用分子模拟方法研究HIV-1整合酶突变体的耐药性机理

二酮酸类化合物(DKAs)是目前最有前景的HIV-1整合酶(integrase, IN)抑制剂．为了解DKAs引起的多种耐药株共有的耐药性机理,选择3种S-1360引起的IN耐药突变体,用分子对接和分子动力学模拟,研究了野生型和突变型IN与S-1360的结合模式,基于该结合模式探讨了3种耐药突变体所共有的耐药性机理．结果表明：在突变体中,S-1360结合到耐药突变IN核心区中的位置靠近功能loop 3区却远离与 DNA结合的关键残基,结合位置不同导致S-1360的抑制作用部分丧失;残基138到166区域的柔性对IN发挥生物学功能很重要,S-1360能与DNA结合的关键残基N155及K159形成氢键,这2个氢键作用降低了该区域的柔性,突变体中无类似氢键,因而该区域柔性增高;在突变体中,S-1360的苯环远离病毒DNA结合区,不能阻止病毒DNA末端暴露给宿主DNA;T66I突变导致残基Ⅰ的长侧链占据IN的活性口袋,阻止抑制剂以与野生型中相同的方式结合到活性中心,这均是产生抗药性的重要原因．这些模拟结果与实验结果吻合,可为抗IN的抑制剂设计和改造提供帮助．     

Efficiency and Fidelity of Full-Site Integration Reactions Using Recombinant Simian Immunodeficiency Virus Integrase

Full-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SIV) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a target substrate. Under optimized conditions, recombinant SIV IN produced donor-target products consistent with full-site (two donor ends) and half-site (one donor end) reactions with equivalent frequency. Restriction enzyme analysis of the 3.8-kbp full-site reaction products confirmed the concerted insertion of two termini from separate donors into a single target molecule. Donor ends carrying the viral U5 termini were preferred over U3 termini for producing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-target junctions of the cloned products were characterized by sequencing. Analysis of 149 recombinants demonstrated approximately 84% fidelity for the appropriate simian retrovirus 5-bp host duplication. As seen previously in similar reactions with human immunodeficiency virus type 1 (HIV-1) IN from lysed virions, approximately 8% of the donor-target recombinants generated with recombinant SIV IN incurred specific 17- to 18- or 27- to 29-bp deletions. The efficiency and fidelity of the full-site integration reaction mediated by the purified, recombinant SIV IN is comparable to that of HIV-1 IN from virions. These observations suggest that a purified recombinant lentivirus IN is itself sufficient to recapitulate the full-site integration process.     

Molecular Modeling for a Comparative Analysis of Interactions Between 2LTRZFP and 2-LTR-Circle Junctions

In eukaryotes, one of the most often noticed DNA-binding motifs are Cys2His2 zinc finger proteins which have been extensively utilized as a framework for designing novel DNA-binding proteins. In this study, the conformational change, and binding stability, affinity and interactions of the 2LTRZFP–HIV-1 2-LTR-circle junction complexes were successfully reproduced with MD simulations, MM-PBSA/GBSA and binding mode analyses. The binding free energies were obtained, and it was found that the calculated binding free energy of Zif1 is lower than Zif2 and together with pharmacophore modeling, these binding results indicate strong affinity of 2LTRZFP for the DNA. Mutants of 2LTRZFP were analyzed. The computed energies provided proof for a relationship between the binding free energy and total electrostatic interaction energy. The mutation in a key amino acid (Glu81Tyr) of the finger 3 in position 6 showed increased binding energy to the DNA. The result agrees with our previous study which showed that the HIV-1 IN may be hindered by the 2LTRZFP. The obtained results may assist in the design of ZFPs depended on the 2-LTR-circle junctions inhibition mechanism.

     

Design, synthesis, and anti-integrase activity of catechol-DKA hybrids

Following the discovery of diketoacid-containing compounds as HIV-1 integrase (IN) inhibitors, a plethora of new molecules have been published leading to four drugs under clinical trial. In an attempt to rationally design new dimeric diketoacids (DKAs) targeting two divalent metal ions on the active site of IN, potent inhibitors against purified IN were found with varied selectivity for strand transfer. In this context, we designed and synthesized a new series of catechol-DKA hybrids. These compounds presented micromolar anti-integrase activities with moderate antiviral properties.     

