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Multifaceted HIV functionalities and therapeutic strategies for their inhibition Published, Papers in Press, August 29, 2019, DOI 10.1074/jbc.REV119.006901 X Alan N. Engelman1 From the Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215 and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115 Edited by Karin Musier-Forsyth

Antiretroviral inhibitors that are used to manage HIV infec- replication cycle (1)(Fig. 1). Unique among animal viruses is the tion/AIDS predominantly target three required for requirement for retroviruses to integrate their genetic informa- virus replication: reverse transcriptase, protease, and integrase. tion into the genome of the host cell that they infect. Integration Although integrase inhibitors were the last among this group to is mediated by the viral protein integrase (IN), which is incor- be approved for treating people living with HIV, they have since porated into fledgling viral particles alongside the other viral risen to the forefront of treatment options. Integrase strand enzymes reverse transcriptase (RT) and protease (PR). PR ini-

transfer inhibitors (INSTIs) are now recommended components tiates virus particle maturation by cleaving viral Gag and Gag- Downloaded from of frontline and drug-switch antiretroviral therapy formula- Pol polyprotein precursors into separate viral structural pro- tions. Integrase catalyzes two successive magnesium-dependent teins and enzymes, which is required to form the viral core -polynucleotidyl transferase reactions, 3؅ processing and strand (reviewed in Ref. 2). The core consists of the viral ribonucleo transfer, and INSTIs tightly bind the divalent metal ions and protein (RNP) complex, which contains two copies of the RNA ؅ viral DNA end after 3 processing, displacing from the integrase genome bound by viral nucleocapsid, IN, and RT proteins, http://www.jbc.org/ active site the DNA 3؅-hydroxyl group that is required for strand encased within a fullerene shell composed of the viral capsid transfer activity. Although second-generation INSTIs present protein (reviewed in Ref. 3). RT converts retroviral RNA into a higher barriers to the development of viral drug resistance than single molecule of linear DNA containing a copy of the viral first-generation compounds, the mechanisms underlying these long terminal repeat (LTR) at each end (Figs. 1 and 2) (reviewed superior barrier profiles are incompletely understood. A sepa- in Ref. 4). The linear DNA, comprised of U3 and U5 terminal rate class of HIV-1 integrase inhibitors, the allosteric integrase sequences in respective upstream and downstream LTRs, is the by guest on October 11, 2019 inhibitors (ALLINIs), engage integrase distal from the substrate for IN-mediated viral DNA insertion into chromo- active site, namely at the binding site for the cellular cofactor somal DNA (5–7). lens epithelium-derived growth factor (LEDGF)/p75 that helps Four classes of antiretroviral drugs, nucleoside RT inhibitors to guide integration into host genes. ALLINIs inhibit HIV-1 rep- (NRTIs), nonnucleoside RT inhibitors (NNRTIs), PR inhibitors lication by inducing integrase hypermultimerization, which (PIs), and IN strand transfer inhibitors (INSTIs) have in recent precludes integrase binding to genomic RNA and perturbs the years comprised frontline cART formulations (8). Highlighting morphogenesis of new viral particles. Although not yet the success of the INSTI drug class, current guidelines recom- approved for human use, ALLINIs provide important probes mend the use of a second-generation INSTI ( that can be used to investigate the link between HIV-1 integrase (DTG) or (BIC)) co-formulated with two NRTIs to and viral particle morphogenesis. Herein, I review the mecha- treat most people living with HIV (PLHIV) who have not pre- nisms of retroviral integration as well as the promises and chal- viously failed an INSTI-containing regimen (9, 10). INSTIs lenges of using integrase inhibitors for HIV/AIDS management. inhibit IN strand transfer activity and thus specifically block the integration step within the HIV-1 life cycle (11)(Fig. 1). A sep- arate class of inhibitors, the allosteric IN inhibitors (ALLINIs), Combination antiretroviral therapy (cART)2 treats patients with a mixture of drugs to inhibit different steps of the HIV-1 genic EM; CTD, C-terminal domain; CSC, cleaved synaptic complex; DTG, dolutegravir; EVG, ; FDA, Food and Drug Administration; IBD, integrase-binding domain; IN, integrase; INSTI, integrase strand transfer This work was supported by National Institutes of Health Grants R37 AI039394 inhibitor; KPN, karyopherin; LA, long-acting; LEDGF, lens epithelium-de- and R01 AI070042. The author has received fees from ViiV Healthcare Co. rived growth factor; LRA, latency reversal agent; LTR, long terminal repeat; within the past 12 months. The content is solely the responsibility of the MMTV, mouse mammary tumor virus; MVV, Maedi-visna virus; NNRTI; non- author and does not necessarily represent the official views of the National nucleoside reverse transcriptase inhibitor NRTI, nucleoside reverse tran- Institutes of Health. scriptase inhibitor; NTD, N-terminal domain; PDB, Protein Data Bank; PFV, 1 To whom correspondence should be addressed: Dept. of Cancer Immunol- prototype foamy virus; PI, protease inhibitor; PIC, preintegration complex; ogy and Virology, Dana-Farber Cancer Institute, Boston, MA 02215. PLHIV, people living with HIV; PPT, polypurine tract; PR, protease; PrEP, Tel.: 617-632-4381; Fax: 617-632-4338; E-mail: alan_engelman@dfci. pre-exposure prophylaxis; RAL, ; RANBP, Ran-binding protein; harvard.edu. RNP, ribonucleoprotein; RT, reverse transcriptase; SHIV, simian-human 2 The abbreviations used are: cART, combination antiretroviral therapy; immunodeficiency virus; SSC, stable synaptic complex; t-BSF, tert-butylsul- ALLINI, allosteric ; BIC, bictegravir; CAB, ; fonamide; TCC, target capture complex; TNPO, transportin; SH3, Src homo- CCD, catalytic core domain; CIC, conserved intasome core; cryo-EM, cryo- logy 3.

J. Biol. Chem. (2019) 294(41) 15137–15157 15137 © 2019 Engelman. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. JBC REVIEWS: HIV integrase mechanisms and inhibition Downloaded from http://www.jbc.org/ by guest on October 11, 2019

Figure 1. HIV replication cycle. After entry into a susceptible target cell, RT converts genomic RNA into linear DNA within the confines of the reverse transcription complex (RTC)(272). Processing of the viral DNA ends by IN yields the PIC (30), which can integrate the endogenous DNA made by reverse transcription into recombinant target DNA in vitro (5). Following nuclear import and integration, the provirus (flanked by composite cyan/yellow/magenta LTRs) serves as a transcriptional template to produce viral mRNAs for translation of viral proteins as well as nascent viral genomes that co-assemble with viral proteins to form immature virions that bud out from the infected cell (2). Shown is a generalized scheme that depicts the major steps of HIV-1 replication, although it is important to note that deviations from this plan exist throughout Retroviridae. Most notably, spumavirus reverse transcription occurs during the second half of the infectious cycle (after integration), and spumaviral particles accordingly predominantly contain dsDNA (104, 273). The primary steps in the HIV-1 replication cycle that are inhibited by the two major classes of IN inhibitors discussed herein are indicated.

