A C-terminal “Tail” Region in the Rous Sarcoma Virus Integrase Provides High Plasticity of Functional Integrase Oligomerization during Intasome Assembly*

The retrovirus integrase (IN) inserts the viral cDNA into the host DNA genome. Atomic structures of five different retrovirus INs complexed with their respective viral DNA or branched viral/target DNA substrates have indicated these intasomes are composed of IN subunits ranging from tetramers, to octamers, or to hexadecamers. IN precursors are monomers, dimers, or tetramers in solution. But how intasome assembly is controlled remains unclear. Therefore, we sought to unravel the functional mechanisms in different intasomes. We produced kinetically stabilized Rous sarcoma virus (RSV) intasomes with human immunodeficiency virus type 1 strand transfer inhibitors that interact simultaneously with IN and viral DNA within intasomes. We examined the ability of RSV IN dimers to assemble two viral DNA molecules into intasomes containing IN tetramers in contrast to one possessing IN octamers. We observed that the last 18 residues of the C terminus (“tail” region) of IN (residues 1–286) determined whether an IN tetramer or octamer assembled with viral DNA. A series of truncations of the tail region indicated that these 18 residues are critical for the assembly of an intasome containing IN octamers but not for an intasome containing IN tetramers. The C-terminally truncated IN (residues 1–269) produced an intasome that contained tetramers but failed to produce an intasome with octamers. Both intasomes have similar catalytic activities. The results suggest a high degree of plasticity for functional multimerization and reveal a critical role of the C-terminal tail region of IN in higher order oligomerization of intasomes, potentially informing future strategies to prevent retroviral integration.

The retrovirus integrase (IN) 3 is responsible for the concerted integration of linear viral cDNA ends into the host DNA genome and has multiple auxiliary roles including reverse transcription and virus particle maturation (for reviews, see Refs. [1][2][3][4]. Reverse transcription of the viral RNA in virus-infected cells results in the cytoplasmic preintegration complex (PIC). The PIC is transported into the nucleus where IN inserts the viral DNA ends into the host genome. To understand the structure and function of IN binding at the viral DNA ends, the crystal structure of the spumaretrovirus prototype foamy virus (PFV) intasome containing recombinant IN and two molecules of viral DNA (19 bp in length) was determined (5). PFV IN is monomeric in solution and assembles into a tetrameric structure complexed with either blunt-ended or 3Ј-OH recessed viral DNA ends, producing a soluble and stable synaptic complex termed the intasome (5,6). PFV IN assembled onto bluntended viral DNA ends can undergo a 3Ј-OH processing reaction, releasing a dinucleotide in the presence of divalent metal ions (7). This primed 3Ј-OH recessed viral DNA is now positioned to be covalently inserted into a bendable target DNA sequence that produces the strand transfer complex (STC) (8 -10).
Atomic structures of other assembled IN-DNA complexes of alpharetrovirus and betaretrovirus were also accomplished (9,11). Purified virus-derived and recombinant avian retrovirus INs (12)(13)(14)(15) as well as the recombinant mouse mammary tumor virus (MMTV) IN are dimeric in solution (11). In contrast to the tetrameric IN in the PFV intasome and STC, the crystal structure of the Rous sarcoma virus (RSV) STC revealed that an octamer of IN was complexed with a branched viral/target DNA substrate that represents the product produced by the concerted integration reaction (9). Cryo-EM analysis of the MMTV intasome with two viral DNA molecules also revealed IN as an octameric structure (11). These above structural studies of retrovirus IN-viral DNA complexes suggest divergent evolution even though all retrovirus INs contain three canonical domains necessary for integration, the N-terminal (NTD), the catalytic core (CCD), and the C-terminal (CTD) domains (16) (Fig. 1). All three domains are necessary for concerted integration.
