Department of Microbiology and Immunology1 and Department of Medicine2, The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033-0850, USA
Author for correspondence: Michael Katzman (at the Department of Medicine). Fax +1 717 531 4633. e-mail mkatzman{at}psu.edu
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Abstract |
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Introduction |
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Methods |
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Oligonucleotides.
All oligodeoxynucleotides used as assay substrates were gel-purified following synthesis and again after being 5'-end-labelled with [-32P]ATP by T4 polynucleotide kinase (Katzman & Sudol, 1994
). Sequences are indicated in the appropriate figures.
In vitro integrase assays.
Double-stranded DNA substrates were prepared by annealing the labelled strand with 4-fold excess unlabelled complementary oligonucleotide (Katzman & Sudol, 1994 ). Standard 10 µl reaction mixtures contained 0·5 pmol of double-stranded DNA, 25 mM TrisHCl (pH 8·0), 10 mM dithiothreitol, 1·0 µl of IN or protein storage buffer, and MnCl2 or MgCl2 as indicated for each experiment. Reaction mixtures were incubated for 90 min at 37 °C, and then stopped by addition of 10 µl loading buffer (95% formamide, 20 mM EDTA, 0·05% bromophenol blue, 0·05% xylene cyanol) and heating at 95 °C for 5 min. Aliquots were loaded onto 20% polyacrylamide (acrylamide to methylene-bisacrylamide ratio, 19:1)7 M urea denaturing gels, followed by electrophoresis at 75 W until the bromophenol blue dye had migrated 21 cm. Wet gels were autoradiographed at -70 °C.
Quantification of results.
The radioactivity of bands in wet gels was quantified with a Betascope (Betagen). Specific viral DNA cleavage, or processing, was calculated as [(c.p.m. of 16-mers)+(c.p.m. of >18-mers)]/(total c.p.m. in lane), with background corrections from analogous parts of a negative control lane; >18-mers refers to products longer than substrate length, which form only following specific cleavage (Katz et al., 1990 ; Craigie et al., 1990
; Bushman & Craigie, 1991
). Use of total c.p.m. in the denominator best reflects conversion of substrate to specific product, but yields lower calculated cleavages than would be suggested by merely comparing 16-mer products to the remaining 18-mer substrates.
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Results |
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Exploration of one other approach to stimulating the Mg2+-dependent activity of these enzymes provided additional important information. Although we confirmed the work of Lee et al. (1995a , b
) that longer oligonucleotide substrates alter the divalent cation preference of HIV-1 IN, a similar result was not obtained for visna virus IN. In particular, double-stranded 18-mers from the HIV-1 U5 end were more susceptible to HIV-1 IN in the presence of Mn2+ than in the presence of Mg2+ (Fig. 3
, lower panel, lanes 2 and 3), whereas 32-mers derived from the same viral DNA end were slightly more susceptible to HIV-1 IN with Mg2+ (Fig. 3
, upper panel, lanes 2 and 3). In contrast, visna virus IN was inactive for processing on either substrate with Mg2+, although it preferred the shorter substrate with Mn2+ (Fig. 3
, lanes 5 and 6). Thus, lengthening the size of the viral DNA substrate is not sufficient to reveal Mg2+-dependent processing by all integrases. However, addition of DMSO dramatically stimulated the Mg2+-dependent activity of visna virus IN on both substrates, especially the 18-mers (Fig. 3
, lane 7). Although DMSO also stimulated the Mg2+-dependent activity of HIV-1 IN on the shorter substrate, it did not do so on the longer one (Fig. 3
, lane 4), perhaps because the level of activity on this substrate was high already. Because our preparations of HIV-1 IN and visna virus IN showed greatest Mg2+-dependent activity when the substrates were 18 base pairs long and DMSO was present (Fig. 3
, lower panel, lanes 4 and 7), we chose these conditions for subsequent experiments directed at analysing the role of subterminal nucleotides in recognition by IN. These conditions have the additional advantage of permitting direct comparisons with our previous results that were obtained using 18-mer substrates in the presence of Mn2+ (Katzman & Sudol, 1996
).
