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Nucleophile Selection for the Endonuclease Activities of Human, Ovine, and Avian Retroviral Integrases*

Open AccessPublished:January 05, 2001DOI:https://doi.org/10.1074/jbc.M007032200
      Retroviral integrases catalyze four endonuclease reactions (processing, joining, disintegration, and nonspecific alcoholysis) that differ in specificity for the attacking nucleophile and target DNA sites. To assess how the two substrates of this enzyme affect each other, we performed quantitative analyses, in three retroviral systems, of the two reactions that use a variety of nucleophiles. The integrase proteins of human immuno- deficiency virus type 1, visna virus, and Rous sarcoma virus exhibited distinct preferences for water or other nucleophiles during site-specific processing of viral DNA and during nonspecific alcoholysis of nonviral DNA. Although exogenous alcohols competed with water as the nucleophile for processing, the alcohols stimulated nicking of nonviral DNA. Moreover, different nucleophiles were preferred when the various integrases acted on different DNA targets. In contrast, the nicking patterns were independent of whether integrase was catalyzing hydrolysis or alcoholysis and were not influenced by the particular exogenous alcohol. Thus, although the target DNA influenced the choice of nucleophile, the nucleophile did not affect the choice of target sites. These results indicate that interaction with target DNA is the critical step before catalysis and suggest that integrase does not reach an active conformation until target DNA has bound to the enzyme.
      IN
      integrase
      HIV-1 and -2
      human immunodeficiency virus types 1 and 2
      RSV
      Rous sarcoma virus
      Integration of a DNA copy of the retroviral genome into cellular DNA is necessary for retrovirus replication and for the development of retrovirus-related diseases. This recombination event is mediated by the retroviral integrase (IN)1 protein. Purified integrase catalyzes one-step transesterifications in which the nucleophilic oxygen of an OH group attacks a DNA phosphodiester bond and covalently joins to the target DNA at the site of nicking (
      • Engelman A.
      • Mizuuchi K.
      • Craigie R.
      ). To mediate integration in vivo, integrase must catalyze two reactions that use distinctly different nucleophiles and targets. The first reaction, termed processing (see Fig. 1 A), prepares the viral DNA ends for subsequent attachment to cellular DNA (
      • Katzman M.
      • Katz R.A.
      • Skalka A.M.
      • Leis J.
      ). The target for this site-specific endonuclease reaction is the phosphodiester linkage immediately 3′ to the A of highly conserved CA bases near the 3′-ends of unintegrated retroviral DNA. Nicking at this site removes the terminal nucleotides (usually two) that follow the CA and creates a recessed 3′-hydroxyl group at each DNA end. In vitro studies indicate that the nucleophile for this reaction can be provided by the OH group of a water molecule (
      • Vink C.
      • Yeheskiely E.
      • van der Marel G.A.
      • van Boom J.H.
      • Plasterk R.H.A.
      ), by certain alcohols (
      • Vink C.
      • Yeheskiely E.
      • van der Marel G.A.
      • van Boom J.H.
      • Plasterk R.H.A.
      ,
      • Katzman M.
      • Mack J.P.G.
      • Skalka A.M.
      • Leis J.
      ), or by the OH group at the 3′-end of the unintegrated DNA itself (
      • Engelman A.
      • Mizuuchi K.
      • Craigie R.
      ) (see Fig. 2). Thus, processing is a site-specific alcoholysis reaction that uses a variety of nucleophiles (
      • Vink C.
      • Yeheskiely E.
      • van der Marel G.A.
      • van Boom J.H.
      • Plasterk R.H.A.
      ). The second reaction, referred to as joining or strand transfer (see Fig.1 B), inserts the processed viral DNA ends into the two strands of cellular DNA at sites that are separated by a few base pairs (
      • Craigie R.
      • Fujiwara T.
      • Bushman F.
      ,
      • Katz R.A.
      • Merkel G.
      • Kulkosky J.
      • Leis J.
      • Skalka A.M.