by contrast inhibit particle maturation (12, 13) (see below). To includes related enzymes such as transposase proteins and fully understand the nature of these different types of inhibi- RNase H (reviewed in Ref. 17). tors, it is important to appreciate the different steps of HIV-1 Two different IN activities, 3Ј processing and strand transfer, replication (Fig. 1) as well as the mechanistic and structural are required for integration (Fig. 2). During 3Ј processing, IN bases of retroviral DNA integration. prepares the linear reverse transcript for integration by hydro- lyzing the DNA ends 3Ј of conserved CA dinucleotides, which Mechanism of retroviral integration most often liberates a dinucleotide from each end (18–20). IN is a polynucleotidyl transferase composed of three con- However, symmetrical DNA processing is not required for inte- served protein domains: the N-terminal domain (NTD) with gration; the upstream terminus of spumaviral DNA is 5Ј-TG, conserved His and Cys residues (HHCC motif) that coordinate Zn2ϩ binding for 3-helix bundle formation; the catalytic core obfuscating the need for U3 end processing by IN (21), whereas domain (CCD), which adopts an RNase H fold and harbors the a trinucleotide is processed from the U5 end of some primate enzyme active site composed of invariant carboxylate residues lentiviruses (22, 23), including HIV-2 (24). During strand trans- Ј (DDE motif); and the C-terminal domain (CTD), which adopts fer, IN uses the CAOH-3 hydroxyl groups to cut chromosomal an SH3 fold (reviewed in Ref. 14). The role of the DDE residues DNA in a staggered fashion, which, due to the nature of SN2 in catalysis is to coordinate the positions of two divalent cat- chemistry, simultaneously joins the viral DNA ends to the ions, which under physiological conditions are almost certainly 5Ј-phosphate groups of the dsDNA cut (6, 7, 15). The resulting magnesium, to deprotonate attacking oxygen nucleophiles and gapped DNA intermediate with unjoined viral DNA 5Ј ends is destabilize scissile phosphodiester bonds for one-step transes- repaired by host cell machineries to yield the integrated provi- terification chemistry (15, 16). Similar functionalities exist rus flanked by the sequence duplication of the host DNA cut, across a large superfamily of polynucleotidyl transferases that which for HIV-1 is most often 5 bp (25, 26)(Fig. 2).

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Intasome structure and function Integration in cells is mediated by the preintegration com- plex (PIC), which is a large nucleoprotein complex derived from the core of the infecting virion (27, 28). Within the confines of the PIC, IN functions as part of the intasome nucleoprotein complex, which is comprised of a multimer of IN and the viral DNA ends (29–35)(Figs. 1 and 3). A series of X-ray crystallo- graphic and single-particle cryogenic electron microscopy (cryo-EM) structures determined over the past decade has clar- ified that the number of IN molecules required to build the intasome differs depending on the type of retrovirus (reviewed in Ref. 36). Seven retroviral genera are grouped into two sub- families of Retroviridae: Spumavirinae, solely harboring the spumaviruses, and Orthoretrovirinae, which encompass the lentiviruses, such as HIV-1, as well as ␣-, ␤-, ␦-, ⑀-, and ␥-ret- roviruses. X-ray crystal structures of the spumavirus prototype foamy virus (PFV) intasome provided initial high-resolution views of the functional IN-viral DNA architecture as well as

critical insight into the mechanism of INSTI action (see below) Downloaded from (35, 37, 38). The PFV intasome is composed of an IN tetramer with the following division of labor. Two extended, intertwined IN mol- ecules (Fig. 3A, blue and green) harbor operational active sites and thus catalyze 3Ј processing and strand transfer activities, http://www.jbc.org/ whereas the other two IN molecules (Fig. 3A, cyan) with non- operational active sites serve as bookends to truss the DNA- bound IN protomers together (35). The interwoven nature of the two catalytically active IN molecules, with their NTDs mutually swapped between CCDs, was observed previously in crystal structures of two-domain lentiviral IN NTD-CCD con- by guest on October 11, 2019 structs in the absence of DNA (39, 40). Prior to these structures, the NTD from one IN protomer had been shown to function in trans with the active site of a separate IN molecule within the active HIV IN multimer (41, 42). The interwoven NTD-CCD arrangement at the heart of the machine leverages the partici- pation of both viral DNA ends in intasome assembly and DNA recombination. Four types of intasomes describe the ground states and prod- uct complexes associated with IN 3Ј processing and strand transfer activities. IN processes the viral DNA ends in the con- text of the initial stable synaptic complex (SSC), yielding the cleaved synaptic complex (CSC) after viral DNA hydrolysis. The target capture complex (TCC) describes the CSC bound to target or host DNA, whereas strand transfer yields the strand transfer complex (16, 35–37, 43). The overall conformation of the PFV intasome structure changes little as the complex morphs from the SSC to the strand transfer complex and pro- motes IN 3Ј processing and strand transfer activities (16, 35, 37, 43, 44). Although integration occurs largely throughout animal cell genomes (45, 46), host DNA sequences that contort to fit the target DNA-binding interface within the CSC are preferred targets (37, 44, 47–49) (for a detailed review, see Ref. 50). Thus, Figure 2. DNA cutting and joining steps of retroviral integration. The linear viral reverse transcript (lavender lines; plus-strands shaded more darkly than same-colored minus-strands throughout the cartoon) contains a copy of the LTR at each end composed of cyan U3, yellow R repeat, and magenta U5 from each end. After nuclear localization, the intasome interacts with host sequences. The upstream LTR is abutted by the primer-binding site (PBS; pur- target DNA (gray lines with targeted green sequence) to promote DNA strand ple box), whereas the downstream element is abutted by the polypurine tract transfer. The DNA gaps that persist after strand transfer are repaired by host (PPT; lavender box). During 3Ј processing, IN hydrolyzes the DNA adjacent to cell machinery to yield a target site duplication (thin green lines) flanking the invariant CA dinucleotides, which for HIV-1 liberates the pGTOH dinucleotide integrated provirus.

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Figure 3. Retroviral intasome structures. A–C, representative intasomes from the spumavirus PFV (A; protein database (PDB) accession code 3OY9), ␤-ret- rovirus MMTV (B; PDB code 3JCA), and lentivirus MVV (C; PDB code 5M0Q) are color-coded to highlight the CIC. Green and blue, catalytically active IN protomers; cyan, supporting IN CCDs; black, DNA strands. Whereas four PFV IN molecules suffice to form the CIC, both MMTV and MVV require six IN protomers. For MMTV, critical CTDs (magenta) are donated by flanking IN dimers, leading to an overall IN octamer. In MVV, flanking IN tetramers provide the critical CTDs, resulting in

an overall IN hexadecamer. Gray coloring in B and C deemphasizes IN elements that do not compose the CIC. D–F, resected CCD and CTD domains from above by guest on October 11, 2019 green IN protomers, oriented to highlight the different CCD-CTD linker regions (dark gray). Associated magenta CTDs from separate IN protomers in E and F assume similar positions as the green CTD in D. Red sticks, DDE catalytic triad residues.