The domain organizations of IN found in four different retroviruses are shown in Fig. 1. All of the INs have similar size and functional NTD, CCD, and CTD domains, whereas PFV IN possesses an additional N-terminal extension domain that binds viral DNA (5). The linkers between the domains are similar in size except for PFV IN that possesses larger size linkers. These INs except recombinant MMTV IN possess a short "tail" region of 18 residues at their very C terminus adjacent to highly conserved ␤-strand-rich region of the SH3 domain fold in the CTD (Fig. 1). The tail region of MMTV is 54 residues. For RSV IN, the viral protease processes a precursor polymerase protein, producing wild type (WT) IN that terminates at residue 286, which includes the 18 residues of the tail region (17,18). The cleaved 37-amino acid polypeptide, whose function is unknown, is not essential for virus replication (19). Approximately 5-6 residues at the C-terminal end of RSV IN (amino acids 1-286) are also not essential for replication (20,21). There are also 10 -12 residues at the very C terminus of the tail region of human immunodeficiency virus type 1 (HIV-1) IN (amino acids 1-288) that are not critical for virus replication but nonetheless enhance the functions of IN with increasing efficiency in accordance with its length (22,23). The structure of MMTV IN in virus particles and whether there is additional proteolytic cleavage of IN to produce a shorter tail region and a separate cleaved protein fragment are unknown.
Biochemical and biophysical studies have suggested that lentivirus HIV-1 IN is tetrameric in the presence of viral DNA or a branched viral/target DNA product, the result of concerted integration (24 -27). Protein-protein cross-linking studies of isolated HIV-1 IN-DNA complexes by agarose gel electrophoresis or visualized by atomic force microscopy suggest the presence of an IN tetramer in these complexes. Recombinant HIV-1 IN forms a variety of multimeric states from monomers to octamers in solution (28 -32). Very recently intasome and STC atomic structures with IN from HIV-1 (33) and maedi-visna virus (ovine lentivirus) (34) were determined by cryo-EM to be tetrameric to hexadecameric in nature.
PFV (10) and RSV (9) INs form insoluble complexes in low salt buffers with a branched viral/target DNA substrate that mimics the product of concerted integration. These insoluble complexes can be dialyzed in higher salt buffers, which produces soluble and stable STCs for crystallographic analysis. Using a similar procedure, recombinant MMTV IN complexed with viral DNA was analyzed by cryo-EM (11). Without going through the first insoluble IN-DNA complex formation step used in the above studies, a C-terminally truncated RSV IN (amino acids 1-269) is directly capable of producing a soluble IN-viral DNA complex in the presence of HIV-1 strand transfer inhibitors (STIs) that stabilize the intasome (14). STIs act as interfacial inhibitors that trap kinetically stabilized intasomes (14,35) by binding to protein-DNA interfaces (5,36). The RSV intasome contains a tetramer of IN determined by absolute molecular mass measurements using size exclusion chromatography-multiangle light scattering (SEC-MALS) analysis. C-terminally truncated RSV INs (residues 1-269 or 1-270) possess concerted integration activity using 3Ј-OH recessed viral DNA substrates (9,14,15), form IN tetrameric structures in STI-trapped intasomes with 3Ј-OH recessed viral DNA substrates (18 -22 bp) (14), and promote STC assembly containing an octameric IN structure (9).
The crystal structure of RSV STC assembled with IN (residues 1-270) shows a pair of IN dimers engaging the two viral DNA ends for catalysis; these are designated "proximal" subunits (9). Another pair of IN dimers bridge between the viral DNA molecules and help capture target DNA; these are termed "distal" subunits. The CTDs of both the proximal and distal IN dimers make direct contacts with the viral DNA. The CCD-CTD configuration for the proximal IN dimers (9) is comparable with that of the DNA-free IN dimers in its native configuration primed for viral DNA binding (15,37). The CCD-CTD configuration for the distal RSV IN dimers deviates from the canonical conformation, allowing the distal IN dimers to fit into the RSV STC without making steric clashes with the proximal IN subunits or the 5Ј-overhang of the viral DNA strand (9).
What controls the assembly of different IN-DNA complexes under different solution conditions? The assembly mechanisms for these complexes are controlled by the structure of the DNA substrates and/or by specific regions of IN. The CTD of WT RSV IN(1-286), which extends from residues 222 to 286, contains a ␤-strand-rich region of the SH3 domain fold (residues 222-268) and a tail region of 18 residues that are flexible ( Fig. 1) (15,37). The tail region has not been resolved by X-ray structural studies for any retrovirus IN by itself (15,(37)(38)(39) or in the presence of viral DNA or branched viral/target DNA substrates (5,8,10,33,34). Previous results suggested that the RSV IN tail region may affect assembly of the intasome using a viral DNA substrate (14).
