The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu

Background

The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial.

Results

Here, we studied the nuclear import pathway of IN by using multiple in vivo and in vitro systems. Nuclear import was not observed in an importin α temperature-sensitive yeast mutant, indicating an importin α-mediated process. Direct interaction between the full-length IN and importin α was demonstrated in vivo using bimolecular fluorescence complementation assay (BiFC). Nuclear import studies in yeast cells, with permeabilized mammalian cells, or microinjected cultured mammalian cells strongly suggest that the IN bears a NLS domain located between residues 161 and 173. A peptide bearing this sequence -NLS-IN peptide- inhibited nuclear accumulation of IN in transfected cell-cycle arrested cells. Integration of viral cDNA as well as HIV-1 replication in viral cell-cycle arrested infected cells were blocked by the NLS-IN peptide.

Conclusion

Our present findings support the view that nuclear import of IN occurs via the importin α pathway and is promoted by a specific NLS domain. This import could be blocked by NLS-IN peptide, resulting in inhibition of viral infection, confirming the view that nuclear import of the viral pre-integration complex is mediated by viral IN.     

Characterization of antibodies elicited by XMRV infection and development of immunoassays useful for epidemiologic studies

Background

An essential event during the replication cycle of HIV-1 is the integration of the reverse transcribed viral DNA into the host cellular genome. Our former report revealed that HIV-1 integrase (IN), the enzyme that catalyzes the integration reaction, is positively regulated by acetylation mediated by the histone acetyltransferase (HAT) p300.

Results

In this study we demonstrate that another cellular HAT, GCN5, acetylates IN leading to enhanced 3'-end processing and strand transfer activities. GCN5 participates in the integration step of HIV-1 replication cycle as demonstrated by the reduced infectivity, due to inefficient provirus formation, in GCN5 knockdown cells. Within the C-terminal domain of IN, four lysines (K258, K264, K266, and K273) are targeted by GCN5 acetylation, three of which (K264, K266, and K273) are also modified by p300. Replication analysis of HIV-1 clones carrying substitutions at the IN lysines acetylated by both GCN5 and p300, or exclusively by GCN5, demonstrated that these residues are required for efficient viral integration. In addition, a comparative analysis of the replication efficiencies of the IN triple- and quadruple-mutant viruses revealed that even though the lysines targeted by both GCN5 and p300 are required for efficient virus integration, the residue exclusively modified by GCN5 (K258) does not affect this process.

Conclusions

The results presented here further demonstrate the relevance of IN post-translational modification by acetylation, which results from the catalytic activities of multiple HATs during the viral replication cycle. Finally, this study contributes to clarifying the recent debate raised on the role of IN acetylated lysines during HIV-1 infection.     

The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

Background

Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.

Results

Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.

Conclusions

Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP³⁵RIW motif of N-terminal Vpr.     

A cooperative and specific DNA-binding mode of HIV-1 integrase depends on the nature of the metallic cofactor and involves the zinc-containing N-terminal domain