strand transfer proceeds without gross rearrangements in inta- IN octamers. The lentiviral IN CCD-CTD linker, although some architecture. composed of ϳ15–20 residues, adopts ␣-helical conformation Studies of additional retroviral intasomes unveiled a com- (51, 55) that likewise imposes a distance constraint to preclude mon structural feature at the hearts of the machines that was proper IN CTD positioning from the catalytically active IN coined the conserved intasome core (CIC) (51)(Fig. 3, A–C). molecules (Fig. 3F). In the MVV intasome, IN tetramers donate However, as mentioned, different viruses utilize different num- the required CTDs, resulting in an overall IN hexadecamer (51). bers of IN protomers to form the CIC. The tetrameric IN archi- The HIV-1 intasome structure employed a fusion composed of tecture of the PFV intasome defines the basic features of the heterologous Sso7d protein appended onto the IN N terminus, CIC, including two active protomers with CCDs and NTDs which significantly improved IN solubility and enzyme activity swapped across a synaptic interface where two CTDs engage (54, 56). Flanking Sso7d-IN dimers were seen to donate the target DNA for integration (37) and two additional IN mole- required CTDs to complete the CIC structure, resulting in an cules that bookend the active subunits (35). Whereas four PFV overall IN dodecamer. On the one hand, it seems likely that IN molecules suffice to build the CIC, ␣- and ␤-retroviral inta- some flexibility is tolerated in terms of the multimeric character somes require eight IN molecules (52, 53), and the lentiviruses of the flanking IN oligomer that donates the CTD to the CIC HIV-1 and Maedi-visna virus (MVV) require 12 and 16 IN structure, minimally requiring an IN dimer. On the other hand, protomers, respectively (51, 54). The requirement for the dif- it is possible that the use of the heterologous protein domain ferent numbers of IN molecules stems from evolutionary con- precluded high occupancy of flanking IN tetramers in the straints on the amino acid composition of the linker that con- Sso7d-IN intasome structure. Additional structures derived nects the CCD and CTD (52). The linker in PFV IN, composed from WT HIV-1 or related primate lentiviral IN proteins may of ϳ50 residues, is sufficiently long to allow the CTDs of the further inform the IN-to-viral DNA stoichiometry necessarily active IN protomers to assume the positions required for host for HIV-1 IN function. DNA binding (Fig. 3D). The analogous linker in ␣- and ␤-ret- A key unanswered question in retroviral integration research roviruses, at only ϳ8 residues, precludes the necessary CTD is the mechanism of intasome assembly. DNA-based tetramer- positioning (Fig. 3E). These viruses accordingly use two flank- ization of the predominant IN species in solution has been pro- ing IN dimers to position the critical CTDs, resulting in overall posed (51) based on observations that PFV IN in the absence of

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DNA is monomeric (35), ␣- and ␤-retroviral INs are predomi- ethidium bromide that would likely score as a hit if test com- nantly dimeric (52, 53), and lentiviral INs are predominantly pounds were comixed together with IN and viral DNA is highly tetrameric although with evidence for additional lower- and unlikely to specifically inhibit integration in infected cells. higher-order forms (40, 42, 51, 57–62). However, the relation- Numerous early compounds accordingly lacked specificity to ship between protein behavior in solution and the multimeric inhibit integration during HIV-1 (reviewed in Ref. 88). state of IN in virions or during reverse transcription is largely A key turning point in IN inhibitor development came from unknown. Because HIV-1 IN binds genomic RNA in virions reformulating the design of the in vitro assay to prebind IN to a (63), it seems possible that RNA-bound IN may transfer to the synthetically preprocessed viral DNA end substrate (87) and DNA ends as they form during reverse transcription to initiate screen for inhibitors of strand transfer activity, which led to the SSC formation. The IN tail region, which is composed of the discovery of first-in-class INSTIs (11). Although these diketo amino acids C-terminal from the CTD SH3 fold, varies in acid compounds were never licensed to treat PLHIV, they nev- length from about 5 residues in the lentivirus equine infectious ertheless served as important molecules with which to probe anemia virus to 55 residues in MMTV. The tail region in ␣-ret- INSTI mechanisms of action. INSTIs harbor two commonali- roviral IN, which is 19 residues, can regulate DNA-dependent ties across otherwise diverse pharmacophores (Figs. 4A and 5). IN octamer formation (64, 65). Although implicating a role for At the hearts of the compounds are three adjacent heteroatoms this region of IN in intasome assembly, tail regions are unre- (most usually oxygen; red in Fig. 5, B and C), whereas a terminal solved in all IN and intasome structures solved to date, limiting halogenated benzene ring connects to the rest of the molecule the interpretation of how the tail might regulate nucleoprotein via a flexible linker (Fig. 4A; blue in Fig. 5, B and C). The com- complex formation. pounds avidly bound IN-viral DNA complexes yet failed to Downloaded from INSTIs appreciably bind HIV-1 IN in the absence of viral DNA (89), and subsequent work revealed the importance of the terminal Ј Research in the mid-1980s first established a role for the 3 deoxyadenylate residue at the 3Ј end of processed viral DNA in region of the pol gene, which encodes for IN, in retroviral rep- the regulation of INSTI binding and dissociation (90, 91). The lication (66–69), and the extension of this requirement to

conserved INSTI heteroatoms engage the divalent metal ions http://www.jbc.org/ HIV-1 highlighted IN as a high-value antiviral target (70). How- that are bound by the DDE active-site residues (35, 92). ever, a scant number of promising preclinical lead compounds Raltegravir (RAL) in 2007 was the first INSTI licensed by the were known by the time RT and PR inhibitors were adminis- United States Food and Drug Administration (FDA) (93) and tered to patients in cART formulations (71–73), calling into elvitegravir (EVG) in 2012 became the second licensed INSTI question whether IN inhibitors would ever make it to the clinic. (94). (Fig. 4A). Although prior work demonstrated the impor- Indeed, around this time, I can recall one of the more promi- by guest on October 11, 2019 tance of divalent metal ion and viral DNA sequence for INSTI nent researchers in our field espousing the view at a national binding (90–92), the field lacked a detailed view of how INSTIs meeting that clinical IN inhibitors were unattainable. The rea- inhibited IN strand transfer activity. Fortuitously, both NRTIs soning here was based on the observation that an equal number and INSTIs, which target respective RT and IN active sites of IN, RT, and PR molecules are packaged into each virion par- ticle, which one can estimate as 120 based on the 20:1 synthesis composed of invariant amino acid residues, inhibit a wide range ratio of Gag to Gag-Pol (74) and circa 2,400 Gag molecules per of retroviruses (95–103) including spumaviruses (104, 105). virion (75). Per replication cycle, RT and PR catalyze roughly Thus, the PFV intasome could serve as a model system to inves- 19,400 and 12,900 chemical reactions, respectively. However, tigate INSTI mechanism of action. Co-crystal structures with the same population of IN molecules performs only four chem- RAL or EVG revealed that the halobenzyl groups assumed the Ј ical reactions. How then could one effectively inhibit IN in the position of the purine rings of the 3 -deoxyadenylate residue, face of this seemingly large excess of available enzyme? Fortu- supplanting the terminal nucleoside from the IN active site (Fig. itously, my colleague turned out to be incorrect. What was 5, A and B). INSTI binding accordingly inactivates the intasome unknown at the time of our discussion was the utility of mole- complex by displacing from the enzyme active site the DNA Ј cules designed to inhibit IN strand transfer activity. Whereas 3 -OH group that is required to cut chromosomal DNA for HIV-1 IN processes the viral DNA ends to yield the CSC con- strand transfer activity (35). comitant with or soon after reverse transcription (30, 76)(Fig. Second-generation INSTI compounds physically expand 1), integration into chromosomal DNA does not occur until upon first generation scaffolds while maintaining both metal- hours to days (76–79) or, in some extreme cases, weeks later chelating and DNA-supplanting drug functions. Such modifi- (80). The comparatively long-lived CSC intasome replication cations include increasing the length of the linker between the intermediate is a pharmacological HIV-1 Achilles’ heel that is metal-chelating and halobenzyl moieties (106–108), increasing leveraged fully by the INSTI class of antiretroviral compounds. the number of central ring moieties to three (106, 107, 109), Because HIV-1 IN purified from recombinant sources dis- and, akin to EVG, derivatization of a second ring that lies distal played 3Ј processing and strand transfer activities in vitro (15, from the halobenzyl group (110–113)(Fig. 4A). Second-gener- 20, 81, 82), systems to search for inhibitory molecules of HIV-1 ation INSTIs more fully occupy the IN active-site region that IN activity were readily scalable (83–85). However, due to sub- spans from the DNA-binding pocket on the one side to the optimal assay designs, few early leads turned out to specifically connector sequence that links IN secondary structural ele- inhibit HIV-1 integration under physiological conditions (86, ments ␤4 and ␣2 on the other (112–115)(Fig. 5C, ␤4-␣2 87). Consider the following example. A compound such as connector).