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Positions 5 and 6 in U3 substrates are recognized differently by the two INs
To ascertain whether positions 5 and 6 played equal roles in Mg2+-dependent recognition and cleavage by IN, these positions were independently exchanged between the HIV-1 and visna virus U3 substrates. The results of testing these substrates revealed that visna virus IN was more sensitive to changes in position 5, immediately adjacent to the invariant CA bases, whereas HIV-1 IN was more sensitive to changes in position 6. Thus, a substrate in which position 5 in the visna virus U3 substrate was substituted by the corresponding HIV-1 U3 base pair (designated visna U3s5) was processed significantly less efficiently by visna virus IN, and a substrate in which position 6 was substituted (designated visna U3s6) remained highly susceptible to this enzyme (Fig. 5B, lanes 14/15 and 19/20 compared with Fig. 5A
, lanes 9/10). In contrast, the analogous HIV-1 U3s5 was not as hindered for susceptibility to HIV-1 IN as was the HIV-1 U3s6 substrate (Fig. 5B
, lanes 2/3 and 7/8 compared with Fig. 5A
, lanes 2/3; the differences are more evident when quantified and presented as in Fig. 5C
, as discussed below). Note that the HIV-1 U3s5 and visna U3s6 substrates share the final nine positions and acted similarly in these assays (Fig. 5B
, lanes 15 and 1620). Similarly, HIV-1 U3s6 and visna U3s5 share their final nine positions and exhibited the same pattern of susceptibility to the two enzymes (Fig. 5B
, lanes 610 and 1115). The amounts (but not the patterns) of strand-transfer products were similar for substrates that matched at the final nine positions also (Fig. 5B
, upper parts of lanes). The sequences of the singly substituted DNA substrates and quantification of multiple replicate reactions performed on different days are shown in Fig. 5C
. This presentation highlights that the patterns of relative susceptibility to the two integrases are similar for HIV-1 U3s5 and visna U3s6 (the fifth and eighth entries in the figure), as well as for HIV-1 U3s6 and visna U3s5 (the sixth and seventh entries). Moreover, comparing results for a single enzyme on different substrates clearly demonstrates that visna virus IN was more dependent on position 5 than position 6 (compare its activity on visna U3, visna U3s5, and visna U3s6), whereas HIV-1 IN interacted more critically with position 6 (compare its activity on HIV-1 U3, HIV-1 U3s5, and HIV-1 U3s6). These results are identical to those obtained with Mn2+ as the divalent cation (Katzman & Sudol, 1996
).
Substitutions in U5 substrates
The above results reveal the importance of the final nine positions at the viral DNA U3 terminus for Mg2+-dependent functional interactions with IN. To assess the importance of the final nine positions in the context of the U5 terminus when Mg2+ serves as the cofactor, we tested an HIV-1 U5-derived oligonucleotide substrate in which positions 1018 were mutated to base pairs found at none of the terminal DNA sequences from either virus (as shown in Fig. 1A). We found that this substrate (designated HIV-1 U5m1018), as was the wild-type HIV-1 U5 sequence, was highly susceptible to both integrases (Fig. 6A
). We then used a substrate in which every position but the final six was mutated (designated HIV-1 U5m718). This substrate also was susceptible to both integrases (Fig. 6B
). Thus, as was observed with Mn2+ (Katzman & Sudol, 1996
), the final six base pairs contain sufficient information for specific processing by either IN. Nonetheless, the latter substrate was less susceptible than the wild-type sequence (compare Fig. 6B
with Fig. 4
, lanes 15, lower panel) and the susceptibility to visna virus IN was affected to a greater extent. Although these observations also were true for reactions conducted with Mn2+ (Katzman & Sudol, 1996
), the difference for visna virus IN was greater with Mg2+. The sequences of the substrates and the results of multiple replicate reactions are summarized in Fig. 6(C)
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Discussion |
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Recently, several groups identified conditions under which HIV-1 IN has enhanced activity with Mg2+. In addition to DMSO, factors reported to increase the Mg2+-dependent activity of HIV-1 IN include: use of longer oligonucleotide substrates (Engelman & Craigie, 1995 ; Lee et al., 1995a
