1 integrase. Increased knowledge of the mechanism of retroviral DNA integration should provide new strategies for the design of effective antivirals that inhibit integrase in the future.

Retroviral integrases are composed of three independently folding domains whose organization relevant to one another is largely unknown. As an approach to understanding its structure, we have investigated the effect of the required metal cofactor(s), Mn2+ or Mg2+, on the conformation of human immunodeficiency virus type 1 (HIV-1) integrase (IN) using monoclonal antibodies (mAbs) that are specific for each of these three domains. Upon the addition of increasing concentrations of the divalent cations to immobilized HIV-1 IN in ELISA assays, binding of mAbs specific for either the C-terminal domain or for an epitope in the catalytic core domain was lost, whereas binding of an N terminus-specific mAb was unaffected. Size exclusion chromatography of a nonaggregating derivative of HIV-1 IN showed that the oligomeric state of the protein did not change under conditions in which recognition of the core and C terminus-specific mAbs was lost. Preincubation with Mn2+ increased the resistance of HIV-1 IN to proteolytic digestion and produced a digestion pattern that was significantly different from that observed with the apoprotein. A derivative that lacked the N-terminal domain, IN(50-288), exhibited the same metal-dependent changes observed with the full-length protein, whereas the isolated catalytic core domain IN(50-212) did not. From this we conclude that the metal-induced conformational change comprises a reorganization of the core and C-terminal domains. Preincubation with Mn2+ increased the specific activity of HIV-1 IN 5-fold. Enzymatic activity was inhibited by the conformation-sensitive C terminus-specific mAb, but this inhibition was reduced greatly if the enzyme was first preincubated with metal ions. Thus, it appears that apo-HIV-1 IN exists predominantly in an inactive conformation that is converted into a catalytically competent form upon the addition of metal ions.