HIV-1 integrase catalyzes the insertion of the viral genome into chromosomal DNA. We characterized the structural determinants of the 3′-processing reaction specificity—the first reaction of the integration process—at the DNA-binding level. We found that the integrase N-terminal domain, containing a pseudo zinc-finger motif, plays a key role, at least indirectly, in the formation of specific integrase–DNA contacts. This motif mediates a cooperative DNA binding of integrase that occurs only with the cognate/viral DNA sequence and the physiologically relevant Mg²⁺ cofactor. The DNA-binding was essentially non-cooperative with Mn²⁺ or using non-specific/random sequences, regardless of the metallic cofactor. 2,2′-Dithiobisbenzamide-1 induced zinc ejection from integrase by covalently targeting the zinc-finger motif, and significantly decreased the Hill coefficient of the Mg²⁺-mediated integrase–DNA interaction, without affecting the overall affinity. Concomitantly, 2,2′-dithiobisbenzamide-1 severely impaired 3′-processing (IC₅₀ = 11–15 nM), suggesting that zinc ejection primarily perturbs the nature of the active integrase oligomer. A less specific and weaker catalytic effect of 2,2′-dithiobisbenzamide-1 is mediated by Cys 56 in the catalytic core and, notably, accounts for the weaker inhibition of the non-cooperative Mn²⁺-dependent 3′-processing. Our data show that the cooperative DNA-binding mode is strongly related to the sequence-specific DNA-binding, and depends on the simultaneous presence of the Mg²⁺ cofactor and the zinc effector.Integration of HIV-1 DNA into the host genome ensures stable maintenance of the viral genome in the host organism and, therefore, is a key process in the virus life cycle. Integrase (IN) is responsible for two distinct, consecutive catalytic steps in the integration process (1). The first of these two reactions is 3′-processing, which corresponds to the specific cleavage of two nucleotides from the 3′-ends of the linear viral DNA. The hydroxyl groups of newly recessed 3′-ends are then used in the second reaction— strand transfer—for the covalent joining of viral and cellular (or target) DNAs, resulting in full-site integration. For both reactions, IN functions as a multimer, most likely a dimer for 3′-processing and a tetramer (dimer of a dimer) for concerted integration (2–7). Two other reactions occur in vitro, a disintegration reaction that represents, in first approximation, the reversal of the half-site integration process (8) and a specific internal cleavage occurring on a symmetrical DNA site (9). All reactions require a metallic cofactor, Mg²⁺ or Mn²⁺, and, except for disintegration (10,11), all reactions require the full-length IN. There are several experimental evidences to suggest that Mg²⁺ is more physiologically relevant as a cofactor, particularly because Mg²⁺-dependent catalysis exhibits weaker non-specific endonucleolytic cleavage and the tolerance of sequence variation at the ends of the viral DNA is much greater in the presence of Mn²⁺ than in the presence of Mg²⁺ (12–15).The emergence of viral strains resistant against available drugs and the dynamic nature of the HIV-1 genome support a continued effort towards the discovery and characterization of novel targets and anti-viral drugs. Due to its central role in the HIV-1 life cycle, IN represents a promising therapeutic target. In the past, in vitro IN assays were extensively used to find IN inhibitors (16). Current inhibitors can be separated into two main classes, depending on their mechanisms of action: (i) Compounds that competitively prevent the DNA binding of IN to the viral DNA. These compounds are mainly directed against the 3′-processing reaction as they bind to the donor site within the catalytic site—i.e. the ‘specific’ DNA-binding site for the viral DNA -. This group is referred to as ‘integrase DNA-binding inhibitors’ (INBI) and includes styrylquinoline compounds (17,18). (ii) The second class includes compounds that cannot bind to the DNA-free IN. They bind to the pre-formed IN–viral DNA complex. These compounds preferentially inhibit strand transfer over the 3′-processing reaction [this family of compounds is referred to as ‘integrase strand transfer inhibitors’ (INSTI)], probably by displacing the viral DNA end from the active site (7,19–21). It is not clear whether this mechanism alone accounts for the inhibitory properties of INSTIs or whether these compounds also prevent the binding of the target DNA to the acceptor site—i.e. the ‘non-specific’ DNA binding site. INSTI compounds have generally good ex vivo activity against HIV replication, probably due to their ability to inhibit pre-assembled viral DNA/IN complexes. Raltegravir which is currently used in clinical treatment of HIV-1 belongs to this class. For both anti-IN classes, resistance mutations were identified (17,20,22,23). Difficulties in deeply understanding their mechanisms of action are closely related to the absence of structural data that clearly delineate the donor and the acceptor DNA binding sites in the active site. Although structural information is now available regarding the IN–viral DNA interaction, based on the recent crystal structure of the