      ). Sequencing of multiple insertion sites has not revealed any target consensus sequence, even though certain sites within any sequence may be preferred during in vivo and in vitro integration (
      • Katzman M.
      • Katz R.A.
      ). Thus, joining can be considered nonspecific with respect to target DNA. However, only one nucleophilic donor,i.e. the processed viral DNA end, can be used for successful integration.
      Figure thumbnail gr1
      Figure 1Endonuclease activities of integrase. Curved arrows denote the coupled nucleophilic cleavage and joining that is common to all actions catalyzed by integrase.Heavy lines represent viral DNA and light linesrepresent any DNA. 5′-Phosphate groups are depicted by closed circles and 3′-hydroxyl groups by open circles. The invariant CA bases near the viral DNA end are in boldface. During processing (A), the terminal nucleotides (NN) can be removed by a variety of nucleophiles (represented as ROH). During joining (B), the recessed 3′-end of the processed viral DNA acts as the nucleophile to nick and join to any site in target DNA (similar reactions occur on each cellular DNA strand). During disintegration (C), which is a reversal of the joining reaction, a juxtaposed 3′-DNA-end attacks after the CA to seal the nick and regenerate the processed viral DNA end. During nonspecific alcoholysis (D), a variety of nucleophiles can nick and join to any internal site in DNA. The first two reactions occur in vivo, whereas all four reactions occur in vitro. Characteristics of the attacking nucleophile and target DNA are indicated at the right.
      Figure thumbnail gr2
      Figure 2Alternative nucleophiles for processing viral DNA ends. Integrase uses the electron pair (depicted as two dots) of certain nucleophilic molecules to nick after invariant CA bases (shown in boldface) near each of the viral DNA 3′-ends. Depending on whether water (HOH), an alcohol (ROH), or the viral DNA 3′-OH end acts as the nucleophilic donor for the nicking reaction (denoted by arrows above the substrate DNA), the terminal nucleotides (NN) leave either as a linear dinucleotide, bound to an alcohol, or circularized, respectively. The products can be distinguished by gel electrophoresis if the CA-containing strand is 32P-labeled near the 3′-end (as indicated by the circle); the products cannot be distinguished if the substrate is labeled at the 5′-end (as indicated by the asterisk).
      In addition to the biologically relevant processing and joining activities, integrase exhibits two other endonuclease activities in vitro. During disintegration (see Fig. 1 C), which is a reversal of the joining reaction, the 3′-OH of an oligonucleotide representing nicked cellular DNA attacks the bond linking the CA at the viral DNA end to nonviral DNA. This action seals the nick in the cellular DNA mimic and releases a processed viral DNA end (
      • Chow S.A.
      • Vincent K.A.
      • Ellison V.
      • Brown P.O.
      ). Because the nucleophile and target for this reaction are determined by the oligonucleotide complex, which juxtaposes the reactive groups, disintegration is specific for both substrates. The final and most recently described endonuclease activity of integrase is nonspecific alcoholysis (see Fig. 1 D). During this activity, integrase uses a variety of small alcohols as nucleophiles that nick and join to any internal 5′-phosphate group of DNA (
      • Katzman M.
      • Sudol M.
      ). As with DNA joining, any position in nonviral DNA can be attacked even though certain sites within a given target are preferred. Both disintegration and nonspecific alcoholysis can be catalyzed by the isolated central domain of the integrase protein, unlike processing and joining, which generally require the complete enzyme (
      • Katzman M.
      • Sudol M.
      ,
      • Bushman F.D.
      • Engelman A.
      • Palmer I.
      • Wingfield P.
      • Craigie R.
      ). The biological significance of disintegration and nonspecific alcoholysis is unknown. However, because integrase has a single catalytic site (
      • Esposito D.
      • Craigie R.
      ), these reactions have proved useful for analyzing the mechanism and substrate interactions of this important enzyme.