Our current study shows that two RSV proximal IN dimers (amino acids 1-269) assemble onto two viral DNA ends at 4°C to produce an STI-trapped intasome containing IN tetramers. Increasing the temperature for assembly to 18°C and the length of the tail region of different IN constructs containing residues  (40) occurs rapidly at 4°C, and the major complex is stable under these assembly conditions for at least 120 h ( Fig.  2A) (14). SEC-MALS analysis established the absolute molecular mass of this major complex as 151,000 Ϯ 2,000 Da, suggesting the presence of an IN tetramer complexed with two viral DNA molecules (14). The appearance of a minor hump on the slower migrating slope of the tetrameric intasome suggested the presence of another intasome species. Increasing the length of RSV IN by 5 residues from 1-269 to 1-274 under the same assembly conditions resulted in the slow conversion of the tetrameric RSV intasome into an independent IN-DNA complex that migrated with the apparent molecular mass of ϳ269,000 Da, consistent with an octameric intasome (Fig. 2B). The conversion of intasomes containing IN tetramers to this slower eluting IN-DNA complex was not complete in this assembly buffer at 4°C. Similar results were obtained using a 19R GU3 substrate. There was no evidence of other larger size IN-DNA complexes produced under these conditions.
Length of Tail Region and Temperature Accelerate Conversion of Tetrameric RSV Intasome to an Octameric Intasome-The tail region of IN was extended from 1-274 to 1-278 to address whether this increased length enhanced the assembly of the octameric intasome. Also, we addressed whether increasing the temperature from 4 to 18°C during assembly would accelerate the conversion of the tetrameric intasome to the octameric IN form. Incubation of IN(1-278) at 18°C for 4 h with 18R GU3 produced both forms of intasomes, whereas further incubation to 24 h essentially converted all of the transient tetrameric intasome to the most stable octameric structure (    (14).
For comparison, we also examined the ability of IN(1-323) to assemble both the intasome and STC. The intasome was assembled in the presence of MK-2048 and 18R GU3 for 48 h at 18°C under standard conditions. The molecular mass of the RSV intasome produced with IN(1-323) was 293,000 Ϯ 3,000 Da (n ϭ 3), and that of the STC was 272,000 Ϯ 20,000 Da (n ϭ 3). The mass of purified dimeric IN(1-323) was 66,000 Ϯ 2,400 Da (n ϭ 3). In summary, these above measurements of different RSV intasomes and STCs produced by different lengths of IN molecules are consistent with an octameric IN structure associated with each complex.  (14). No intasomes were produced with a 21R-NSP ODN containing nonspecific DNA sequences using IN  in the presence of MK-2048 (Fig. 5). Similar STI and viral DNA requirements to produce the RSV intasome containing two proximal IN dimers using IN(1-269) were observed previously (14).
STI binding to the intasome requires 3Ј-OH processing of the dinucleotide from the catalytic DNA strand by IN prior to displacement of the terminal 3Ј-deoxyadenosine from the active site by the STI (5, 7). The STI is in equilibrium with the assembled intasomes (14). We investigated whether intasome assembly occurred with blunt-ended 20B GU3 in the presence of MK-2048 at 18°C for 72 h under standard assembly conditions.  Close Proximity of Viral DNA Ends in Intasomes-The ability of STIs to trap the RSV (14) and HIV-1 (26, 35) intasomes containing a tetramer of WT IN suggests close proximity (from ϳ10 to 100 Å) of the 5Ј-ends of the two viral DNA molecules within the complex as measured by fluorescence resonance energy transfer (FRET) (41). We determined that this is also the case with the RSV tetrameric intasome produced by IN  and the octameric intasome produced by IN(1-278) (Fig. 7). The 5Ј-terminal nucleotide (A) of a 2-bp overhang of the noncatalytic strand of 20R GU3 was labeled with either Cy3 (donor) or Cy5 (acceptor) fluorophore. Each assembled intasome mixture was diluted to 0.5 M DNA in the assembly buffer containing 1 M MK-2048 prior to FRET measurements at 10°C (Fig.   7). FRET was also observed with assembly mixtures that were diluted to 0.1 M DNA (data not shown). The results suggest that both STI-trapped intasomes maintain the positions of the 5Ј-ends of two viral DNA molecules in close proximity.