full-length primate foamy virus (PFV-1) IN in complex with its cognate processed viral DNA, the target DNA binding mode and the precise location of the acceptor site remains open to debate (7). Moreover, it is a difficult task to experimentally discriminate between the two DNA binding sites and no significant or only modest difference can be evidenced in vitro (depending on the method used for monitoring IN–DNA interactions) between the HIV-1 IN binding to the cognate viral DNA sequence and a non-specific random sequence in terms of overall affinity, suggesting that the specific and the non-specific DNA-binding modes display similar binding free energies (5,24). The basis of DNA binding specificity remains essentially unknown.HIV-1 IN (288 amino acids) contains three functional domains. The central domain or catalytic core domain (IN^50–213 or CC) contains the catalytic triad (DDE) that coordinates one or two metallic cofactors [probably a pair coordinated by three carboxylate groups of the triad, based on the X-ray structure of the PFV-1 IN (7)] and is essential for enzymatic activity; this domain alone can perform the disintegration reaction (10,11). This domain is flanked by the N-terminal (IN^1-49) and the C-terminal (IN^214–288) domains. The C-terminal domain is involved in IN–DNA contacts, together with the CC domain (25,26). The N-terminal domain contains a conserved non-conventional HHCC motif that binds zinc to ensure proper domain folding and promotes IN multimerization (27–29). It is worth noting that the integrity of the HHCC motif is crucial in the stringent Mg²⁺-context but appears dispensable under the less stringent Mn²⁺ condition (30), suggesting, at least, an indirect role of the zinc-binding domain in the establishment of specific and physiologically relevant IN–DNA complexes. In the structure of the PFV-1 IN–viral DNA complex, the N-terminal domain is also involved in the interaction with DNA (7).In this article, we found that IN binds cooperatively to the cognate viral DNA sequence only in the presence of Mg²⁺. The presence of Mn²⁺ or, most importantly, the use of non-specific random sequences, regardless of the metallic cofactor, dramatically reduced the Hill coefficient. This finding suggests that the cooperative DNA-binding mode of IN is strongly related to the formation of specific IN–DNA contacts. To gain deeper insight into the role of the zinc-binding domain in the cooperative/multimerization process, in relationship with the establishment of specific protein–DNA contacts, we studied the effect of DIBA-1 (2,2′-dithiobisbenzamide-1) (Figure 1A) on IN activity. This compound is a zinc ejector affecting many proteins containing zinc fingers, including HIV-1 nucleocapsid or estrogen receptor (31–34). Here, we found that DIBA-1 induced zinc ejection from the IN N-terminal domain by covalently targeting the HHCC motif. In the presence of Mg²⁺, DIBA-1 did not affect significantly the overall affinity of IN for the DNA substrate but dramatically reduced the Hill coefficient. Concomitantly, DIBA-1 strongly inhibited the catalytic step, with IC₅₀ values against the 3′-processing reaction of 11–15 nM. Interestingly, we found a secondary DIBA-1 binding site in the catalytic core (involving residue Cys 56), suggesting a second mechanism of action of DIBA-1, independent of zinc ejection. The prevalence of the two distinct mechanisms was dependent on the cofactor context, with the second one accounting for the weaker DIBA-1 inhibitory effect under Mn²⁺ conditions (IC₅₀ = 115–126 nM). DIBA-1 behaves as a non-competitive/catalytic inhibitor that did not disturb the fractional saturation of DNA sites, regardless of the mechanism considered. Open in a separate windowFigure 1.DIBA-1 induces the ejection of zinc from IN. (A) Structure of 2,2′-dithiobisbenzamide-1 (DIBA-1) (MW, 614 Da). (B) Ejection of zinc was measured by optical absorbance at 495 nm using a sample containing full-length IN (1 µM) and PAR (10⁻⁴ M) in a Tris buffer (20 mM pH 7.0) containing 15% DMSO (v/v) in the absence of reducing agent (black squares) or in the presence of 4 mM β-mercaptoethanol (white circles). The concentration of DIBA-1 for complete zinc release—[DIBA-1]_eq—was estimated graphically. This value was determined as a function of the initial concentration of IN (C). The slope of the straight line indicates that the zinc ejection coincides with the reaction of two DIBA-1 molecules per IN protomer.Altogether, our results show that, although it is a difficult task to discriminate between the specific viral sequence and a non-specific random sequence in terms of overall affinity, these sequences lead to distinct DNA-binding properties in terms of cooperativity. Moreover, our results highlight that the Mg²⁺-dependent catalytic activity of IN is strongly sensitive to the loss of cooperative DNA binding. Such a cooperative DNA-binding mode accounts for specific activity and requires: (i) the cognate viral DNA sequence, (ii) Mg²⁺ as a catalytic cofactor and (iii) zinc which can be considered as a positive allosteric effector. Development of non-competitive compounds acting on the N-terminal domain may be of interest for anti-IN pharmacology.     

