Anna Marie Skalka

Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro.

The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle. The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN). Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA. In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA. These results demonstrate that the retroviral IN protein is an integrase.

HIV-1 integrase: structural organization, conformational changes, and catalysis.

Integrase comprises three domains capable of folding independently and whose three-dimensional structures are known. However, the manner in which the N-terminal, catalytic core, and C-terminal domains interact in the holoenzyme remains obscure. Catalytically active recombinant IN can exist in a dynamic equilibrium of monomers, dimers, tetramers, and higher order species. Numerous studies indicate that the enzyme functions as a multimer, minimally a dimer. The IN proteins from HIV-1 and ASV have been studied most carefully with respect to the structural basis of catalysis. Although the active site of ASV IN does not undergo significant conformational changes on binding the required metal cofactor, that of HIV-1 IN does. The reversible, metal-induced conformational change in HIV-1 IN impairs the binding of some anti-HIV-1 IN monoclonal antibodies to the enzyme and results in differential susceptibility of the protein to proteolysis. This active site-mediated conformational change reorganizes the catalytic core and C-terminal domains and appears to promote an interaction that is favorable for catalysis. Other metal-dependent structural changes in HIV-1 IN include the promotion of interactions between the N terminal and the catalytic core domains and the induction of tetramers by zinc ions. The end result of these metal-induced changes is apparently the induction of an activated holoenzyme that can form a stable ternary integrase-metal-DNA complex. These structural changes, which appear to be crucial for optimum catalysis in HIV-1 IN, do not occur in ASV IN. The structural changes observed in HIV-1 IN may serve to recruit the catalytic machinery in this enzyme to a conformation that is native for ASV IN.

The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations

BACKGROUND Members of the structurally-related superfamily of enzymes that includes RNase H, RuvC resolvase, MuA transposase, and retroviral integrase require divalent cations for enzymatic activity. So far, cation positions are reported in the X-ray crystal structures of only two of these proteins, E. coli and human immunodeficiency virus 1 (HIV-1) RNase H. Details of the placement of metal ions in the active site of retroviral integrases are necessary for the understanding of the catalytic mechanism of these enzymes. RESULTS The structure of the enzymatically active catalytic domain (residues 52-207) of avian sarcoma virus integrase (ASV IN) has been solved in the presence of divalent cations (Mn2+ or Mg2+), at 1.7-2.2 A resolution. A single ion of either type interacts with the carboxylate groups of the active site aspartates and uses four water molecules to complete its octahedral coordination. The placement of the aspartate side chains and metal ions is very similar to that observed in the RNase H members of this superfamily; however, the conformation of the catalytic aspartates in the active site of ASV IN differs significantly from that reported for the analogous residues in HIV-1 IN. CONCLUSIONS Binding of the required metal ions does not lead to significant structural modifications in the active site of the catalytic domain of ASV IN. This indicates that at least one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a catalytically competent active site. Only a single divalent cation was observed even at extremely high concentrations of the metals. We conclude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein. The unexpected differences between the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 IN, or a tendency for structural polymorphism.

Genome-Wide Analyses of Avian Sarcoma Virus Integration Sites

ABSTRACT 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.

Molecular mechanisms in retrovirus DNA integration

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 enzymes 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 Integrase, Putting the Pieces Together