      Despite their mechanistic similarities, the four reactions catalyzed by integrase represent all possible combinations of specificity for the attacking nucleophile and the target DNA (see Fig. 1). In particular, processing uses various nucleophiles and a specific target site, joining uses a specific nucleophile and various target sites, disintegration uses a specific nucleophile and a specific target site, and nonspecific alcoholysis uses various nucleophiles and various target sites. How a single enzyme accomplishes this feat is unclear, but study of the interactions between integrase and its various substrates should shed light on its catalytic mechanism and may identify potential antiviral targets. In the current report, we describe quantitative analyses, in three retroviral systems, of the two integrase reactions that use a variety of nucleophiles (Fig. 1,A and D). The experiments tested two hypotheses: (i) that the target DNA influences the choice of attacking nucleophile and (ii) that the nature of the nucleophile influences the choice of target DNA sites. The data show that the integrase proteins of human immunodeficiency virus type 1 (HIV-1), visna virus, and Rous sarcoma virus (RSV) exhibit distinct preferences for water or other nucleophiles during processing and nonspecific alcoholysis assays. Moreover, although the target DNA influenced the choice of nucleophile, the nucleophile did not affect the choice of target sites. These results have implications for models of integration.

      DISCUSSION

      Retroviral integrase is a multifunctional enzyme that is responsible for the permanent incorporation of a DNA copy of the retroviral genome into cellular DNA. Knowledge of the biochemistry of how integrase functions is critical for obtaining a more complete view of the virus life cycle and for new treatments for the acquired immunodeficiency syndrome and other retrovirus-related diseases. In particular, how integrase interacts with its various substrates and how these substrates interact with each other are poorly understood. To examine how the target DNA and nucleophile affect each other during reactions catalyzed by integrase, the experiments in this report compared nucleophile selection by different integrases during the activities that accommodate a variety of nucleophiles.
      Processing of viral DNA ends in preparation for insertion into cellular DNA is the first enzymatic action required of integrase. In vitro studies have identified three types of nucleophiles that can provide the OH group for nicking near the viral DNA ends,i.e. water (
      • Vink C.
      • Yeheskiely E.
      • van der Marel G.A.
      • van Boom J.H.
      • Plasterk R.H.A.
      ), the 3′-end of the unintegrated DNA (
      • Engelman A.
      • Mizuuchi K.
      • Craigie R.
      ), and certain alcohols. Exogenous alcohols shown previously to participate in this reaction either have OH groups on adjacent carbon atoms, including glycerol, 1,2-ethanediol, and 1,2-propanediol (
      • Vink C.
      • Yeheskiely E.
      • van der Marel G.A.
      • van Boom J.H.
      • Plasterk R.H.A.
      ), or have OH and amino groups on adjacent carbons, such as the amino acids serine and threonine (
      • Vink C.
      • Yeheskiely E.
      • van der Marel G.A.
      • van Boom J.H.
      • Plasterk R.H.A.
      ,
      • Katzman M.
      • Mack J.P.G.
      • Skalka A.M.
      • Leis J.
      ). In contrast to a report that 1,3-diols do not participate, we found that the retroviral integrases from two lentiviruses (HIV-1 and visna virus) and an oncovirus (RSV) can use 1,3-propanediol for processing, although less efficiently than 1,2-diols (Fig. 6). It is likely that the structure of acceptable nucleophiles reflects the configuration of the site on the protein that interacts with the nucleophilic donor before or during catalysis.
      We found that the three integrases studied in this report exhibited different nucleophile selectivities during processing. Consistent with previously published data for Mn2+-dependent reactions (
      • Engelman A.
      • Mizuuchi K.
      • Craigie R.
      ,
      • Vink C.
      • Yeheskiely E.
      • van der Marel G.A.
      • van Boom J.H.
      • Plasterk R.H.A.
      ,
      • Katzman M.
      • Sudol M.
      ,
      • Muller B.
      • Jones K.S.
      • Merkel G.W.
      • Skalka A.M.
