George Merkel

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.

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.

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.

Activities and substrate specificity of the evolutionarily conserved central domain of retroviral integrase

The retroviral integrase (IN) is a virus-encoded enzyme that is essential for insertion of viral DNA into the host chromosome. In order to map and define the properties of a minimal functional domain for this unique viral enzyme, a series of N- and C-terminal deletions of both Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) INs were constructed. The RSV IN deletion mutants were first tested for their ability to remove two nucleotides from the end of a substrate representing the terminus of viral DNA in order to assess the contribution of N and C regions towards this reaction, referred to as processing. The results suggest that C-terminal amino acids of the intact RSV protein are required to maintain specificity of the processing reaction. Though deficient for processing, the RSV deletion mutants exhibited a secondary endonucleolytic activity that was indistinguishable from that of wild-type IN, demonstrating that all retained some enzymatic activity. RSV, and a larger set of HIV-1, IN deletion mutants were then tested for their ability to perform an intramolecular, concerted cleavage-ligation reaction using an oligodeoxynucleotide substrate that mimics the intermediate viral-host DNA junction found prior to the final step of covalent closure. The composite results from such analyses define a minimal functional central region of approximately 140 amino acids for each enzyme that includes the highly conserved D,D(35)E domain. Results with HIV-1 and HIV-2 IN also indicate that the efficiency of concerted cleavage-ligation depends upon the presence of CA/GT base pairs within the viral component of the DNA substrate at the reaction site. Even the isolated central region of HIV-1 IN exhibited this sequence requirement for optimal activity. We conclude that this evolutionarily conserved central region of IN not only encodes residues that are required for the catalytic activity of the enzyme but also harbors some or all of the determinants responsible for recognition of the CA/GT dinucleotides that are present at the ends of all retroviral DNAs.

Wortmannin Potentiates Integrase-Mediated Killing of Lymphocytes and Reduces the Efficiency of Stable Transduction by Retroviruses

ABSTRACT 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-PKCS) 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 2killing 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.

Atomic resolution structures of the core domain of avian sarcoma virus integrase and its D64N mutant.

Six crystal structures of the core domain of integrase (IN) from avian sarcoma virus (ASV) and its active-site derivative containing an Asp64 --> Asn substitution have been solved at atomic resolution ranging 1.02-1.42 A. The high-quality data provide new structural information about the active site of the enzyme and clarify previous inconsistencies in the description of this fragment. The very high resolution of the data and excellent quality of the refined models explain the dynamic properties of IN and the multiple conformations of its disordered residues. They also allow an accurate description of the solvent structure and help to locate other molecules bound to the enzyme. A detailed analysis of the flexible active-site region, in particular the loop formed by residues 144-154, suggests conformational changes which may be associated with substrate binding and enzymatic activity. The pH-dependent conformational changes of the active-site loop correlates with the pH vs activity profile observed for ASV IN.

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

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.

Targeting Tn5 Transposase Identifies Human Immunodeficiency Virus Type 1 Inhibitors

ABSTRACT Human immunodeficiency virus (HIV) type 1 (HIV-1) integrase is an underutilized drug target for the treatment of HIV infection. One limiting factor is the lack of costructural data for use in the rational design or modification of integrase inhibitors. Tn5 transposase is a structurally well characterized, related protein that may serve as a useful surrogate. However, little data exist on inhibitor cross-reactivity. Here we screened 16,000 compounds using Tn5 transposase as the target and identified 20 compounds that appear to specifically inhibit complex assembly. Six were found to also inhibit HIV-1 integrase. These compounds likely interact with a highly conserved region presumably within the catalytic core. Most promising, several cinnamoyl derivatives were found to inhibit HIV transduction in cells. The identification of integrase inhibitors from a screen using Tn5 transposase as the target illustrates the utility of Tn5 as a surrogate for HIV-1 integration even though the relationship between the two systems is limited to the active site architecture and catalytic mechanism.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates

Background: No full-length structure of HIV integrase alone has been reported. Results: We elucidated the architectures of dimers and tetramers of full-length HIV-1 integrase in solution. Conclusion: HIV apo-integrase can assemble in two alternate dimer forms: a reaching and a core-core dimer. The tetramer comprises two stacked reaching dimers, stabilized by core-core interactions. Significance: New insights into HIV integrase architecture and its inhibition are suggested. 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.