The TRIM family protein KAP1 inhibits HIV-1 integration

The integration of viral cDNA into the host genome is a critical step in the life cycle of HIV-1. This step is catalyzed by integrase (IN), a viral enzyme that is positively regulated by acetylation via the cellular histone acetyl transferase (HAT) p300. To investigate the relevance of IN acetylation, we searched for cellular proteins that selectively bind acetylated IN and identified KAP1, a protein belonging to the TRIM family of antiviral proteins. KAP1 binds acetylated IN and induces its deacetylation through the formation of a protein complex which includes the deacetylase HDAC1. Modulation of intracellular KAP1 levels in different cell types including T cells, the primary HIV-1 target, revealed that KAP1 curtails viral infectivity by selectively affecting HIV-1 integration. This study identifies KAP1 as a cellular factor restricting HIV-1 infection and underscores the relevance of IN acetylation as a crucial step in the viral infectious cycle.     

Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells

Retrovirus preintegration complexes (PIC) in virus-infected cells contain the linear viral DNA genome (approximately 10 kbp), viral proteins including integrase (IN), and cellular proteins. After transport of the PIC into the nucleus, IN catalyzes the concerted insertion of the two viral DNA ends into the host chromosome. This successful insertion process is termed "full-site integration." Reconstitution of nucleoprotein complexes using recombinant human immunodeficiency virus type 1 (HIV-1) IN and model viral DNA donor substrates (approximately 0.30 to 0.48 kbp in length) that are capable of catalyzing efficient full-site integration has proven difficult. Many of the products are half-site integration reactions where either IN inserts only one end of the viral donor substrate into a circular DNA target or into other donors. In this report, we have purified recombinant HIV-1 IN at pH 6.8 in the presence of MgSO4 that performed full-site integration nearly as efficiently as HIV-1 PIC. The size of the viral DNA substrate was significantly increased to 4.1 kbp, thus allowing for the number of viral DNA ends and the concentrations of IN in the reaction mixtures to be decreased by a factor of approximately 10. In a typical reaction at 37 degrees C, recombinant HIV-1 IN at 5 to 10 nM incorporated 30 to 40% of the input DNA donor into full-site integration products. The synthesis of full-site products continued up to approximately 2 h, comparable to incubation times used with HIV-1 PIC. Approximately 5% of the input donor was incorporated into the circular target producing half-site products with no significant quantities of other integration products produced. DNA sequence analysis of the viral DNA-target junctions derived from wild-type U3 and U5 coupled reactions showed an approximately 70% fidelity for the HIV-1 5-bp host site duplications. Recombinant HIV-1 IN successfully utilized a mutant U5 end containing additional nucleotide extensions for full-site integration demonstrating that IN worked properly under nonideal active substrate conditions. The fidelity of the 5-bp host site duplications was also high with these coupled mutant U5 and wild-type U3 donor ends. These studies suggest that recombinant HIV-1 IN is at least as capable as native IN in virus particles and approaching that observed with HIV-1 PIC for catalyzing full-site integration.     

Efficient concerted integration by recombinant human immunodeficiency virus type 1 integrase without cellular or viral cofactors