      ), HIV-1 integrase and visna virus integrase primarily used water rather than the viral DNA ends as the nucleophile, whereas RSV integrase had a strong preference for using the 3′-OH at the viral DNA end (Table I). These results have now been documented in reactions performed in the absence of exogenous nucleophiles by using glycerol-free integrase preparations, which had previously been done only for the HIV-1 enzyme (
      • Vink C.
      • Yeheskiely E.
      • van der Marel G.A.
      • van Boom J.H.
      • Plasterk R.H.A.
      ). Moreover, when reactions were supplemented with optimal concentrations of alcohols, HIV-1 integrase used the alcohols almost as often as water, whereas visna virus integrase used the alcohols somewhat less often than water. In marked contrast, RSV integrase used the alcohols inefficiently. When processing reactions were conducted with Mg2+ as the divalent cofactor, the preference of the HIV-1 and visna virus integrases for water became even stronger and minimal amounts of the other products were detected, consistent with previous reports for HIV-1 or HIV-2 integrase (
      • Vink C.
      • Yeheskiely E.
      • van der Marel G.A.
      • van Boom J.H.
      • Plasterk R.H.A.
      ,
      • van Gent D.C.
      • Oude Groeneger A.A.M.
      • Plasterk R.H.A.
      ,
      • Mazumder A.
      • Gupta M.
      • Pommier Y.
      ). Although the use of water by RSV integrase also was facilitated in reactions with Mg2+, as described by others (
      • Muller B.
      • Jones K.S.
      • Merkel G.W.
      • Skalka A.M.
      ), we found that the avian enzyme made cyclic products and alcohol adducts even with this cation. The distinct preferences of the three enzymes are unlikely to be due to membership in different retrovirus subfamilies, because preferential usage of the 3′ viral DNA end as the nucleophile for processing has also been described for murine leukemia virus (
      • Dotan I.
      • Scottoline B.P.
      • Heuer T.S.
      • Brown P.O.
      ) and feline immunodeficiency virus integrases (
      • Vink C.
      • van der Linden K.H.
      • Plasterk R.H.A.
      ). Rather, these preferences must reflect subtle differences in the structure of the various proteins, as suggested by crystallographic studies of the HIV-1 and RSV enzymes (
      • Andrake M.D.
      • Skalka A.M.
      ,
      • Goldgur Y.
      • Dyda F.
      • Hickman A.B.
      • Jenkins T.M.
      • Craigie R.
      • Davies D.R.
      ,
      • Maignan S.
      • Guilloteau J.-P.
      • Zhou-Liu Q.
      • Clément-Mella C.
      • Mikol V.
      ). This conclusion is consistent with the finding that certain amino acid substitutions in the central region of HIV-1 or HIV-2 integrase affect the relative amounts of the various dinucleotide products created by processing (
      • van Gent D.C.
      • Oude Groeneger A.A.M.
      • Plasterk R.H.A.
      ,
      • Engelman A.
      • Craigie R.
      ). Similarly, the influence of the divalent cation on nucleophile selection for processing may reflect metal-induced conformational changes of the enzyme (
      • Asante-Appiah E.
      • Seeholzer S.H.
      • Skalka A.M.
      ). Steric effects and electrostatic interactions between integrase and substrate DNA also have been suggested to contribute to the choice of nucleophile during processing (
      • Mazumder A.
      • Gupta M.
      • Pommier Y.
      ).
      Nonspecific alcoholysis, the recently discovered fourth activity of integrase, is the most potent action of several integrase proteins (
      • Katzman M.
      • Sudol M.
      ,
      • Katzman M.
      • Sudol M.
      ). This activity uses a variety of nucleophiles (as does processing), nicks almost any target DNA site (as does joining), and only requires the central domain of the protein (as does disintegration). Thus, nonspecific alcoholysis has a unique combination of characteristics that makes it a useful tool for understanding how integrase selects nucleophiles and target sites for catalysis. We have previously emphasized the relationship of this activity to joining, because both activities attack multiple DNA sites, lack a target consensus sequence, avoid 5′-ends of substrates, and display site preferences that are a function of the target DNA sequence and the viral source of integrase (
      • Katzman M.