Further Mapping of the Tail Region Residues That Participate in Assembly of Octameric Intasome-We determined whether WT RSV IN(1-286) without and with a S282D mutation and IN(1-323), which extended an additional 37 residues past the terminal Ala (Fig. 1), were also capable of producing the octameric intasome. Standard assembly conditions were used. The yield and assembly efficiency were nearly the same for WT and IN(1-323) after 24 h at 18°C, whereas IN S282D was the least efficient (Fig. 8, A, B, and C, respectively). In addition, the transition of the tetrameric intasome to the octameric form was    (Fig. 9). The concerted integration activity of these IN constructs using 18R GU3 were essentially equivalent except for IN(1-323) under the same conditions (Fig. 9, C and D). The results suggest that the tail region may facilitate stabilization of the complex for coupling the 3Ј-OH processing reaction of blunt-ended DNA by IN for concerted integration activity. All of the IN constructs were inhibited by STIs (data not shown) as shown previously (14).
We had shown that WT RSV IN(1-286), IN(1-274), and IN(1-269) had equivalent 3Ј-OH processing activities at 37°C for 30 min using 32 P-labeled blunt-ended 4.6-kbp viral DNA (14). Similar results were obtained with these same IN constructs including IN(1-278) and IN S282D but not IN(1-323), which had significantly lower processing activity (Table 1)   these insoluble complexes are dialyzed against higher salt buffers they produce either a PFV tetrameric STC (10), an RSV octameric STC (9), or an MMTV octameric intasome for atomic resolution studies (11). PFV IN is capable of directly producing a soluble tetrameric intasome without first producing insoluble IN-DNA complexes (5, 7, 8). Likewise, RSV IN is capable of directly producing a soluble STI-trapped intasome that contains an IN tetramer (14). Using SEC analysis, a direct comparison of these two different assembly methods using RSV IN(1-269) and a branched viral/target DNA substrate (Fig.  10A, see inset) was performed in the absence of STIs. Both methods are essentially equivalent for producing soluble RSV STC (Fig. 10A) that contains an octameric IN structure (9).
We wanted to determine whether all of the RSV IN constructs were capable of forming the STC in contrast to the inability of only IN(1-269) to produce the octameric intasome containing only viral DNA ( Figs. 2A and 3C) (14). Using the direct assembly method, WT RSV IN and all of the modified C-terminal IN constructs essentially produce nearly equivalent quantities of soluble STCs at 4°C under identical assembly conditions (Fig. 10B). The data suggest that both viral and target DNA sequences play key roles in forming the RSV STC (9), and its assembly is independent of the C-terminal tail. In summary, our studies suggest that residues particularly between 269 and 278 of IN as well as the entire 18-residue tail region of IN(1-286) play a critical role in prompting the assem-

TABLE 1 The 3-OH processing activities of RSV INs
The 3Ј-OH processing activity of the RSV IN constructs was determined as described earlier (15) in the presence of Mg 2ϩ at 37°C for the indicated times. The standard deviation was determined from at least three independent experiments.

Discussion
The specific functions for the tail region extending beyond the ␤-strand-rich region of the SH3 domain fold in the CTD of RSV IN or similar tail regions of HIV-1 and PFV INs have not been determined (Fig. 1). Our C-terminal RSV IN truncation studies suggest that these 18 residues are critical for assembly of the STI-trapped intasome containing an octameric IN structure with 18R GU3 DNA but not for the intasome containing only a tetrameric IN structure (14). The assembly conditions for the most efficiently produced and stable RSV intasome containing the octameric IN structure is with IN(1-278) at 18°C (Fig. 6), suggesting that an optimal kinetic threshold for this specific assembly is met with the viral DNA substrate.
The two proximal IN dimers engage the viral DNA ends for catalysis, whereas the distal IN dimers bridge between the two GU3 DNA molecules and engage the target DNA substrate (9).  IN(1-278) produced intasomes that maintain the viral DNA ends in close proximity (Fig.  7), also previously demonstrated with WT RSV IN without STI present (41). The presence of an STI in an intasome formed with HIV-1 IN (26) or PFV IN (5) does not drastically alter its structure. An alternative possibility of the tetramer consisting of two DNA molecules bound to a proximal and distal dimer was also considered. However, this possibility was ruled out as proximal and distal dimers cannot simultaneously take the native (and DNA-binding compatible) conformation (15). An IN dimer can bind the viral DNA but cannot dimerize unless they swap NTDs to form the core tetramer of the intasome (9). The results suggest the additional tail residues in IN(1-278) are necessary and sufficient for interacting with viral DNA and/or other residues for assembly of the stable octameric intasome without target DNA being present.