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Figure 4. INSTI structures and ALLINI chemotypes. A, diagrams of the four FDA-licensed INSTIs as well as investigational second-generation compound 6p http://www.jbc.org/ (113, 119 ). B, representative ALLINI chemotypes. Asterisks mark common positions of t-butoxyacid moieties.

Overlaying the structures of INSTI-bound PFV intasomes to HIV/AIDS treatment. Although DTG was initially deemed safe those of the SSC and TCC yielded important insight into the for pregnant women (120), follow-up work highlighted a

mechanism of drug action (16, 112, 113). The RAL metal- greater frequency of neural tube defect in infants born to by guest on October 11, 2019 chelating oxygen atom distal from the halobenzyl group coin- Botswanan mothers who were taking DTG-containing cART cided with the nucleophilic water molecule for IN 3Ј processing since the time of conception (4 of 426; 0.94%) versus frequencies activity (red sphere in Fig. 5D), whereas the RAL-chelating oxy- observed in the general population (86 of 87,755; 0.1%) or in gen proximal to the halobenzyl coincided with the scissile phos- infants from mothers taking other cART regimens (14 of phodiester bond in viral DNA (Fig. 5D). For strand transfer, the 11,300; 0.12%) (121). Such observations prompted several reg- halobenzyl-proximal oxygen coincided with the nucleophilic ulatory agencies including the FDA and the World Health 3Ј-oxygen of processed viral DNA, whereas the distal RAL oxy- Organization in 2018 to issue alerts regarding possible gen overlapped with the scissile phosphodiester bond in target increased risk of neural tube defect in infants born to mothers DNA (16)(Fig. 5E). These observations first identified INSTIs taking DTG-containing cART at the time of conception (122). as IN substrate mimics, which was subsequently expanded Subsequent retrospective analyses have failed to detect a link through the broader concept of substrate envelope. Previously between DTG usage and neural tube birth defect, although espoused for HIV-1 PR and the mechanism of PI action, the such studies were generally limited by sample size (123, 124). substrate envelope is defined as the space occupied by the sub- Retrospective analysis has also failed to detect an increase in the strate (peptide in the case of PR; DNA for IN) in an enzyme frequency of neural tube defect in infants born to mothers on active site. Because the enzyme must interact with the substrate RAL-based drug regimens (125). Recent follow-up work that for catalysis, drugs that interfere with enzyme–substrate inter- increased the number of patients from 426 to 1,683 in the actions should be inhibitory and might impart relatively high Botswanan cohort revised the neural tube defect frequency resistance barriers (116–118). Indeed, second-generation from 0.94% to 0.3%, which was still 0.2% greater than the fre- INSTI elements distal from the halobenzyl groups coincide quencies observed in control populations (126). It will be with the position of host DNA in PFV intasome structures (16, informative to ascertain whether BIC, which is the other 112–114)(Fig. 5E), likely accounting for the competition licensed second-generation INSTI and is structurally related to between target DNA and INSTIs for binding to HIV-1 IN-viral DTG (Fig. 4A), also influences neural tube defect frequency DNA complexes (89). Compound modifications that further versus control populations. interfere with HIV-1 IN–substrate interactions could improve INSTI potency and increase the barrier to acquire drug-resist- Mechanisms of HIV resistance to INSTIs ant mutations (119). Substitutions of HIV-1 IN residues Gln-148, Asn-155, or Second-generation INSTIs are currently undergoing exten- Tyr-143 were recognized early on as separate genetic pathways sive safety evaluation due to their planned global rollout for to clinical RAL resistance (127), and mutant viral strains har-

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Figure 5. Mechanisms of INSTI action. A, close-up view of one PFV IN active site in the CSC intasome structure (PDB code 3OY9) with IN secondary elements labeled. Additional labels highlight the conserved CA dinucleotide of the transferred DNA strand (magenta sticks) and associated G nucleotide of the non- transferred strand (orange), the 3Ј-OH nucleophile of the terminal deoxyadenylate used by IN to cut chromosomal DNA, as well as IN residues (in sticks) that compose the catalytic triad (Asp-128, Asp-185, and Glu-221) and that when changed confer INSTI resistance (Tyr-212 and Asn-224). Blue and red stick colors ␣ ␤ ␩ ␣ ␤ denote nitrogen and oxygen atoms, respectively. Gray spheres, divalent metal ions; , , and denote -helix, -strand, and 310-helix, respectively. B, RAL (cyan sticks)-bound PFV intasome structure (PDB code 3OYA) oriented as in A to highlight the mode of INSTI binding. RAL binding results in a greater than 6-Å displacement of the 3Ј-OH of the terminal deoxyadenylate from the IN active site. The position of the RAL methyl-oxadiazole group is highlighted in the cartoon on the left as well as the chemical diagram on the right, which was reconfigured from Fig. 4A to accentuate the position within the crystal structure. Other colors and labeling are the same as in A or as described under “INSTIs.” C, same as in B, except with DTG bound (PDB code 3S3M). The position of the IN ␤4-␣2 connector is noted; other labeling is the same as in A and B. D, RAL (magenta) from the drug-bound PFV CSC structure (PDB code 3OYA) is overlaid with the PFV SSC structure (green; PDB code 4E7I) to highlight mimicry between drug oxygen atoms and oxygen atoms critical for IN 3Ј processing activity (red sphere, nucleophilic water (W) molecule; red bridge in viral DNA (vDNA), scissile phosphodiester bond). E, similar to D; RAL is superimposed onto the PFV TCC structure (IN and target DNA in cyan and vDNA in blue; PDB code 4E7K) to highlight similarly positioned drug oxygen atoms with the vDNA 3Ј-oxygen and scissile phosphodiester bond in target DNA (tDNA) critical for strand transfer activity. Other labeling is the same as in A and B. boring changes such as Q148H/G140S or N155H conveyed sig- oxadiazole constituent of this drug stacked against the p-cresol nificant resistance to EVG as well (reviewed in Ref. 128). side chain of IN residue Tyr-212 (Fig. 5B). Changes that would Although structure-based studies with PFV intasomes in- reduce the aromatic nature of HIV-1 IN residue Tyr-143 would formed the mechanisms of drug resistance (35, 38), partial accordingly result in loss of an important RAL binding contact. amino acid identity between PFV and HIV-1 INs limits the The structure accounted for the relative specificity of RAL resis- extent of information that can be gleaned from the model sys- tance to Tyr-143 changes in IN (129, 130), as only RAL among tem. PFV and HIV-1 IN are overall 18.4% identical, whereas the clinical INSTIs harbors the methyl-oxadiazole constituent their respective CCDs share 22.4% identity (35). Fortuitously, (Fig. 4A). Although Asn-224 similarly resides near the INSTI- two of the three clinically relevant amino acids, Tyr-143 and binding pocket, it does not directly contact bound drugs. The Asn-155, are conserved as Tyr-212 and Asn-224 in PFV IN (Fig. mutant His side chain in the intasome structure derived from 5A). The binding mode of RAL to the PFV intasome in partic- PFV IN N224H interacted with the 3Ј-deoxyadenylate– ular informed the Tyr-143 resistance pathway, as the methyl- bridging phosphate, which was disrupted by second-generation