, b
), high enzyme:substrate ratios (Engelman & Craigie, 1995
; Esposito & Craigie, 1998
), or the presence of PEG (Engelman & Craigie, 1995
), zinc (Zheng et al., 1996
; Lee & Han, 1996
; Lee et al., 1997
), dioxane (Goodarzi et al., 1995
), or the HIV-1 nucleocapsid protein (Carteau et al., 1997
). Our results indicate that these conditions may not be uniformly effective for revealing activity by different integrases or enzyme preparations with Mg2+. Although we confirmed that longer substrates enhance the Mg2+-dependent activity of HIV-1 IN, a similar effect was not observed for visna virus IN (Fig. 3
). We also were unable to stimulate the Mg2+-dependent activity of our HIV-1 or visna virus enzyme preparations with PEG or Zn2+ and found that these reagents inhibited activity in some reactions. Others have presented evidence that PEG can inhibit activity with Mn2+ (Engelman & Craigie, 1995
; Lee & Han, 1996
) and that Zn2+ inhibits the DNA joining activity of HIV-1 IN (Wolfe et al., 1996
) and a nicking activity of avian sarcoma virus IN (Bujacz et al., 1997
). In contrast, the organic solvent DMSO dramatically stimulated the Mg2+-dependent processing and joining activity of both of our enzymes. In fact, 3040% DMSO supported activity with Mg2+ comparable to what is typically obtained with Mn2+ (Fig. 2
). These data represent the first demonstration that Mg2+ can support activity by visna virus IN, a protein that has proven useful for constructing chimeric enzymes with HIV-1 IN (Katzman & Sudol, 1995
, 1998
). Others have noted that 1020% DMSO can slightly enhance the Mg2+-dependent processing activity of HIV-1 IN (Engelman & Craigie, 1995
; Lee & Han, 1996
) and feline immunodeficiency virus IN (Shibagaki et al., 1997
). Interestingly, the HIV-1 IN preparation used by Esposito & Craigie (1998)
readily exhibited Mg2+-dependent processing with only 5% DMSO (along with 5% PEG). DMSO has also been reported to stimulate the Mg2+-dependent integration activity of HIV-1 IN (Goodarzi et al., 1995
; Miller et al., 1995
), avian myeloblastosis virus IN (Fitzgerald et al., 1992
; Vora & Grandgenett, 1995
) and murine leukaemia virus IN (Fujiwara & Craigie, 1989
).
How DMSO (or any of the other manoeuvres) promotes IN activity with Mg2+ is unknown. DMSO can affect protein secondary structure by two possible mechanisms (Jackson & Mantsch, 1991 ; Huang et al., 1995
). When DMSO replaces solvent water molecules, the proteins environment becomes less polar and hydrophobic interactions between nonpolar amino acid residues may be weakened. In addition, DMSOIN interactions may replace hydrogen bonds at the protein surface or within interior cavities. Resulting effects on protein conformation may influence metal coordination by the enzymes active site residues (Maignan et al., 1998
; Goldgur et al., 1998
) or the stability of INsubstrate interactions in the presence of Mg2+ (Pemberton et al., 1996
). Whatever the mechanism, it is significant that IN retains high specificity for the processing site near viral DNA ends in reactions conducted with DMSO and Mg2+ (Fig. 2
). The true biological relevance of various reaction conditions is unclear because the environment in which IN operates within the cell is poorly defined. For example, the dilute aqueous reaction conditions typically used in vitro are unlikely to mimic intracellular conditions (Fulton, 1982
; Zimmerman & Minton, 1993
; Knull & Minton, 1996
). Although we do not claim that the presence of 3040% DMSO is physiological, it is noteworthy that proteins may constitute a similar percentage of the weight of the cytoplasm in some cells (Fulton, 1982
). Given the complexity of the intracellular milieu, the effects of molecular crowding on IN function deserve further study (Zimmerman & Minton, 1993
; Minton, 1998
). Nonetheless, the important point is that conditions were identified that provided a useful and productive means to investigate the role of subterminal viral DNA nucleotides as specific recognition signals for the HIV-1 and visna virus integrases in the presence of Mg2+. The data demonstrate that this particular parameter (i.e. the choice of divalent cation) does not affect recognition of certain key elements of the viral DNA substrate.