Functional insights into how the 18-residue length of the tail region in HIV-1 IN affects the replication and integration properties of HIV-1 IN mutants in cell culture and their biochemical properties in vitro have been investigated (22,23). Single amino acid truncations of the CTD, starting from residues 265 to 277 of HIV-1, revealed several interesting results (23). Truncated "tail-less" CTD HIV-1 IN(1-269) mutant accommodated approximately half of the WT integration frequencies without and with viral complementation in vivo. Truncations from residues 273 to 282 in IN mutants produced near WT HIV-1 replication capabilities. The results suggested that the increased length of the tail region stabilizes the viral DNA ends for 3Ј-OH processing in vivo, thus promoting concerted integration (23). Similar results were obtained using other sets of C-terminal HIV-1 IN truncations (22). Significantly, the tail region fragment (residues 271-288) of HIV-1 IN cross-linked to viral and target DNA sequences with a hybrid dumbbell DNA substrate used to investigate disintegration activity (42). These HIV-1 IN studies suggest that the tail region is not critical for replication but enhances IN functions with increasing efficacy with its length.
The C terminus on WT IN(1-286) in avian retroviruses is produced by viral protease p15 cleavage of a polymerase precursor, and the cleaved 37-amino acid fragment is not essential for virus replication (Fig. 1) (12,17,19,(43)(44)(45). Further natural partial proteolytic processing of the IN tail region results in approximately 5-6 residues being removed from its C termi- nus, which does not grossly affect replication of the virus; partial phosphorylation of RSV IN at Ser-282 in vivo (Fig. 1) prevents proteolytic processing of the tail region at the very C terminus (20,21,43). Mutagenesis studies both in vitro and in vivo of the ␤-strand region of the SH3 domain in RSV IN showed that this region is essential for virus replication and concerted integration (13,15,20,46,47). These results suggest that both HIV-1 and RSV INs can tolerate partial removal of their tail regions without affecting reverse transcription and integration in vivo. There are no apparent amino acid sequence or charge similarities between the tail regions of PFV, HIV-1, and RSV INs (Fig. 1).
The efficiency for assembling the RSV octameric intasome at 18°C increased with the length of the tail region to residue 278 and was directly related to the complete disappearance of the GU3 substrate in the assembly mixture in the presence of IN(1-278) (Figs. 3 and 6). Increasing or decreasing the length of the tail region of IN(1-278) hindered the efficient use of the 18R GU3 substrate for assembly (Fig. 6). Further insights into the functions of the tail region of RSV IN were observed. We addressed whether residues beyond Ile-269 increased the ability of IN to couple 3Ј-OH processing of blunt-ended DNA to promote concerted integration in vitro similarly to what was observed with HIV-1 IN in vivo (23). Increasing the length from IN(1-269) to either IN(1-274) or IN(1-278) also increased the initial rate of concerted integration activity several times in the presence of 20B GU3 substrate (Fig. 9A). This initial faster rate essentially disappeared after 15-min incubation at 37°C for catalysis in comparison with IN . It is noteworthy that IN(1-286) containing the S282D mutation also had an initial slower rate of concerted integration activity relative to WT IN (Fig. 9), which is consistent with the slower replication rate of the virus containing this mutation relative to RSV (20). Lastly, the attachment of the 37-amino acid fragment (not essential for replication) to WT IN(1-286) to produce IN(1-323) effectively prevented IN(1-323) to promote concerted integration activity with blunt-ended viral DNA (Fig. 9). Apparently, the positioning of the blunt ends of the GU3 are not correct in this intasome produced by IN(1-323) to allow proper positioning of these ends for coupling 3Ј-OH processing and concerted integration. The 3Ј-OH processing of the 32 P-labeled blunt-ended 4.6-kbp DNA by IN(1-323) is also defective ( Table 1). The results suggest that observed proteolytic processing of polymerase polyprotein in RSV particles to WT IN(1-286) is essential for correct assembly of the intasome and for concerted integration activity in vivo.