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INSTI MK-2048 binding (38). Although disruption of the His– upstream viral DNA terminus after reverse transcription. DNA interaction could contribute to the mechanism of clinical Selection of DTG resistance in cell culture has revealed changes INSTI resistance to HIV-1 N155H (38), intasome structures in the HIV-1 3Ј PPT, which was unexpected because this with INs that share greater amino acid identity to HIV-1 are sequence abuts the downstream LTR distal from the viral DNA expected to more completely inform INSTI resistance mecha- termini (Fig. 2)(146). One possible explanation is that the alter- nisms outside of the Tyr-143 pathway. Of note, although MVV ations lead to misprocessing of the PPT during reverse tran- is a lentivirus, its IN also shares limited amino acid sequence scription and accordingly extend the U3 DNA terminus, which identity with HIV-1 IN (27.4% overall; 34.3% between CCDs). would be a suboptimal sequence for IN binding (147). However, Given fast-paced advancements in single-particle cryo-EM sequencing of 2-LTR circles, which form at low frequency in the (131, 132), one can optimistically expect comparatively high- cell nucleus via DNA ligation and thus provide a snapshot of resolution structures of INSTIs bound to the HIV-1 intasome viral DNA end sequences, failed to identify the hypothesized in the not too distant future. Such structures should critically PPT extension (148). PPT mutations have been recorded in one inform mechanisms of INSTI drug resistance as well as how to patient who received DTG monotherapy (149), indicating that potentially improve INSTI potencies moving forward. such changes may very well be clinically relevant. Additional Clinical (133–135) as well as in vitro (136) studies have high- work is required to more fully document the frequency of PPT lighted the superior resistance profiles of second-generation changes in patients that fail DTG therapy as well as how such INSTIs such as DTG compared with predecessor first-genera- changes engender drug resistance. tion compounds. Whereas selection of HIV-1 resistance in cell Other changes outside of the IN coding region, including the culture invariably leads to changes in IN that can confer Ͼ100- HIV-1 env gene, can confer resistance to DTG (150). HIV-1 can Downloaded from fold resistance to RAL and EVG, such resistance is much harder infect cells through fusing directly with the cellular plasma to come by for DTG, and the selected changes in IN, such as membrane or through the virological synapse that forms R263K, engender just a few-fold resistance to the compound between an infected cell and an uninfected cell (151) (reviewed (137). Whereas cART formulations typically comprise three in Ref. 152). The env mutations effectively increased the multi- distinct compounds, such observations inspired clinical evalu- plicity of HIV-1 infection by significantly increasing the effi- ation of DTG as a monotherapy or as dual therapy in conjunc- ciency of cell-to-cell infection (150). This mechanism of drug http://www.jbc.org/ tion with an NRTI, NNRTI, or PI (138–143). Based on rates of resistance, which is indirect because it is highly unlikely to virological failure, the use of DTG as a monotherapy for PLHIV influence the dissociative half-life of DTG from the HIV-1 inta- is contra-indicated, whereas the evaluation of dual therapy some, is reminiscent of prior reports that HIV-1 infection options is ongoing (143) (reviewed in Ref. 144). Current guide- through the virological synapse reduced the efficacy of certain

lines recommend the use of DTG, BIC, or RAL with two NRTIs cART compounds (153, 154). Additional work is required to by guest on October 11, 2019 for most PLHIV (9, 10). determine whether changes in HIV-1 env can confer resistance The rates at which INSTIs dissociate from IN-viral DNA to DTG in the clinical setting (150). complexes in vitro have informed the mechanisms of drug The investigational second-generation INSTI cabotegravir action and drug resistance. Consistent with its comparatively (CAB) formulated as a crystalline nanoparticle conferred long- high resistance barrier, the dissociative half-life of DTG, 71 h, acting (LA) protection against challenge by chimeric simian- was significantly longer than the corresponding RAL and EVG HIV (SHIV) in the macaque model of HIV/AIDS (155–157). values of about 9 and 3 h, respectively (145). Although analyses LA-CAB administered as a monotherapy or in combination of mutant IN-viral complexes failed to identify a direct correla- with LA-, which is a long-acting NNRTI, is being tion between drug dissociation half-life and antiviral potency evaluated as a pre-exposure prophylaxis (PrEP) to prevent and resistance, HIV-1 was generally sensitive to INSTIs when HIV-1 infection (reviewed in Ref. 158). One of the biggest fac- compound dissociative half-life was greater than 4 h and resist- tors contributing to the emergence of anti-HIV drug resistance ant to inhibition when half-lives were less than1h(145). Thus, is dosing protocol compliance, which for oral cART formula- INSTI dissociative half-life is a useful predictor of drug potency tions is one to several pills daily. LA regimens largely obfuscate and drug resistance. Whereas some IN amino acid substitu- the need for end-user dose monitoring, which could increase tions, such as Y143R/K/C, increase dissociation by altering a compliance, although at the same time such regimens require direct IN-INSTI contact (35), changes such as Q148H/G140S regular injections to maintain plasma trough concentrations seemingly act indirectly by altering the conformation of the IN above values required to inhibit HIV-1 replication. Macaques active-site region (38). that were positive for SHIV RNA but seronegative at the time of Alterations of viral DNA sequence, especially the terminal infection could develop resistance to LA-CAB, and the associ- deoxyadenylate residue, also alter INSTI dissociative half-life ated IN changes conferred potent cross-resistance to all (91), although to date, LTR sequence changes have not been licensed INSTIs (159). Such observations highlight the need to implicated in INSTI resistance. Reverse transcription initiates carefully monitor patients to avoid initiating PrEP during with minus-strand DNA synthesis via a co-packaged host unrecognized acute HIV-1 infection. CAB in cell culture is mar- tRNALys3 primer that engages the primer-binding site near the ginally less effective at inhibiting infection by certain INSTI- 5Ј end of the viral RNA genome (see Ref. 4 for review). Synthesis resistant viruses than is either DTG or BIC (160), and both DTG of the plus-strand of retroviral DNA is primed via an oligonu- (161) and BIC (162) have been formulated as LA compounds. cleotide derived from the 3Ј polypurine (PPT) tract. In the RNA Future research will evaluate the efficacy of LA INSTIs to treat genome, the 3Ј PPT abuts the U3 sequence that will form the at risk patients with PrEP as well as PLHIV.