      • Sudol M.
      • Pufnock J.S.
      • Zeto S.
      • Skinner L.M.
      ). We have now found that HIV-1 integrase and visna virus integrase exhibit similar patterns of nucleophile selection during nonspecific alcoholysis reactions, whereas RSV integrase performs greater hydrolysis. The high baseline level of hydrolysis by RSV integrase was not due to contaminating nucleases, because it demonstrated the signature nicking pattern (
      • Katzman M.
      • Sudol M.
      ) characteristic of the avian enzyme (Figs.8 C and 9 C). In fact, the patterns of preferential nicks created by each enzyme were the same whether the enzymes were catalyzing hydrolysis or alcoholysis, consistent with the idea that these reactions reflect one catalytic mechanism. Moreover, the nicking patterns were independent of which exogenous alcohol was used. Thus, the nucleophile did not affect the choice of target DNA sites.
      In contrast to the above conclusion, the target DNA did affect selection of the nucleophile. In fact, several differences in nucleophile usage were noted between processing and nonspecific alcoholysis, reactions that differ merely by the DNA that is presented to integrase. Most striking was the finding that exogenous nucleophiles stimulated the nonspecific nuclease activity of integrase. Much of the additional nicking of nonviral DNA was due to the use of exogenous alcohols as the attacking nucleophile (Fig. 9), whereas the same alcohols only competed with water during processing (e.g.Fig. 5). In addition, the various 1,2-diols were used more equivalently during nonspecific alcoholysis than during processing (Figs. 6 and 8). Moreover, the difference in usage of a 1,3-diol compared with the 1,2-diols appeared to be less during nonspecific alcoholysis than during processing. Finally, RSV integrase used water relatively inefficiently as the nucleophile for processing viral DNA ends (TableI) but performed high levels of hydrolysis on nonviral DNA (Figs. 8 and9).
      The ability to accommodate a diversity of nucleophiles suggests considerable flexibility for the active site of integrase (
      • Engelman A.
      • Mizuuchi K.
      • Craigie R.
      ). Indeed, we observed that the use of exogenous alcohols could exceed the use of water during processing or nonspecific alcoholysis (Figs. 5 and 9). Although the alcohol concentrations reached in these experiments are relatively high (40% glycerol or propylene glycol corresponds to 5.4m and 40% ethylene glycol corresponds to 7.2m), they are much less than the 33 mconcentration of water in a solution that is 60% water. How integrase chooses various nucleophiles for these reactions is unclear, but current evidence indicates that the nucleophiles interact with integrase as specific substrates rather than merely as solvent molecules attacking DNA that is appropriately positioned by the enzyme. Most importantly, use of the nucleophiles involves specificity because not all compounds are used equivalently. For example, ethanol and 2-propanol were not used by integrase (
      • Vink C.
      • Yeheskiely E.
      • van der Marel G.A.
      • van Boom J.H.
      • Plasterk R.H.A.
      ,
      • Katzman M.
      • Sudol M.
      ). Moreover, many compounds that inhibit integrase in vitro have adjacent OH groups (
      • LaFemina R.L.
      • Graham P.L.
      • LeGrow K.
      • Hastings J.C.
      • Wolfe A.
      • Young S.D.
      • Emini E.A.
      • Hazuda D.J.
      ,
      • Pommier Y.
      • Neamati N.
      ), as do the most active alcohols in these assays, suggesting that these agents act at a similar protein site.