Multimerization and DNA Binding Properties of INI1/hSNF5 and Its Functional Significance

INI1/hSNF5/BAF47/SMARCB1 is an HIV-1 integrase (IN)-binding protein that modulates viral replication in multiple ways. A minimal IN-binding domain of INI1, S6 (amino acids 183–294), transdominantly inhibits late events, and down-modulation of INI1 stimulates early events of HIV-1 replication. INI1 both stimulates and inhibits in vitro integration depending on IN concentration. To gain further insight into its role in HIV-1 replication, we purified and biochemically characterized INI1. We found that INI1 forms multimeric structures. Deletion analysis indicated that the Rpt1 and Rpt2 motifs form the minimal multimerization domain. We isolated mutants of INI1 that are defective for multimerization using a reverse yeast two-hybrid system. Our results revealed that INI1 residues involved in multimerization overlap with IN-binding and nuclear export domains and are required for nuclear retention and co-localization with IN. Multimerization-defective mutants are also defective for mediating the transdominant effect of INI1-S6-(183–294). Furthermore, we found that INI1 is a minor groove DNA-binding protein. Although IN binding and multimerization are required for INI1-mediated inhibition, the acceptor DNA binding property of INI1 may be required for stimulation of in vitro strand transfer activities of IN. Binding of INI1 to IN results in the formation of presumably inactive high molecular weight IN-INI1 complexes, and the multimerization-defective mutant was unable to form these complexes. These results indicate that the multimerization and IN binding properties of INI1 are necessary for its ability to both inhibit integration and influence assembly and particle production, providing insights into the mechanism of INI1-mediated effects in HIV-1 replication.HIV-1³ replication is a dynamic process that is modulated by the interaction of several host cellular proteins (1). A genome-wide siRNA-mediated knockdown indicated that hundreds of host factors are involved in the stimulation or inhibition of HIV-1 replication (2). Understanding the interplay between the host proteins and the HIV-1 viral proteins is essential to fully comprehend the dynamic relationship between the virus and the host.INI1/hSNF5/BAF47/SMARCB1 is a core component of the SWI/SNF chromatin-remodeling complex. It interacts directly with the HIV-1-encoded integrase (IN) required for the integration of the viral DNA into the host chromosome (3, 4). IN mediates the insertion of viral cDNA into host chromosomal DNA by sequential steps of 3′ processing and strand transfer (or joining) (4, 5). INI1 binds directly to HIV-1 IN in vitro and in vivo and modulates several steps of HIV-1 replication (3, 6–8). The ectopically expressed minimal IN-binding domain of INI1 transdominantly and potently inhibits HIV-1 assembly and particle production (8). The inhibitory effect is dependent on IN-INI1 interaction and is abrogated when an IN mutant defective for interaction with INI1 is used (8). Furthermore, particle production is minimal in cells lacking INI1, and reintroduction of INI1 into these cells can partially correct the defect (6). These results indicate that INI1 is required for HIV-1 late events. Additional studies have indicated that INI1 is selectively incorporated into HIV-1 but not other retroviral and lentiviral particles (9). Virally encapsidated INI1 is required for post-entry early events of HIV-1 replication prior to integration (6). These studies indicate that producer cell-associated as well as virion-associated INI1 is required for HIV-1 replication. Contrary to these proviral functions of INI1, siRNA-mediated knockdown studies indicate that INI1 in the target cells inhibits early events of HIV-1 replication (7). These studies indicate that whereas INI1 in the target cells may act as an antiviral host protein, HIV-1 may subvert the INI1 antiviral effect, and HIV-1 may utilize this host factor for late events in the producer cells and for early preintegration events in the target cells. Interestingly, in an earlier study, we demonstrated that partially purified INI1 both inhibits and stimulates in vitro integration in a manner dependent on IN concentration (3). Although INI1 stimulates in vitro strand transfer reactions at low IN concentrations, it inhibits the reaction at high concentrations (3). Further structure-function analysis of INI1 is required to understand this complex and dual role of INI1 during HIV-1 replication.INI1 gene is also a tumor suppressor that is biallelically deleted in aggressive pediatric cancers known as rhabdoid tumors (10). INI1 mutations have been found in other soft tissue cancers (11–13). The mechanism of INI1-mediated tumor suppression is not fully understood. INI1 protein has two highly conserved domains that are imperfect direct repeats (termed Rpt1 and Rpt2) of each other and a third conserved coiled coil domain (termed homology region 3 or HR3) at the C terminus. The Rpt1 and Rpt2 domains appear to be involved in protein-protein interaction with various cellular and viral proteins (3, 14–18). Additionally, the Rpt2 domain harbors a masked nuclear export signal, and the C-terminal domain is involved in inhibiting the nuclear export of the protein in the steady state. INI1 exhibits nonspecific DNA binding activity (18). The cancer-associated mutations occur throughout the open reading frame of the INI1 gene, suggesting that mutation in any one of the INI1 domains may inactivate the protein and that multiple domains are required for its function (19–21).To gain further insight into the mechanism of its action, we purified INI1 protein to homogeneity and characterized it biochemically. Here we report, for the first time, that INI1 forms dimeric and higher order multimeric structures. We have characterized the multimerization domain of INI1 and found that multimerization and IN binding activities of INI1 are required for inhibition of in vitro integration. Furthermore, we found that the multimerization, IN binding, and nuclear export properties of INI1 are important for transdominant effects. In addition, we found that INI1 possesses a minor groove DNA binding activity and that the nonspecific acceptor DNA binding activity of INI1 may be required for stimulation of in vitro integration. Finally, we found that multimerization of the full-length protein is necessary for its ability to be retained in the nucleus and to co-localize with HIV-1 IN in the nucleus. Thus, our studies provide novel insights into the mechanism by which INI1 regulates HIV-1 replication.     