DNase I footprint protection studies using virion-purified avian myeloblastosis virus or recombinant RSV IN(1-286) with larger size DNA substrates (ϳ1 kbp) demonstrated that IN is capable of fully protecting the GU3 or WT U3 non-catalytic DNA strand to ϳ20th nucleotide from the DNA end, and this protection is directly coupled to concerted integration activity catalyzed by the intasome (46,48,49). IN(1-269) produces the tetrameric intasome (14), whereas the addition of 5 residues or 9 residues to IN(1-269) culminates in the formation of the octameric intasome (Fig. 3). This suggests that the C-terminal tail interacts with viral DNA as well as with adjacent IN molecules to stabilize the octamer.
What are the mechanisms responsible for assembly of the RSV STC and the two different STI-trapped intasomes containing either a tetrameric or octameric IN structure? The branched viral/target DNA is a substrate for RSV IN  to assemble the STC containing the octameric IN structure for crystallization (9) and formation of the STC in solution (Fig.  10). There are multiple protein-DNA and protein-protein contacts that play various roles in the RSV STC structure. All of the INs in this current study can utilize the branched viral/target DNA substrate (Fig. 10B) to assemble the STC containing IN octamers, suggesting that both viral and target DNA sequences play roles in these assembly processes.
The atomic structure of IN in the intasome without the target DNA is probably similar to what is observed in the RSV STC including the CTD interactions of the distal IN dimers with viral DNA (9). The RSV octameric STC structure modeled with the C-terminal tails (residues 270 -286) suggests important roles of the different tail regions for assembly and stability of the octameric intasome (Fig. 11). The positioning of the C-terminal tails (residues 270 -286) in the STC complex viewed from the viral DNA side is shown in Fig. 11A. Six of the total eight subunits, from both proximal and distal IN dimers, have their well ordered CTD residue Ile-269 (9) in close proximity with the viral DNA, and subsequently the C-terminal tails potentially have extensive interactions with the viral DNA molecules. Rotating the view of Fig. 11A for 180°in Fig. 11B (shows the view from the target DNA side) demonstrates that the two inner subunits of distal dimers C and G chains have no interactions with DNA molecules. Instead, the three neighboring IN subunits form a pocket where each C-terminal tail fits snugly into the pocket. Fig. 11C shows the side view of the assembled STC and the fitting of the C-terminal tail of C chain (inner subunit of a distal dimer) in the pocket. Fig. 11, D and E, show the detailed conformation of C-terminal tails of D chain (outer subunit of a distal dimer) and C chain with three lysine residues emphasized. There are numerous other potential interactions like the N atom of Lys-277 forming a salt bridge with Asp-7 of the E chain (Fig. 11E). All of the interchain interactions could facilitate the assembly of the octameric complex by incorporating IN subunits into the tetrameric complex. Our model offers the opportunity to investigate the role of various residues in the assembly of both the tetrameric and the octameric intasome.
In conclusion, our studies suggest that residues between 269 and 278 of RSV IN in the 18-residue tail region of IN(1-286) play a critical role in prompting the assembly of the STItrapped octameric intasome containing viral DNA. Both HIV-1 and RSV IN biological data support a functional role for the tail region, although atomic structural information is lacking to support a complete understanding. The differential observations to assemble the tetrameric and octameric intasomes between IN(1-269) and IN (1-278), respectively, suggest critical roles of residues 270 -278 in recruitment of distal IN dimers. Assembly of the two proximal IN dimers with viral DNA is presumably the first step in the cytoplasmic PIC of infected cells, paralleling the assembly of the intasome in vitro. These observations suggest that a tetramer of IN is the basic catalytic unit for concerted integration for PFV, HIV-1, and RSV INs supported by biological, structural, and biochemical data. The auxiliary role or indeed a possible transient role performed by the tail region of the two proximal and distal IN dimers may provide additional stabilization to the octameric intasome and thus provide a platform for target binding (9). The observations that HIV-1 (50) and murine leukemia virus (51) INs protect the very termini as well as extended regions of the viral DNA ends to ϳ200 bp in the cytoplasmic PIC of virus-infected cells suggest that further oligomerization properties of IN are to be discovered.

