Study on the inhibitory mechanism and binding mode of the hydroxycoumarin compound NSC158393 to HIV‐1 integrase by molecular modeling

Human immunodeficiency virus type 1 integrase (IN) is an essential enzyme in the life cycle of this virus and also an important target for the study of anti‐HIV drugs. In this work, the binding modes of the wild type IN core domain and the two mutants, that is, W132G and C130S, with the 4‐hydroxycoumarin compound NSC158393 were evaluated by using the “relaxed complex” molecular docking approach combined with molecular dynamics (MD) simulations. Based on the monomer MD simulations, both of the two substitutions affect not only the stability of the 128–136 peptides, but also the flexibility of the functional 140s loop. In principle, NSC158393 binds the 128–136 peptides of IN; however, the specific binding modes for the three systems are various. According to the binding mode of NSC158393 with WT, NSC158393 can effectively interfere with the stability of the IN dimer by causing a steric hindrance around the monomer interface. Additionally, through the comparative analysis of the MD trajectories of the wild type IN and the IN‐NSC158393 complex, we found that NSC15893 may also exert its inhibitory function by diminishing the mobility of the function loop of IN. Three key binding residues, that is, W131, K136, and G134, were discovered by energy decomposition calculated with the Molecular Mechanics Generalized Born Surface Area method. Characterized by the largest binding affinity, W131 is likely to be indispensable for the ligand binding. All the above results are consistent with experiment data, providing us some helpful information for understanding the mechanism of the coumarin‐based inhibitors. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 700–709, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

A structural model of the HIV-1 Rev-integrase complex: the molecular basis of integrase regulation by Rev

The HIV-1 Rev and integrase (IN) proteins control important functions in the viral life cycle. We have recently discovered that the interaction between these proteins results in inhibition of IN enzymatic activity. Peptides derived from the Rev and IN binding interfaces have a profound effect on IN catalytic activity: Peptides derived from Rev inhibit IN, while peptides derived from IN stimulate IN activity by inhibiting the Rev-IN interaction. This inhibition leads to multi integration, genomic instability and specific death of virus-infected cells. Here we used protein docking combined with refinement and energy function ranking to suggest a structural model for the Rev-IN complex. Our results indicate that a Rev monomer binds IN at two sites that match our experimental binding data: (1) IN residues 66-80 and 118-128; (2) IN residues 174-188. According to our model, IN binds Rev and its cellular cofactor, lens epithelium derived growth factor (LEDGF), through overlapping interfaces. This supports previous observations that IN is regulated by a tight interplay between Rev and LEDGF. Rev may bind either the IN dimer or tetramer. Accordingly, Rev is suggested to inhibit IN by two possible mechanisms: (i) shifting the oligomerization equilibrium of IN from an active dimer to an inactive tetramer; (ii) displacing LEDGF from IN, resulting in inhibition of IN binding to the viral DNA. Our model is expected to contribute to the development of lead compounds that inhibit the Rev-IN interaction and thus lead to multi-integration of viral cDNA and consequently to apoptosis of HIV-1 infected cells.     

Interaction between HIV-1 Rev and integrase proteins: a basis for the development of anti-HIV peptides

Human immunodeficiency virus 1 (HIV-1) Rev and integrase (IN) proteins are required within the nuclei of infected cells in the late and early phases of the viral replication cycle, respectively. Here we show using various biochemical methods, that these two proteins interact with each other in vitro and in vivo. Peptide mapping and fluorescence anisotropy showed that IN binds residues 1-30 and 49-74 of Rev. Following this observation, we identified two short Rev-derived peptides that inhibit the 3'-end processing and strand-transfer enzymatic activities of IN in vitro. The peptides bound IN in vitro, penetrated into cultured cells, and significantly inhibited HIV-1 in multinuclear activation of a galactosidase indicator (MAGI) and lymphoid cultured cells. Real time PCR analysis revealed that the inhibition of HIV-1 multiplication is due to inhibition of the catalytic activity of the viral IN. The present work describes novel anti-HIV-1 lead peptides that inhibit viral replication in cultured cells by blocking DNA integration in vivo.     

Peptide inhibitors of HIV-1 integrase: From mechanistic studies to improved lead compounds

The HIV-1 integrase enzyme (IN) catalyzes integration of viral DNA into the host genome. We previously developed peptides that inhibit IN in vitro and HIV-1 replication in cells. Here we present the design, synthesis and evaluation of several derivatives of one of these inhibitory peptides, the 20-mer IN1. The peptide corresponding to the N-terminal half of IN1 (IN1 1–10) was easier to synthesize and much more soluble than the 20-mer IN1. IN1 1–10 bound IN with improved affinity and inhibited IN activity as well as HIV replication and integration in infected cells. While IN1 bound the IN tetramer, its shorter derivatives bound dimeric IN. Mapping the peptide binding sites in IN provided a model that explains this difference. We conclude that IN1 1–10 is an improved lead compound for further development of IN inhibitors.     