Structural determinants for HIV-1 integrase inhibition by beta-diketo acids.

Among all the HIV-1 integrase inhibitors, the beta-diketo acids (DKAs) represent a major lead in anti-HIV-1 integrase drug design. These derivatives inhibit the integration reaction in vitro with a strong specificity for the 3'-end joining step. They are also antiviral and inhibit integration in vivo. The aim of the present study has been to investigate the molecular interactions between DKAs and HIV-1 integrase. We have compared 5CITEP with one of the most potent DKAs reported by the Merck group (L-708,906) and found that 5CITEP inhibits 3'-processing at concentrations where L-708,906 is only active on strand transfer. We also report a novel bifunctional DKA derivative that inhibits 3'-processing even more effectively than 5CITEP. The interactions of these inhibitors with the viral DNA donor ends have been studied by performing experiments with oligonucleotides containing defined modifications. We propose that the bifunctional DKA derivative binds to both the acceptor and donor sites of HIV-1 integrase, whereas the monofunctional L-708,906 derivative binds selectively to the acceptor site.     

The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains

Background

Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear.

Results

Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues ⁷⁵GCRHSRIGVTRQRRAR⁹⁰, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr^75-90 R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA.

Conclusions

For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the identified N- and C-terminal binding domains of the protein to CypA.     

The contribution of peroxynitrite generation in HIV replication in human primary macrophages

Background

The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN) (IN inhibitors, IINs) has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp) thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA) derivatives active on the HIV-1 IN strand transfer (ST) step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR) reversing ability.

Results

The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells.

Conclusion

To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.     