Experimental Procedures
Production and Purification of RSV IN Constructs-WT RSV Prague A IN (amino acids 1-286) was produced as described (52). C-terminal IN truncation mutants are designated by numbers from the N terminus to the terminating amino acid in the construct. C-terminal truncations 1-269, 1-274, 1-278, and full-length IN containing a single point mutation (S282D) were produced by site-directed mutagenesis of WT RSV IN. The addition of 37 residues to RSV IN resulted in IN(1-323), whose sequence was derived from the polymerase sequence of RSV PrA and PrC strains (53,54). Negatively charged residues Asp and Glu comprise 27% of these shared protein sequences in this 37-residue fragment. The sequence of this 37-amino acid protein is GISDWIPWEDEQEGLQGETASNKQER-PGEDTLAANES. The IN clones were verified by DNA sequence analysis. RSV IN constructs were expressed in Escherichia coli BL21 (DE3)pLysS, purified, and concentrated to 10 -30 mg/ml using Amicon Ultra-15 centrifugal filters as described previously (14). All purified INs were free of contaminating DNA endonuclease activities using supercoiled DNA as substrate.
Concerted Integration and 3Ј-OH Processing Assays-The concerted integration assay using blunt-ended and 3Ј-OH recessed ODN viral DNA substrates with RSV IN was described previously (14). The concentrations of IN and the viral ODNs were 2 and 1 M, respectively. The strand transfer products were separated on a 1.8% agarose gel, stained with SYBR Gold (Invitrogen), and analyzed by a Typhoon 9500 laser scanner (GE Healthcare).
The 3Ј-OH processing of 32 P-labeled blunt-ended viral 4.6kbp DNA at 37°C was described previously (15). The time of incubation was varied as indicated. Concentrations of the various purified RSV INs and DNA in the assay mixture were 20 and 0.5 nM, respectively.
Viral DNA and Viral DNA/Target ODNs for Assembly of IN-DNA Complexes-Double-stranded blunt-ended and 3Ј-OH recessed ODNs containing RSV GU3 long terminal repeat sequences from 18 to 22 nucleotides in length were synthesized by Integrated DNA Technologies (14). The DNA substrates were recessed by 2 nucleotides on the catalytic strand and designated with an R or were blunt-ended and designated with a B. The identified length of the ODN denotes the non-catalytic strand. The sequences were as follows: 18R, 5Ј-ATTGCATAA-GACAACA-3Ј and 5Ј-AATGTTGTCTTATGCAAT-3Ј; 20B, 5Ј-GTATTGCATAAGACAACATT-3Ј and 5Ј-AATGTTG-TCTTATGCAATAC-3Ј. The following 3Ј-OH recessed ODN with nonspecific sequences was also prepared: 21R-NSP, 5Ј-GCAATGATACCGCGAGACC-3Ј and 5Ј-TGGGTCTC-GCGGTATCATTGC-3Ј.
The 5Ј-end on the non-catalytic strand of GU3 was also labeled with either Cy5 or Cy3 and synthesized by Integrated DNA Technologies. The RSV branched viral/target DNA substrate mimicking the product of concerted integration was used for the assembly of the STC (9).
Assembly Protocols for Intasomes and STC-There are two methods for assembly of stable RSV IN-DNA complexes. In the first method, intasomes produced with viral DNA require the presence of STIs to form kinetically "trapped" intasomes (14). The standard direct assembly buffer was 20 mM HEPES, pH 7.5, 100 mM ammonium sulfate, 100 mM NaCl, 1 M non-detergent sulfobetaine-201, 10% dimethyl sulfoxide (DMSO), 10% glycerol, and 1 mM tris(2-carboxyethyl)phosphine (TCEP). IN (as monomers), 3Ј-OH recessed DNA ODN, and STI concentrations were generally set at 45, 15, and 125 M, respectively, unless otherwise indicated. After addition of DNA to the assembly mixture and subsequently IN, the samples were incubated at 4 or 18°C for various times as indicated. The STIs used were raltegravir, MK-2048, MK-0536, dolutegravir, and elvitegravir. The direct assembly condition to produce the RSV STC using a branched viral/target DNA substrate was the same as described above for the intasomes except the NaCl concentration was 0.4 M, a 42-bp STC DNA substrate was used (15 M) (9), and no STIs were present.