Replication of retroviruses requires integration of the linear viral DNA genome into the host chromosomes. Integration requires the viral integrase (IN), located in high-molecular-weight nucleoprotein complexes termed preintegration complexes (PIC). The PIC inserts the two viral DNA termini in a concerted manner into chromosomes in vivo as well as exogenous target DNA in vitro. We reconstituted nucleoprotein complexes capable of efficient concerted (full-site) integration using recombinant wild-type human immunodeficiency virus type I (HIV-1) IN with linear retrovirus-like donor DNA (480 bp). In addition, no cellular or viral protein cofactors are necessary for purified bacterial recombinant HIV-1 IN to mediate efficient full-site integration of two donor termini into supercoiled target DNA. At about 30 nM IN (20 min at 37 degrees C), approximately 15 and 8% of the input donor is incorporated into target DNA, producing half-site (insertion of one viral DNA end per target) and full-site integration products, respectively. Sequencing the donor-target junctions of full-site recombinants confirms that 5-bp host site duplications have occurred with a fidelity of about 70%, similar to the fidelity when using IN derived from nonionic detergent lysates of HIV-1 virions. A key factor allowing recombinant wild-type HIV-1 IN to mediate full-site integration appears to be the avoidance of high IN concentrations in its purification (about 125 microg/ml) and in the integration assay (<50 nM). The results show that recombinant HIV-1 IN may not be significantly defective for full-site integration. The findings further suggest that a high concentration or possibly aggregation of IN is detrimental to the assembly of correct nucleoprotein complexes for full-site integration.     

Comparative molecular dynamics simulations of HIV-1 integrase and the T66I/M154I mutant: binding modes and drug resistance to a diketo acid inhibitor

HIV-1 IN is an essential enzyme for viral replication and an interesting target for the design of new pharmaceuticals for use in multidrug therapy of AIDS. L-731,988 is one of the most active molecules of the class of beta-diketo acids. Individual and combined mutations of HIV-1 IN at residues T66, S153, and M154 confer important degrees of resistance to one or more inhibitors belonging to this class. In an effort to understand the molecular mechanism of the resistance of T66I/M154I IN to the inhibitor L-731,988 and its specific binding modes, we have carried out docking studies, explicit solvent MD simulations, and binding free energy calculations. The inhibitor was docked against different protein conformations chosen from prior MD trajectories, resulting in 2 major orientations within the active site. MD simulations have been carried out for the T66I/M154I DM IN, DM IN in complex with L-731,988 in 2 different orientations, and 1QS4 IN in complex with L-731,988. The results of these simulations show a similar dynamical behavior between T66I/M154I IN alone and in complex with L-731,988, while significant differences are observed in the mobility of the IN catalytic loop (residues 138-149). Water molecules bridging the inhibitor to residues from the active site have been identified, and residue Gln62 has been found to play an important role in the interactions between the inhibitor and the protein. This work provides information about the binding modes of L-731,988, as well as insight into the mechanism of inhibitor-resistance in HIV-1 integrase.     

Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.

The integration of linear retrovirus DNA by the viral integrase (IN) into the host chromosome occurs by a concerted mechanism (full-site reaction). IN purified from avian myeloblastosis virus and using retrovirus-like DNA restriction fragments (487 bp in length) as donors and circular DNA (pGEM-3) as the target can efficiently catalyze that reaction. Nonionic detergent lysates of purified human immunodeficiency virus type 1 (HIV-1) virions were also capable of catalyzing the concerted integration reaction. The donor substrates were restriction fragments (469 bp) containing either U3-U5 (H-2 donor) or U5-U5 (H-5 donor) long terminal repeat sequences at their ends. As was shown previously with bacterially expressed HIV-1 IN, the U5 terminus of H-2 was preferred over the U3 terminus by virion-associated IN. The reactions involving two donors per circular target by HIV-1 IN preferred Mg2+ over Mn2+. Both metal ions were equally effective for the circular half-site reaction involving only one donor molecule. The linear 3.8-kbp recombinant products produced from two donor insertions into pGEM were genetically selected, and the donor-target junctions of individual recombinants were sequenced. A total of 55% of the 87 sequenced recombinants had host site duplications of between 5 and 7 bp, with the HIV-1 5-bp-specific duplication predominating. The other recombinants that migrated at the linear 3.8-kbp position were mainly small deletions that were grouped into four sets of 17, 27, 40, and 47 bp, each having a periodicity mimicking a turn of the DNA helix. Aprotic solvents (dimethyl sulfoxide and 1,4-dioxane) enhanced both the half-site and the linear 3.8-kbp strand transfer reactions which favored low-salt conditions (30 mM NaCl). The order of addition of the donor and target during preincubation with HIV-1 IN on ice did not affect the quantity of linear 3.8-kbp recombinants relative to that of the circular half-site products that were produced; only the quantity of donor-donor versus donor-target recombinants was affected. The presence of Mg2+ in the preincubation mixtures containing donor and target substrates was not necessary for the stability of preintegration complexes on ice or at 22 degrees C. Comparisons of the avian and HIV-1 concerted integration reactions are discussed.     