Multi-stage Friend murine erythroleukemia: molecular insights into oncogenic cooperation

Background

In order to determine whether human prostate can be productively infected by HIV-1 strains with different tropism, and thus represent a potential source of HIV in semen, an organotypic culture of prostate from men undergoing prostatic adenomectomy for benign prostate hypertrophy (BPH) was developed. The presence of potential HIV target cells in prostate tissues was investigated using immunohistochemistry. The infection of prostate explants following exposures with HIV-1 R5, R5X4 and X4 strains was analyzed through the measure of RT activity in culture supernatants, the quantification of HIV DNA in the explants and the detection of HIV RNA+ cells in situ.

Results

The overall prostate characteristics were retained for 2^1/2 weeks in culture. Numerous potential HIV-1 target cells were detected in the prostate stroma. Whilst HIV-1 R5_SF162 strain consistently productively infected prostatic T lymphocytes and macrophages, the prototypic X4_IIIB strain and a primary R5X4 strain showed less efficient replication in this organ.

Conclusion

The BPH prostate is a site of HIV-1 R5 replication that could contribute virus to semen. A limited spreading of HIV-1 X4 and R5X4 in this organ could participate to the preferential sexual transmission of HIV-1 R5 strains.     

Specific and Independent Recognition of U3 and U5 att Sites by Human Immunodeficiency Virus Type 1 Integrase In Vivo

The retroviral attachment (att) sites at viral DNA ends are cis-acting regions essential for proviral integration. To investigate the sequence features of att important for human immunodeficiency virus type 1 (HIV-1) integration in vivo, we generated a series of 25 att mutants of HIV-1 by mutagenesis of the U3, U5, or both boundaries of att. Our results indicated that the terminal 11 or 12 bp of viral DNA are sufficient for specific recognition by HIV-1 integrase (IN) and suggested that IN might recognize each att site independently in vivo.     

Defining the DNA Substrate Binding Sites on HIV-1 Integrase

A tetramer model for human immunodeficiency virus type 1 (HIV-1) integrase (IN) with DNA representing long terminal repeat (LTR) termini was previously assembled to predict the IN residues that interact with the LTR termini; these predictions were experimentally verified for nine amino acid residues [Chen, A., Weber, I. T., Harrison, R. W. & Leis, J. (2006). Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends. J. Biol. Chem., 281, 4173-4182]. In a similar strategy, the unique amino acids found in avian sarcoma virus IN, rather than HIV-1 or Mason-Pfizer monkey virus IN, were substituted into the structurally related positions of HIV-1 IN. Substitutions of six additional residues (Q44, L68, E69, D229, S230, and D253) showed changes in the 3′ processing specificity of the enzyme, verifying their predicted interaction with the LTR DNA. The newly identified residues extend interactions along a 16-bp length of the LTR termini and are consistent with known LTR DNA/HIV-1 IN cross-links. The tetramer model for HIV-1 IN with LTR termini was modified to include two IN binding domains for lens-epithelium-derived growth factor/p75. The target DNA was predicted to bind in a surface trench perpendicular to the plane of the LTR DNA binding sites of HIV-1 IN and extending alongside lens-epithelium-derived growth factor. This hypothesis is supported by the in vitro activity phenotype of HIV-1 IN mutant, with a K219S substitution showing loss in strand transfer activity while maintaining 3′ processing on an HIV-1 substrate. Mutations at seven other residues reported in the literature have the same phenotype, and all eight residues align along the length of the putative target DNA binding trench.     

The (52-96) C-terminal domain of Vpr stimulates HIV-1 IN-mediated homologous strand transfer of mini-viral DNA