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ALLINIs integration (192, 198), an effect that was exacerbated signifi- Despite relatively high barriers, second-generation INSTIs cantly by RNAi-mediated knockdown of LEDGF/p75 expres- do select for resistance (149, 159, 163). As exemplified by the sion (199). Such observations highlighted the antiviral potential clinical successes of the NRTIs and NNRTIs, it would accord- of small molecules designed to inhibit the interaction between ingly be highly beneficial to have additional drug classes that HIV-1 IN and LEDGF/p75. Although inhibition of IN-LEDGF/ inhibit IN activity through novel mechanisms of action. p75 binding was initially espoused as the antiviral mechanism Whereas a number of different types of IN-targeting mole- of action (167), there has since been little evidence to suggest cules have been described in the literature, the class of com- that inhibition of the protein–protein interaction significantly pounds collectively known as ALLINIs has advanced the contributes to ALLINI potency. furthest. Predecessor compounds of potent ALLINIs were dis- ALLINI compounds are built around heterocyclic cores, covered via two different means, including a high-throughput such as pyridine (171, 200), thiophene (201), quinoline (165, screen for inhibitors of IN 3Ј processing activity (164–166) and 167, 168, 170, 197, 202–204), isoquinoline (205), thienopyri- dine (167, 206)(Fig. 4B), or naphthyridine (207); additional che- structure-guided modeling of the amino acid contacts that motypes have been described in the patent literature (reviewed mediate the interaction of HIV-1 IN with the host integration in Ref. 208). Potent compounds contain a 2-carbon arm harbor- targeting cofactor lens epithelium-derived growth factor ing t-butoxy and carboxylic acid that is commonly connected to (LEDGF)/p75 (167). In addition to ALLINI (168), such com- the ring two positions from the heteroatom (Fig. 4B; also see pounds have been referred to as LEDGIN for LEDGF-interac- Fig. 6, B and C). As espoused in the initial LEDGIN paper (167), tion site (167), NCINI for noncatalytic site IN inhibitor (165, the binding modes of these compounds to HIV-1 IN in large 169), IN-LAI for IN-LEDGF allosteric inhibitor (170), and Downloaded from part mimic LEDGF/p75 binding. Co-crystal structures with the MINI for multimeric IN inhibitor (171). HIV-1 IN CCD dimer revealed that ALLINI carboxylic acids Although HIV-1 in large part integrates throughout the mimic the carboxylate side chain of LEDGF/p75 residue Asp- human genome (46), it does so in nonrandom fashion, on aver- 366 by making similar contacts with the backbone amides of IN age favoring active genes that reside within relatively gene- residues Glu-170 and His-171 (Fig. 6, cyan IN monomer). The dense regions of chromosomes (172). This targeting preference http://www.jbc.org/ t-butoxy moiety additionally interacts with IN residue Thr-174 is largely dictated through specific interactions of two PIC-as- from this same monomer. ALLINI central rings mimic LEDGF/ sociated proteins, IN and capsid, with respective host factors p75 residue Ile-365 by occupying a hydrophobic pocket com- LEDGF/p75 and cleavage and polyadenylation specificity factor posed of residues from both IN monomers. The benzimidazole 6(173, 174) (for a recent review, see Ref. 50). LEDGF/p75 is a moiety in pyridine ALLINI KF116 additionally interacts with chromosome-associated (59, 175, 176) transcriptional co-acti- IN residue Thr-125 of the green IN monomer (Fig. 6C). Similar by guest on October 11, 2019 vator (177) that harbors two globular domains, an N-terminal binding modes can explain why quinoline ALLINIs effectively PWWP (for Pro-Trp-Trp-Pro) chromatin reader with affinity inhibited the LEDGF/p75-IN interaction in vitro (167, 170, 202, for histone 3 Lys-36 trimethylation (178–180), and a down- 204, 206). By contrast, pyridine (171) and isoquinoline (205) stream region that was termed the IN-binding domain (IBD) ALLINIs are comparatively weak inhibitors of the virus–host because it mediated the binding of LEDGF/p75 to HIV-1 IN in interaction. vitro (181). The LEDGF/p75 IBD is a PHAT domain (for pseu- ALLINIs can inhibit HIV-1 IN 3Ј processing and strand do-HEAT repeat analogous topology) composed of two helix- transfer activities in vitro in a LEDGF/p75-independent man- hairpin-helix HEAT repeats (182)(Fig. 6A, left). The IN CCD ner (168, 170, 202, 205, 206). Accordingly, quinoline ALLINIs dimerizes via an extensive interface with DDE catalytic triads inhibited HIV-1 infection at the integration step when the com- positioned at distal apices (183). LEDGF/p75 hotspot interac- pounds were added to susceptible target cells at the time of tion residues Ile-365, Asp-366, and Phe-406 within the IBD virus infection (12, 167, 170, 202). However, inhibition of inte- hairpins engage both IN monomers at the CCD dimer interface gration is arguably a side effect of ALLINI antiviral potency (13, (182, 184). Whereas Asp-366 hydrogen-bonds with the back- 169, 171), as the compounds display much greater potencies bone amides of residues Glu-170 and His-171 within one IN during the late phase of HIV-1 replication (12, 169–171, 204, monomer, Ile-365 occupies a hydrophobic pocket composed of 209, 210). Retroviral RNP complexes appear electron-dense in IN residues from both IN monomers (Fig. 6A, right). Electro- negatively stained thin sections due to the comparative inability positive residues within IBD ␣1 make additional contacts with of electrons to pass through these structures. The underlying electronegative residues within the HIV IN NTD (185). HIV-1 basis for ALLINI antiviral activity is IN hypermultimerization IN subunits undergo dynamic exchange in solution (61), and (12, 169–171, 204, 209–214), which inhibits IN binding to LEDGF/p75 binding accordingly stabilized HIV-1 IN dimers RNA in virions (63) and elicits the formation of eccentric HIV-1 and tetramers (40, 61, 186, 187) and significantly stimulated IN particles with viral RNP complexes situated outside of compar- catalytic activities in vitro (40, 59, 61, 185, 188). The LEDGF/ atively electron-translucent and often deformed capsid shells p75-IN interaction is specific to the lentivirus genus of Retro- (12, 13, 169–171, 201, 209, 210, 212, 215). The morphology viridae (189–191). defect is reminiscent of what is seen via a variety of mutations in Cellular depletion of LEDGF/p75 predominantly limits the IN region of HIV-1 pol that yield so-called class II IN HIV-1 infection by reducing the level of integrated viral DNA mutant viruses (216) (reviewed in Refs. 217 and 218). Such (192–197). Expression of fusion proteins in susceptible target eccentric viral particles are noninfectious due to their inability cells comprised of GFP and the LEDGF/p75 IBD also inhibited to promote reverse transcription in target cells (12, 13, 169–