Molecular dissection of an inhibitor targeting the HIV integrase dependent preintegration complex nuclear import

Human immunodeficiency virus (HIV) continues to be a major contributor to morbidity and mortality worldwide, particularly in developing nations where high cost and logistical issues severely limit the use of current HIV therapeutics. This, combined HIV's high propensity to develop resistance, means that new antiviral agents against novel targets are still urgently required. We previously identified novel anti‐HIV agents directed against the nuclear import of the HIV integrase (IN) protein, which plays critical roles in the HIV lifecycle inside the cell nucleus, as well as in transporting the HIV preintegration complex (PIC) into the nucleus. Here we investigate the structure activity relationship of a series of these compounds for the first time, including a newly identified anti‐IN compound, budesonide, showing that the extent of binding to the IN core domain correlates directly with the ability of the compound to inhibit IN nuclear transport in a permeabilised cell system. Importantly, compounds that inhibited the nuclear transport of IN were found to significantly decrease HIV viral replication, even in a dividing cell system. Significantly, budesonide or its analogue flunisolide, were able to effect a significant reduction in the presence of specific nuclear forms of the HIV DNA (2‐LTR circles), suggesting that the inhibitors work though blocking IN, and potentially PIC, nuclear import. The work presented here represents a platform for further development of these specific inhibitors of HIV replication with therapeutic and prophylactic potential.     

Identification and mapping of inhibitory sequences in the human immunodeficiency virus type 2 vif gene.

Human immunodeficiency virus (HIV) regulates the expression of its genes temporally at the mRNA processing step. A subset of the mRNA species which encode the structural and some accessory genes contains inhibitory sequences (INS or CRS elements) which prevent nuclear export of the RNA or its utilization in the cytoplasm. Such inhibition is overridden by the interaction of a viral protein, Rev, with its RNA target sequence, RRE. The vif gene product, which is essential for virus replication in vivo, is encoded by a singly spliced mRNA, and its expression is dependent on rev in infected cells. However, INS elements have not been found in the HIV-1 vif gene itself, although such elements have been observed in Gag, Pol, and Env coding sequences. We have now identified an INS within the 5' half of HIV-2 vif which does not show any homology with cellular mRNAs or other previously identified INS and CRS elements of HIV. These results suggest that retroviral mRNAs have novel labile sequences different from those of cellular mRNAs.     

Viral structural transition mechanisms revealed by multiscale molecular dynamics/order parameter extrapolation simulation

On the basis of an all‐atom multiscale analysis theory of nanosystem dynamics, a multiscale molecular dynamics/order parameter extrapolation (MD/OPX) approach has recently been developed. It accelerates MD for long‐time simulation of large bionanosystems and addresses rapid atomistic fluctuations and slowly varying coherent dynamics simultaneously. In this study, MD/OPX is optimized and implemented to simulate viral capsid structural transitions. Specifically, 200 ns MD/OPX simulation of the swollen state of cowpea chlorotic mottle virus capsid reveals that it undergoes significant energy‐driven shrinkage in vacuum, which is a symmetry‐breaking process involving local initiation and front propagation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 61–73, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

HIV-1 integrase is capable of targeting DNA to the nucleus via an importin alpha/beta-dependent mechanism

In addition to its well-documented role in integration of the viral genome, the HIV-1 enzyme IN (integrase) is thought to be involved in the preceding step of importing the viral cDNA into the nucleus. The ability of HIV to transport its cDNA through an intact nuclear envelope allows HIV-1 to infect non-dividing cells, which is thought to be crucial for the persistent nature of HIV/AIDS. Despite this, the mechanism utilized by HIV-1 to import its cDNA into the nucleus, and the viral proteins involved, remains ill-defined. In the present study we utilize in vitro techniques to assess the nuclear import properties of the IN protein, and show that IN interacts with members of the Imp (Importin) family of nuclear transport proteins with high affinity and exhibits rapid nuclear accumulation within an in vitro assay, indicating that IN possesses potent nucleophilic potential. IN nuclear import appears to be dependent on the Imp alpha/beta heterodimer and Ran GTP (Ran in its GTP-bound state), but does not require ATP. Importantly, we show that IN is capable of binding DNA and facilitating its import into the nucleus of semi-intact cells via a process that involves basic residues within amino acids 186-188 of IN. These results confirm IN as an efficient mediator of DNA nuclear import in vitro and imply the potential for IN to fulfil such a role in vivo. These results may not only aid in highlighting potential therapeutic targets for impeding the progression of HIV/AIDS, but may also be relevant for non-viral gene delivery.     