A major result from these experiments is that exchange of positions 5 and 6 between U3 substrates switched the patterns of susceptibility to two lentiviral integrases (Fig. 5). Positions 5 and 6 were also important for Mg2+-dependent oligonucleotide processing by avian retroviral IN (Katzman et al., 1989
; Cobrinik et al., 1991
). Thus, interaction with these positions may be a general feature of retroviral integrases. However, integrases can differ with respect to the importance of these positions, as HIV-1 IN was more sensitive to substitution of U3 position 6 and visna virus IN was more dependent on position 5. These results complement those of Esposito & Craigie (1998)
, who found that the two base pairs just internal to the conserved CA bases play an important role in processing of U5 substrates by HIV-1 IN, that this effect was independent of the choice of divalent cation, and that position 6 had a greater influence on susceptibility to HIV-1 IN. Thus, IN likely interacts with positions 5 and 6 at both viral DNA termini in a similar fashion. Of note, Esposito & Craigie (1998)
found that positions approximately one turn along the DNA helix from the conserved CA also affected susceptibility, but only when Mg2+ was the divalent cation. Their results from an in vitro selection assay and photocross-linking studies also suggested roles for these more-internal positions only when Mg2+ was present. Our data also suggest that positions 718 of viral DNA can influence the activity of IN, an effect that was greatest for visna virus IN with Mg2+ (Fig. 6
) compared with Mn2+ (Katzman & Sudol, 1996
). One difference between our results is that Esposito & Craigie found a large effect on Mg2+-dependent processing when HIV-1 U5 position 12 or 13 (using our numbering) was substituted, whereas we found only minor effects when these positions, along with several others, were simultaneously replaced in the HIV U5m1018 substrate (Fig. 6
). This result suggests that other positions within this region can compensate for any effects on susceptibility to IN. It is not surprising that positions outside the terminal six base pairs can affect the extent of the reaction because at least 15 base pairs of substrate DNA are needed for optimal IN activity (Katzman et al., 1989
; LaFemina et al., 1991
; Sherman et al., 1992
). Nonetheless, the final six U5 base pairs of viral DNA contain sufficient sequence information for specific recognition and cleavage in reactions conducted with Mg2+ (Fig. 6
), Mn2+ (Katzman & Sudol, 1996
) or both Mg2+ and Mn2+ (Sherman et al., 1992
).
In contrast to the above findings, the choice of divalent cation appears to have a larger effect on interactions with the target for viral DNA insertion. Thus, the patterns of joined products for a given enzyme, donor DNA and target DNA sequence differed as a function of the metal (Fig. 2). The data of other groups also demonstrate that the strand-transfer patterns created by HIV-1 IN depend on whether Mg2+ or Mn2+ serves as the cofactor (Engelman & Craigie, 1995
; Lee et al., 1995b
; Lee & Han, 1996
); a similar result occurred with feline immunodeficiency virus IN (Shibagaki et al., 1997
). Moreover, when we used a PCR-based assay to monitor insertion of processed viral DNA ends into a plasmid DNA target, the insertion patterns created by HIV-1 IN and visna virus IN differed for reactions conducted with Mn2+ compared with those conducted with Mg2+ (data not shown). That the nature of the divalent cation differentially affects interactions with viral DNA and host DNA suggests that IN has different binding sites for these substrates.
The most important finding in this study is that the role of subterminal viral DNA positions in recognition by the integrases of HIV-1 and visna virus was confirmed when Mg2+ substituted for Mn2+ during in vitro reactions. Thus, the choice of divalent cation is not critical for interactions between IN and positions close to the conserved CA bases at the viral DNA termini. The generalizability of our findings is strengthened by the use of substrates that contained single and multiple base substitutions, sequences derived from either end of unintegrated viral DNA, and two different retroviral systems. Thus, the large body of Mn2+-dependent biochemical data identifying terminal viral DNA positions that are important in substrate recognition by various integrases likely describe biologically relevant interactions. The only caveat, as originally suggested by Esposito & Craigie (1998) , is that the role of more-internal viral DNA positions may have been underestimated in reactions conducted with Mn2+. These functional data will permit correlations with structural information when the viral DNA binding site of IN is identified.
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Acknowledgments |
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References |
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Received 2 September 1999;
accepted 8 November 1999.