Azido-containing aryl beta-diketo acid HIV-1 integrase inhibitors

Aryl beta-diketo acids (ADK) comprise a general class of potent HIV-1 integrase (IN) inhibitors, which can exhibit selective inhibition of strand transfer reactions in extracellular recombinant IN assays and provide potent antiviral effects in HIV-infected cells. Recent studies have shown that polycyclic aryl or aryl rings bearing aryl-containing substituents are components of potent members of this class. Reported herein is the first use of azido functionality as an aryl replacement in beta-diketo acid IN inhibitors. The ability of azido-containing inhibitors to exhibit potent inhibition of IN and antiviral protection in HIV-infected cells, renders the azide group of potential value in the further development of ADK-based IN inhibitors.     

Contribution of host nucleoporin 62 in HIV-1 integrase chromatin association and viral DNA integration

HIV-1 integration is promoted by viral integrase (IN) and its cellular cofactors. The lens epithelium-derived growth factor (LEDGF/p75), an IN interacting cellular cofactor, has been shown to play an important role in HIV-1 chromatin targeting and integration. However, whether other cellular cofactors are also involved in viral replication steps is still elusive. Here, we show that nucleoporin 62 (Nup62) is a chromatin-bound protein and can specifically interact with HIV-1 IN in both soluble nuclear extract and chromatin-bound fractions. The knockdown of Nup62 by shRNA reduced the association of IN with host chromatin and significantly impaired viral integration and replication in HIV-1-susceptible cells. Furthermore, the expression of the IN-binding region of Nup62 in CD4(+) T cells significantly inhibited HIV-1 infection. Taken together, these results indicate that the cellular Nup62 is specifically recruited by HIV-1 IN and contribute to an efficient viral DNA integration.     

A quantum mechanic/molecular mechanic study of the wild-type and N155S mutant HIV-1 integrase complexed with diketo acid

Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg²⁺ cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg²⁺ cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure.     

New class of HIV-1 integrase (IN) inhibitors with a dual mode of action

tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction.     

HIV-1 capture and antigen presentation by dendritic cells: enhanced viral capture does not correlate with better T cell activation

During HIV-1 infection, dendritic cells (DC) facilitate dissemination of HIV-1 while trying to trigger adaptive antiviral immune responses. We examined whether increased HIV-1 capture in DC matured with LPS results in more efficient Ag presentation to HIV-1-specific CD4(+) and CD8(+) T cells. To block the DC-mediated trans-infection of HIV-1 and maximize Ag loading, we also evaluated a noninfectious integrase-deficient HIV-1 isolate, HIV(NL4-3ΔIN). We showed that higher viral capture of DC did not guarantee better Ag presentation or T cell activation. Greater HIV(NL4-3) uptake by fully LPS-matured DC resulted in higher viral transmission to target cells but poorer stimulation of HIV-1-specific CD4(+) and CD8(+) T cells. Conversely, maturation of DC with LPS during, but not before, viral loading enhanced both HLA-I and HLA-II HIV-1-derived Ag presentation. In contrast, DC maturation with the clinical-grade mixture consisting of IL-1β, TNF-α, IL-6, and PGE(2) during viral uptake only stimulated HIV-1-specific CD8(+) T cells. Hence, DC maturation state, activation stimulus, and time lag between DC maturation and Ag loading impact HIV-1 capture and virus Ag presentation. Our results demonstrate a dissociation between the capacity to capture HIV-1 and to present viral Ags. Integrase-deficient HIV(NL4-3ΔIN) was also efficiently captured and presented by DC through the HLA-I and HLA-II pathways but in the absence of viral dissemination. HIV(NL4-3ΔIN) seems to be an attractive candidate to be explored. These results provide new insights into DC biology and have implications in the optimization of DC-based immunotherapy against HIV-1 infection.