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Figure 6. ALLINI mimicry of LEDGF/p75 binding to the HIV-1 IN CCD dimer. A, solution structure of the LEDGF/p75 IBD (left; PDB code 1Z9E) and IBD-CCD co-crystal structure (right; PDB code 2B4J) highlight the locations of hotspot-interacting residues Ile-365 and Asp-366 in the hairpin that connects ␣-helices 1 and 2 and Phe-406 in the ␣4-␣5 hairpin (left). Whereas Asp-366 interacts with the backbone amide groups of IN residues Glu-170 and His-171 of the cyan IN monomer (dashed lines), Ile-365 occupies a hydrophobic pocket composed of IN residues from each monomer (i.e. Trp-132 of the green IN monomer and Met-178 of the cyan monomer; right). Other colorings denote atoms of interacting amino acid residues: nitrogen (blue), sulfur (yellow), and oxygen (red). B, quinoline ALLINI BI 224436 (left, chemical diagram) bound to the IN CCD dimer (right, PDB code 6NUJ). The interactions between the compound carboxylic acid and backbone amides within the IN cyan monomer are analogous to those shown in A for LEDGF/p75 residue Asp-366. Thr-174 of the IN cyan monomer additionally interacts with the t-butoxy moiety of the drug. Other labeling is the same as in A. C, binding of pyridine ALLINI KF116 (left, chemical structure) to the IN CCD dimer (right, PDB code 4O55). This view, rotated down ϳ90° from A and B, is shown to accentuate the drug-binding pocket. In addition to the contacts described in B, Thr-125 of the green IN monomer interacts with the benzimidazole moiety of KF116. Other labeling is as defined in A and B. 171, 209, 217, 218). Both IN and viral RNA are rapidly degraded ing genes linked in some cases to cellular proliferation (223– after cell entry, likely due to their exposure to the cell cytoplasm 225) (reviewed in Ref. 226). Because LEDGF/p75 depletion outside the confines of the protective capsid shell (214, 219). results in global shifts of HIV-1 integration sites toward gene 5Ј ALLINI potency during the late phase of HIV-1 replication is end regions (173, 195, 227), treating patients with quinoline independent of cellular LEDGF/p75 content (12, 169, 220). ALLINIs could cause unwanted side effects from promoter- Remarkably, LEDGF/p75 depletion significantly increased the proximal integration if growth promotion was sufficiently up- potency at which the quinoline ALLINI BI-D inhibited HIV-1 regulated to seed tumorigenesis. However, preliminary work in integration (12, 197). Instead of being the antiviral target, this area has revealed that HIV-1 proviruses formed in the engagement of IN by LEDGF/p75 during the early phase of absence of LEDGF/p75 are transcriptionally repressed. The use HIV-1 infection protects the virus from the inhibitory action of of HIV-1 reporter vectors that express two different fluoro- the compounds (221). Accordingly, quinoline and thienopyri- phores—one from the LTR and another from a constitutively dine ALLINIs can significantly diminish the extent of HIV-1 active internal promoter—indicated that LEDGF/p75 deple- integration into genes in LEDGF/p75-expressing cells (171, tion or treatment with thienopyridine ALLINI CX014442 spe- 204, 221, 222). cifically decreased LTR activity and accordingly increased the Cells that comprise the latent HIV-1 reservoir show variable proportion of latent proviruses that form during the early phase growth characteristics, with integrations near growth-promot- of HIV-1 infection in cell culture (222). Moreover, such provi-

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ruses were refractory to transcriptional activation by latency necessitated by such approaches will likely require chimeric reversal agents (LRAs) (222). Such observations have prompted SHIV strains that carry HIV-1 IN (232). the notion that quinoline or thienopyridine ALLINIs could be The role of IN in HIV-1 particle morphogenesis is an ongoing used as part of PrEP regimens to limit the size of the latent viral area of investigation. IN, as a free protein or as part of Gag-Pol, reservoir that forms during the acute phase of HIV-1 infection could nucleate the formation of the capsid shell around the (228). Curiously, LEDGF/p75 can repress the transcription of RNP (13). Disruption of IN–RNA binding (63) and/or IN–IN established HIV-1 proviruses (229). The mechanistic connec- dynamics through mutations or ALLINIs would then yield non- tion between the transcriptional competency of newly formed infectious particles with eccentric electron density. Such mod- HIV-1 proviruses and IN-LEDGF/p75 binding is currently els invoke IN as a molecule tether or communicator between unclear. It should be informative to fine map the positions of the RNP and the capsid; although IN can directly interact with these proviruses as well as their responses to LRA treatment. different components of the RNP, including RT (233–237) and Both an X-ray crystal structure (212) and molecular model- genomic RNA (63), interactions with the virus capsid protein ing (213, 214) have yielded clues as to the nature of ALLINI- have not been reported. ALLINIs are important compounds induced IN hypermultimerization. Quinoline ALLINIs bound with which to further probe the role of IN in HIV-1 particle at the LEDGF/p75-binding pocket of an IN dimer engaged the morphogenesis. CTD of a separate IN dimer, thereby templating the polymeri- zation of IN dimers through successive interdimeric CTD- HIV-1 resistance to ALLINI compounds ALLINI-CCD bridge contacts (212, 213). Whereas quinoline Resistance against quinoline and thienopyridine ALLINIs is ALLINIs similarly hypermultimerized IN dimers and tetram- readily selected in cell culture, with most changes mapping to Downloaded from ers, the pyridine ALLINI KF116 specifically multimerized IN IN residues in the vicinity of the LEDGF/p75-binding pocket tetramers (214). Modeling revealed that hypermultimerization (165, 167, 201–203, 212). Whereas some of these, such as Thr- in this case occurred through successive CTD-ALLINI-CCD 174, are invariant among circulating HIV-1 strains, others, such bridge contacts whose formation specifically required IN as Ala-124 and Ala-125, are highly polymorphic in nature tetramers (214). KF116 potency tracked hand-in-hand with HIV-1 IN tetramerization, indicating that the tetramer is the (238–241) (reviewed in Ref. 242). Because Asn is often found at http://www.jbc.org/ predominant form of IN in HIV-1 virions (214). position 124 and A124D conferred significant resistance to the Cell-free virions are recalcitrant to ALLINI treatment, quinoline ALLINI BI-D (165), recent studies have tested anti- revealing that inhibition requires HIV-1 exposure to ALLINIs viral activities of compounds against HIV-1 strains harboring in the confines of virus-producing cells (12, 169, 170). Thien- representative 124/125 polymorphisms, such as Thr/Thr, Thr/

opyridine ALLINI CX05045 enhanced the multimerization of Ala, Ala/Thr, Ala/Ala, Asn/Thr, and Asn/Ala (200, 207, 215). by guest on October 11, 2019 ϳ purified HIV-1 Pol protein in vitro, indicating that the antiviral Whereas Asn-124 and Ala-125 each conferred 50-fold resis- target during HIV-1 infection could be Gag-Pol (209). Genetic tance to pyridine ALLINI compound 20 (200) and thiophene experiments, however, revealed that this need not be the case. ALLINI MUT-A (215), respectively, naphthyridine ALLINI Although usually incorporated into HIV-1 particles via Gag- compound 23 remained active against such strains (207). Coun- Pol, IN can be supplied in trans as a fusion protein with the terscreening against representative polymorphic strains such as accessory protein Vpr (230). HIV-1 harboring IN expressed these, as well as viruses containing changes such as A128T and solely from Vpr-IN remained fully sensitive to ALLINI inhibi- T174I that are commonly selected during virus passage (165, tion, revealing that Gag-Pol need not be the initial site of 167, 171, 201–203, 212), is important to the ongoing develop- ALLINI engagement (12, 13). ALLINI-mediated multimeriza- ment of the ALLINI drug class. tion of Pol protein in vitro minimally suggests that the CCD Similar to INSTI resistance (35, 38), some changes in IN that dimer interface within IN is present in Pol. Additional work is confer ALLINI resistance, such as T174I, alter a direct binding required to ascertain whether ALLINIs may first gain access to contact (Fig. 6, B and C), whereas others work via less direct IN via engaging Gag-Pol under normal infection conditions. mechanisms. The A128T change in IN conferred significant Conceivably, initial engagement via Gag-Pol or IN could resistance to the hypermultimerization activities of quinoline depend on the type of compound, as the pyridine ALLINI ALLINIs without affecting the binding of LEDGF/p75 to the KF116 specifically targeted tetrameric IN (214), and it is mutant IN protein (211). IN CCD co-crystal structures revealed unknown whether IN could tetramerize in the context of that the bulky Thr substituent shifted the position of ALLINI Gag-Pol. binding, lowering its propensity to hypermultimerize the Similar to the NNRTIs, which engage an allosteric binding mutant IN (211). On the flip side, thiophene ALLINI MUT-A pocket on RT (reviewed in Ref. 231), ALLINIs are specific for efficiently inhibited LEDGF/p75 binding to Ala-125– HIV-1 and do not inhibit closely related primate lentiviruses containing IN yet in large part lost the ability to hypermultim- such as HIV-2 or simian immunodeficiency virus from rhesus erize this polymorphic variant. In this case, downstream inter- macaques (167, 201, 209). Eccentric HIV-1 particles produced actions of the CCD-engaged ALLINI with the CTD of another by exposure to the thiophene ALLINI MUT-A displayed IN oligomer seemed to underlie the loss of hypermultimeriza- immunoreactivity characteristics similar to mock-treated viri- tion activity (215). Thus, resistance to ALLINIs can be instilled ons, indicating a potential novel avenue for chemically inacti- by loss of direct IN binding contact, a shift in compound posi- vated immunogens as vaccine candidates (201). Given the spec- tion within the CCD-binding pocket, or inability of bound com- ificity of ALLINIs for HIV-1 IN, intensive safety evaluations pound to mediate downstream interactions.