Viral integrase (IN) and Vpr are both components of the human immunodeficiency virus type 1 (HIV-1) pre-integration complex. To investigate whether these proteins interact within this complex, we investigated the effects of Vpr and its subdomains on IN activity in vitro. When a 21mer oligonucleotide was used as a donor and acceptor, both Vpr and its C-terminal DNA-binding domain [(52–96)Vpr] inhibited the integration reaction, whereas the (1–51)Vpr domain did not affect IN activity. Steady-state fluorescence anisotropy showed that both full-length and (52–96)Vpr bind to the short oligonucleotide, thereby extending previous observations with long DNA. The concentrations of the two proteins required to inhibit IN activity were consistent with their affinities for the oligonucleotide. The use of a 492 bp mini-viral substrate confirmed that Vpr can inhibit the IN-mediated reaction. However, the activity of (52–96)Vpr differed notably since it stimulated specifically integration events involving two homologous mini-viral DNAs. Order of addition experiments indicated that the stimulation was maximal when IN, (50–96)Vpr and the mini-viral DNA were allowed to form a complex. Furthermore, in the presence of (50–96)Vpr, the binding of IN to the mini-viral DNA was dramatically enhanced. Taken together, these data suggest that (52–96)Vpr stimulates the formation of a specific complex between IN and the mini-viral DNA.     

Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

Background

Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1.

Results

A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank^GAG1D4 (16.5 kDa) was isolated. Ank^GAG1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K _d ~ 1 ??M, and the Ank^GAG1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank^GAG1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank^GAG1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank^GAG1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank^GAG1D4-CA with the Gag assembly and budding pathway.

Conclusions

The resistance of Ank^GAG1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank^GAG1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules.     

Intracardiac injection of a capsid-modified Ad5/35 results in decreased heart toxicity when compared to standard Ad5

Background

Human immunodeficiency virus type 1 (HIV-1) Nef-encoded protein plays key functions at almost all stages of the viral life cycle, but its role in translation is largely unknown.

Methods

To determine the effect of Nef on translation we used an in vitro translation assay. The detection of Nef/RPS10 complexes and the presence of 18S rRNA and tRNAs in the complexes were performed by coimmunoprecipitation and RT-PCR assay.

Results

We observed that the HIV-1 Nef protein specifically impaired translation in vitro. We observed the interaction of Nef with RPS10 by coimmunoprecipitation assay. In addition 18S rRNA and tRNAs were present in the Nef/RPS10 complexes.

Conclusions

Our results are consistent with a model in which the Nef protein by binding to two components of the 40S small ribosomal subunit, RPS10 and 18S rRNA, and to a lesser extent to tRNAs, could lead to decreased protein synthesis.     

HIV-1 Accessory Protein Vpr: Relevance in the pathogenesis of HIV and potential for therapeutic intervention

Background

Non-neutralising antibodies to the envelope glycoprotein are elicited during acute HIV-1 infection and are abundant throughout the course of disease progression. Although these antibodies appear to have negligible effects on HIV-1 infection when assayed in standard neutralisation assays, they have the potential to exert either inhibitory or enhancing effects through interactions with complement and/or Fc receptors. Here we report that non-neutralising antibodies produced early in response to HIV-1 infection can enhance viral infectivity.

Results

We investigated this complement-mediated antibody-dependent enhancement (C'-ADE) of early HIV infection by carrying out longitudinal studies with primary viruses and autologous sera derived sequentially from recently infected individuals, using a T cell line naturally expressing the complement receptor 2 (CR2; CD21). The C'-ADE was consistently observed and in some cases achieved infection-enhancing levels of greater than 350-fold, converting a low-level infection to a highly destructive one. C'-ADE activity declined as a neutralising response to the early virus emerged, but later virus isolates that had escaped the neutralising response demonstrated an increased capacity for enhanced infection by autologous antibodies. Moreover, sera with autologous enhancing activity were capable of C'ADE of heterologous viral isolates, suggesting the targeting of conserved epitopes on the envelope glycoprotein. Ectopic expression of CR2 on cell lines expressing HIV-1 receptors was sufficient to render them sensitive to C'ADE.

Conclusions

Taken together, these results suggest that non-neutralising antibodies to the HIV-1 envelope that arise during acute infection are not 'passive', but in concert with complement and complement receptors may have consequences for HIV-1 dissemination and pathogenesis.     

A laser pointer driven microheater for precise local heating and conditional gene regulation in vivo. Microheater driven gene regulation in zebrafish

Background

Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys) WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro.

Results

We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required.

Conclusion

Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.