      It is important to note that the cyclic dinucleotide and alcohol adducts described in these experiments are stable products that do not convert to hydrolysis products. This fact was confirmed by purifying the different products from gels, incubating them under various conditions, including exposure to alkali, and re-examining them by gel electrophoresis (data not shown). Thus, our measurements reflect the true distribution of the various products. Moreover, the relative distribution of the various products was not affected by the extent of the reaction. In particular, time-course studies of the processing reaction showed similar kinetics of appearance of the three types of dinucleotide products. In addition, the relative amounts of the various products were not affected by the concentration of substrate DNA when initial rates of product formation were measured during the early, linear phase of the time course. Analogous results were obtained for the nonspecific alcoholysis reaction when the kinetics of appearance of the hydrolysis and alcoholysis products and the initial rate of formation of these products as a function of DNA concentration were compared. Furthermore, the concentrations of integrase used in this report were within the linear part of the reaction curve for both types of assays when initial rates of product formation were compared with enzyme concentration (data not shown). Interestingly, the amount of product formed in these experiments was always less than the amount of HIV-1 and visna virus integrase present and only a low level of turnover was detected for RSV integrase (and only during Mn2+-dependent reactions). Although the fraction of integrase that was catalytically active is unknown, these results are consistent with the findings of others for HIV-1 and RSV integrase during processing (
      • Hawkins M.E.
      • Pfleiderer W.
      • Mazumder A.
      • Pommier Y.G.
      • Balis F.M.
      ,
      • Jones K.S.
      • Coleman J.
      • Merkel G.W.
      • Laue T.M.
      • Skalka A.M.
      ,
      • Lee S.P.
      • Kin H.G.
      • Censullo M.L.
      • Han M.K.
      ,
      • Tramontano E.
      • La Colla P.
      • Cheng Y.-C.
      ). Low or absent turnover may reflect stable complex formation between integrase and substrate DNA (
      • Bushman F.D.
      • Wang B.
      ,
      • Ellison V.
      • Brown P.O.
      ,
      • Vink C.
      • Puras-Lutzke R.A.
      • Plasterk R.H.A.
      ,
      • Yoshinaga T.
      • Kimura-Ohtani Y.
      • Fujiwara T.
      ,
      • Miller M.D.
      • Bor Y.-C.
      • Bushman F.
      ,
      • Yi J.
      • Asante-Appiah E.
      • Skalka A.M.
      ), which is mechanistically appealing because processing can occur in the cytoplasm of a cell but DNA joining does not occur until after the preintegration complex has entered the nucleus. Moreover, multiple turnovers are unnecessary, because integrase is only required to nick two viral DNA ends and two strands of host DNA. In vivo, the ratio of integrase molecules to viral DNA ends is about 75:2 (
      • Jones K.S.
      • Coleman J.
      • Merkel G.W.
      • Laue T.M.
      • Skalka A.M.
      ), which is approximated by typical in vitroconditions. Together, these additional experiments and observations strengthen the validity of our conclusions regarding the choice of nucleophile by the various integrases.
      The most important new findings from this work are that the target DNA influenced the choice of nucleophile during reactions catalyzed by integrase, but the nucleophile did not affect the choice of target DNA sites. These functional observations suggest that integrase achieves an active conformation only after DNA becomes bound to the protein. A similar suggestion has been made from recent structural information (
      • Chen Z.
      • Yan Y.
      • Munshi S.
      • Li Y.
      • Zugay-Murphy J.
      • Xu B.
      • Witmer M.
      • Felock P.
      • Wolfe A.
      • Sardana V.
      • Emini E.A.
      • Hazuda D.
      • Kuo L.C.
      ,
      • Yang Z.-N.
      • Mueser T.C.
      • Bushman F.D.
      • Hyde C.C.
      ), although crystals of integrase with bound DNA have not been described. In the context of retroviral integration, this conclusion suggests that: 1) regardless of which nucleophile for processing has been bound, integrase binds a viral DNA end and reaches an active conformation poised to nick this target at the site 3′ to the CA, and 2) whether or not the processed viral DNA end has been repositioned as the attacking nucleophile for joining, integrase binds cellular DNA and reaches an active conformation poised to nick this target at certain sites. Although the data cannot order the integrase-nucleophile and integrase-target DNA interactions, they suggest that interaction with target DNA is the critical step before each catalytic event. Thus, these results highlight an aspect of retroviral integration that should serve as the focus for efforts to develop antiretroviral agents.

      Acknowledgments

      We thank Ross Shiman for discussions about kinetic aspects of integrase reactions.

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