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Development of resistance to the pyridine ALLINI KF116 protein as the key mediator of HIV-1 PIC nuclear import (258) required successive changes in IN that initiated with T124N, (reviewed in Ref. 3), and numerous follow-up studies have followed by T174I, and culminated with T124N/V165I/T174I questioned purported roles for IN nuclear localization signals (171). Whereas recombinant T124N virus was, as expected, (259–262) and/or IN–host factor interactions (263–267)in similarly infectious as the WT, the infectivity of T124N/T174I HIV-1 nuclear import. Compounds that inhibited IN binding was reduced almost 1,000-fold, and T124N/T174I virions failed to TNPO3 (268) or KPNA2/KPNB1 (269) in vitro displayed to mature due to processing defects in both Gag and Gag-Pol comparatively weak antiviral activities of less than 50% inhibi- precursor proteins (243). These data highlight how a single tion at 100 ␮M and 50% inhibition at ϳ50–100 ␮M, respectively. amino acid substitution in IN can exert catastrophic conse- To establish IN-KPN interactions as bona fide antiviral targets, quences on the late stages of HIV-1 replication. Remarkably, it will be important to show that resistance to compounds with the added V165I change restored polyprotein processing and 10–100-fold greater potencies maps to the IN region of HIV-1 boosted infectivity about 120-fold, to 17% of the level of the WT pol. (243). Because resistance required the virus to pass through a Novel tert-butylsulfonamide (t-BSF) compound 1 inhibited nearly noninfectious mutational bottleneck, KF116 harbors a the late phase of HIV-1 infection 6-fold more potently than the higher genetic barrier to resistance compared with predecessor early phase, indicating bona fide ALLINI activity (270). quinoline ALLINIs (171, 243). Assessment of genetic resistance Whereas the T174I substitution in the LEDGF/p75 IBD bind- barrier is another important consideration in the development ing pocket conferred about 500-fold resistance to a control of clinical ALLINI compounds. quinoline ALLINI compound, the mutant virus was 5-fold Antiretroviral compounds display class-specific slopes in more sensitive to inhibition by t-BSF ALLINI compound 1, Downloaded from their dose–response curves. For example, whereas INSTIs and indicating engagement of the IN CCD dimer at a location other NRTIs display slopes close to 1, NNRTIs and PIs display steeper than the LEDGF/p75-binding pocket (270). The binding of a slopes of ϳ1.7 and from 1.8 to 4.5, respectively (244). Slope is predecessor ALLINI compound, which inhibited IN subunit related to the Hill coefficient, which is a measure of the intra- exchange in solution, mapped to the CCD dimer interface adja- molecular cooperativity of ligand binding to a multivalent cent to the LEDGF/p75-binding pocket (271). These studies receptor. Although ϳ120 molecules of IN enter a susceptible established that regions of the CCD dimer interface outside of http://www.jbc.org/ target cell, only the two that engage the ends of HIV-1 DNA the LEDGF/p75-binding pocket are potential targets for novel within the confines of a single intasome are sensitive to INSTI ALLINI development. Structural determination of the IN action. Likewise, bystander RT molecules that are not actively region within Pol or the tetrameric form of IN within virions engaged in DNA synthesis are unseen by NRTI compounds. (214) may reveal additional IN-IN interfaces for the develop-

The limited number of enzyme–substrate targets per replica- ment of novel ALLINI compounds. by guest on October 11, 2019 tion cycle accounts for the comparatively shallow slopes of INSTI and NRTI dose–response curves (244). By contrast, Conclusions NNRTIs and PIs target apoenzymes. Reasons for steeper dose– IN inhibitors have come a long way since the early days when response curve slopes in these cases include the requirement to some of the most notable individuals in the field felt the goal of inhibit the full complement of viral enzymes, which in this clinical IN inhibition was unattainable. The impressive poten- sense likens the enzyme population to a multivalent receptor cies and resistance barriers of second-generation INSTIs have (244), or the inhibition of multiple steps within the viral repli- prompted worldwide rollouts, although safety profiles for preg- cation cycle (245). ALLINIs like PIs display comparatively steep nant women at the time of conception require careful monitor- dose–response curve slopes (m Х 4) (168, 203). Because eccen- ing and comprehensive follow-up. The assessment of second- tric HIV-1 particles made in the presence of ALLINIs are defec- generation INSTI-containing dual therapy regimens is ongoing tive for reverse transcription, the compounds potently inhibit for both oral administration for PLHIV and LA formulations minimally two steps of the viral replication cycle, which could for PrEP. The odds-on bet is that second-generation INSTIs account for the steep slopes. Determining ALLINI-to-IN stoi- will be a mainstay part of cART formulations for the foreseeable chiometries required to inhibit HIV-1 replication would be future. expected to further inform the cooperative nature of drug The elucidation of retroviral intasome structures by X-ray action. crystallography and single-particle cryo-EM over the past dec- ade has provided unprecedented insight into the mechanism of Other IN functionalities retroviral integration. The initial PFV intasome structures IN has been proposed to play additional roles in the HIV-1 additionally provided important insight into the mechanisms of lifecycle, including virus particle uncoating after cell entry (246) INSTI action, and ongoing cryo-EM work with HIV-1 and and PIC nuclear import (247, 248). IN accordingly has been related primate lentiviral intasomes is expected to further shown to interact with numerous karyopherin (KPN) nuclear advance our understanding of drug action and drug resistance transport receptors, including KPNA2/importin ␣1(247, 249– mechanisms. Such high-resolution structures should critically 251), KNPB1/importin ␤1(249, 251), KPNB2/transportin-1 inform the future development of these drugs to deal with resis- (TNPO1) (249), KPNA4/importin ␣3(252, 253), importin 7 tance mutations prevalent from prior failure to first-generation (249, 254), Ran-binding protein (RANBP) 9/importin 9 (255), INSTI-containing regimens as well as de novo resistance that RANBP4/importin 4 (256), and TNPO3 (257). Genetic map- will inevitably arise from global second-generation INSTI ping experiments at the same time have highlighted the capsid rollouts.

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J. Biol. Chem. (2019) 294(41) 15137–15157 15157 Multifaceted HIV integrase functionalities and therapeutic strategies for their inhibition Alan N. Engelman J. Biol. Chem. 2019, 294:15137-15157. doi: 10.1074/jbc.REV119.006901 originally published online August 29, 2019

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