Catalytic antibodies from HIV-infected patients specifically hydrolyzing viral integrase suppress the enzyme catalytic activities

Human immunodeficiency virus type 1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. It was shown previously that IN preincubation with various oligodeoxynucleotides (ODNs) induces formation of dimers and oligomers of different gyration radii; only specific ODNs stimulate the formation of catalytically active dimers. Here we have shown that preincubation of IN with specific and nonspecific ODNs leads to a significant and comparable decrease in its hydrolysis by chymotrypsin, while nonspecific ODNs protect the enzyme from the hydrolysis by trypsin worse than specific ODNs; all ODNs had little effect on the IN hydrolysis by proteinase K. In contrast to canonical proteweases, IgGs from HIV‐infected patients specifically hydrolyze only IN. While d(pT)_n markedly decreased the IgG‐dependent hydrolysis of IN, d(pA)_n and d(pA)_n?d(pT)_n demonstrated no detectable protective effect. The best protection from the hydrolysis by IgGs was observed for specific single‐ and especially double‐stranded ODNs. Although IN was considerably protected by specific ODNs, proteolytic IgGs and IgMs significantly suppressed both 3′‐processing and integration reaction catalyzed by IN. Since anti‐IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. Copyright © 2011 John Wiley & Sons, Ltd.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The cell cycle independence of HIV infections is not determined by known karyophilic viral elements

Human immunodeficiency virus and other lentiviruses infect cells independent of cell cycle progression, but gammaretroviruses, such as the murine leukemia virus (MLV) require passage of cells through mitosis. This property is thought to be important for the ability of HIV to infect resting CD4+ T cells and terminally differentiated macrophages. Multiple and independent redundant nuclear localization signals encoded by HIV have been hypothesized to facilitate migration of viral genomes into the nucleus. The integrase (IN) protein of HIV is one of the HIV elements that targets to the nucleus; however, its role in nuclear entry of virus genomes has been difficult to describe because mutations in IN are pleiotropic. To investigate the importance of the HIV IN protein for infection of non-dividing cells, and to investigate whether or not IN was redundant with other viral signals for cell cycle-independent nuclear entry, we constructed an HIV-based chimeric virus in which the entire IN protein of HIV was replaced by that of MLV. This chimeric virus with a heterologous IN was infectious at a low level, and was able to integrate in an IN-dependent manner. Furthermore, this virus infected non-dividing cells as well as it infected dividing cells. Moreover, we used the chimeric HIV with MLV IN to further eliminate all of the other described nuclear localization signals from an HIV genome--matrix, IN, Viral Protein R, and the central polypurine tract--and show that no combination of the virally encoded NLS is essential for the ability of HIV to infect non-dividing cells.     

Characterization of the minimal DNA-binding domain of the HIV integrase protein.

The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.     

Incorporation of functional human immunodeficiency virus type 1 integrase into virions independent of the Gag-Pol precursor protein.

Retroviral integrase (IN) is expressed and incorporated into virions as part of the Gag-Pol polyprotein precursor. IN catalyzes integration of the proviral DNA into host cell chromosomes during the early stages of the virus life cycle, and as a component of Gag-Pol, it is involved in virion morphogenesis during late stages. It is unknown whether the scheme, conserved among retroviruses, for expressing and incorporating IN as a component of the Gag-Pol precursor protein is necessary for its function in the infected cell after viral entry. We have developed human immunodeficiency virus (HIV) virion-associated accessory proteins (Vpr and Vpx) as vehicles to deliver both foreign and viral proteins into the virus particle by their expression in trans as heterologous fusion proteins (X. Wu, et al., J. Virol. 69:3389-3398, 1995; X. Wu, et al., J. Virol. 70:3378-3384, 1996; X. Wu, et al., EMBO J. 16:5113-5122, 1977). To analyze IN function independent of its expression as a part of Gag-Pol, we expressed and incorporated IN into HIV type 1 (HIV-1) virions in trans as a fusion partner of Vpr (Vpr-IN). Our results demonstrate that the Vpr-IN fusion protein is efficiently incorporated into virions and then processed by the viral protease to liberate the IN protein. Virus derived from IN-minus provirus is noninfectious. However, this defect is overcome by trans complementation with the Vpr-IN fusion protein. Moreover, complemented virions are able to replicate through a complete cycle of infection, including formation of the provirus (integration). These results show, for the first time, that full IN function can be provided in trans, independent of its expression and incorporation into virions as a component of Gag-Pol. This finding also indicates that the IN domain of Gag-Pol is not required for the formation of infectious virions when IN is provided in trans. The ability to incorporate functional IN into retroviral particles in trans will provide unique opportunities to explore the function of this critical enzyme in a biologically relevant context, i.e., in infected cells as part of the nucleoprotein/preintegration complex.     

Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

Background

Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE).

Results

MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export.

Conclusion

These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.