Retroviral integrase (IN) mediates retroviral DNA integration, a critical step in viral replication that ensures stable expression of proviral genes in the infected cell and perpetuation of the viral genome in all the host cell progeny. The IN protein is both necessary and sufficient for the integration of a linear DNA with viral end sequences into a target DNA in vitro (1, 2). The integration reaction is known to take place in two distinct steps (see Fig. 1). IN specifically recognizes sequences at both ends of newly synthesized viral DNA, most likely as a component of a large subviral preintegration complex. The first step in integration, a processing reaction, can take place in the cytoplasm of infected cells (3). This reaction produces site-specific cuts near the viral DNA 39-ends, adjacent to a conserved CA dinucleotide, removing (generally) two nucleotides and exposing new 39-hydroxyl ends. The second step, a joining reaction, is a concerted cleavage-ligation reaction (4), which produces a staggered cut in cellular DNA when the newly exposed 39-hydroxyls of the viral DNA ends attack the phosphate bonds at the cellular DNA cleavage site. The product is an intermediate in which the 39-ends of viral DNA are covalently linked to cellular DNA and the 59-ends of viral DNA are flanked by short gaps (5, 6). Repair of the gaps and completion of integration, which can be accomplished by cellular enzymes, produce a short direct repeat of host target DNA. The length of this repeat is characteristic for each virus. For example, HIV-1 proviruses are flanked by 5-base pair repeats and ASV by 6-base pair repeats. These features are likely to reflect subtle differences in the structure or multimeric organization of the two integrases. Details of the biochemistry of IN and its catalytic mechanism have been uncovered mainly through the use of in vitro, reconstructed systems, which employ purified enzymes and model DNA substrates that consist of short oligodeoxynucleotide duplexes (1, 2, 7, 8). Unlike several site-specific recombinases that utilize a protein-DNA covalent intermediate, the cleavage-ligation reaction is a direct transesterification. The in-line nucleophilic attack by the processed viral 39-hydroxyls on the phosphate bond of the target DNA occurs with chiral inversion when appropriate substrates are used for detection (4). It has recently been discovered that a similar, but intramolecular, transesterification is catalyzed by another eukaryotic recombinase complex (RAG1 and RAG2) in the early steps of V(D)J recombination of immunoglobulin genes (9). Retroviral integrases range from 270 to 350 amino acids in length. Various lines of evidence indicate that both ASV and HIV-1 IN function as multimers (minimally, dimers) in vitro (10–12). However, the number and arrangement of IN protomers in the active in vivo complex are still unknown. Deduced amino acid sequence alignments, limited proteolysis, site-directed mutagenesis studies, and complementation experiments (reviewed in Refs. 13 and 14) have revealed the presence of three distinct domains that form independent folding units within each monomer. The first two of these are highly conserved among retroviral and retrotransposon integrases (Fig. 2). Here we will review what is known about the structure of each domain and discuss current ideas concerning domain interactions and multimerization, as they relate to function.

Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity.

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called “processing,” and the second is a polynucleotide transfer or “joining” step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.

Evidence that the retroviral DNA integration process triggers an ATR-dependent DNA damage response

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 integrase in 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.

Retroviral proteases: First glimpses at the anatomy of a processing machine

Anna Marie Skalka Institute for Cancer Research Fox Chase Cancer Center 7701 Burholme Avenue Philadelphia, Pennsylvania 19111 The recent surge of interest in the molecular biology of retroviruses is due in large part to the urgency of identify- ing specific targets for antiviral therapy for diseases such as AIDS. In addition to uncovering genes and control mechanisms that are unique to the human retrovirus strains and their relatives, these studies have also fo- cused attention on specific steps in the replication path- way that are common to all retroviruses. Our understand- ing of one of these steps, the proteolytic processing of viral precursor polyproteins by a virus-encoded protease (PR), has been greatly enhanced by recent studies, not only of the human immunodeficiency virus (HIV) but also of the avian sarcomalleukosis viruses, including avian myelo- blastosis virus (AMV) and Rous sarcoma virus (RSV). The PR proteins of the latter two differ by only two amino acids. Retrovird PR Structures The three-dimensional structure of the mature, 124 amino acid PR purified from RSV particles is the first report of a crystal structure for any retroviral protein (Miller et al., 1989). The structure shows a nearly symmetric dimer simi- lar in conformation to that reported for the single polypep- tide chain-containing microbial aspartic proteases, rhizo- puspepsin and endothiapepsin. This result was quickly supported by a report of similar conclusions based on analysis of the crystal structure of HIV PR protein pro- duced in Escherichia coli (Navia et al., 1989). These find- ings verify earlier hypotheses based on the identification of a conserved Asp-Thr(Ser)-Gly sequence found in one copy in retroviral PRs but present in each of the two do- mains that constitute an active site in cell-derived aspartic proteases (Toh et al., 1985). Biochemical studies have re- cently shown that the active forms of the AMV and HIV PRs behave in gel filtration as expected for dimers of mo- lecular weight similar to that of the cell-derived aspartic protease monomers (Kotler et al., 1989; Hansen et al., 1988). It is significant that the conditions reported to be most favorable for RSV PR crystallization (Miller et al., 1988)-low pH and high salt concentrations-are also op- timal for AMV enzyme activity, and that the crystallized RSV protein retains activity. The two retroviral PR structures are similar in overall to- pology to a model for HIV PR that was proposed by Pearl and Taylor (1987b) based on comparison with the reported structures of the two microbial pepsins cited above. The structures are most conserved at the loops that contain the active site, as illustrated in Figure