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PubMed2005

A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons.

A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins. We report here that while double-mutant UBR1(-/-) UBR2(-/-) mice die as early embryos, the rescued UBR1(-/-) UBR2(-/-) fibroblasts still retain the N-end rule pathway, albeit of lower activity than that of wild-type fibroblasts. An affinity assay for proteins that bind to destabilizing N-terminal residues has identified, in addition to UBR1 and UBR2, a huge (570 kDa) mouse protein, termed UBR4, and also the 300-kDa UBR5, a previously characterized mammalian E3 known as EDD/hHYD. UBR1, UBR2, UBR4, and UBR5 shared a approximately 70-amino-acid zinc finger-like domain termed the UBR box. The mammalian genome encodes at least seven UBR box-containing proteins, which we propose to call UBR1 to UBR7. UBR1(-/-) UBR2(-/-) fibroblasts that have been made deficient in UBR4 as well (through RNA interference) were significantly impaired in the degradation of N-end rule substrates such as the Sindbis virus RNA polymerase nsP4 (bearing N-terminal Tyr) and the human immunodeficiency virus type 1 integrase (bearing N-terminal Phe). Our results establish the UBR box family as a unique class of E3 proteins that recognize N-degrons or structurally related determinants for ubiquitin-dependent proteolysis and perhaps other processes as well.

Amino Acid Motifs↗
PubMed1999

Retroviral DNA integration.

DNA integration is a unique enzymatic process shared by all retroviruses and retrotransposons. During integration, double-stranded linear viral DNA is inserted into the host genome in a process catalyzed by the virus-encoded integrase (IN). The mechanism involves a series of nucleophilic attacks, the first of which removes the terminal 2 bases from the 3' ends of the long terminal repeats and of the second which inserts the viral DNA into the host genome. IN specifically recognizes the DNA sequences at the termini of the viral DNA, juxtaposing both ends in an enzyme complex that inserts the viral DNA into a single site in a concerted manner. Small duplications of the host DNA, characteristic of the viral IN, are found at the sites of insertion. At least two host proteins, HMG-I(Y) and BAF, have been shown to increase the efficiency of the integration reaction.

Avian Sarcoma Viruses↗
PubMed2004

Genomic comparison of archaeal conjugative plasmids from Sulfolobus.

All of the known self-transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence analysis with two earlier sequenced plasmids, pNOB8 and pING1. The analyses revealed three conserved and functionally distinct sections in the genomes. Section A is considered to encode the main components of the conjugative apparatus, where two genes show low but significant sequence similarity to sections of genes encoding bacterial conjugative proteins. A putative origin of replication is located in section B, which is highly conserved in sequence and contains several perfect and imperfect direct and inverted repeats. Further downstream, in section C, an operon encoding six to nine smaller proteins is implicated in the initiation and regulation of replication. Each plasmid carries an integrase gene of the type that does not partition on integration, and there is strong evidence for their integration into host chromosomes, where they may facilitate intercellular exchange of chromosomal genes. Two plasmids contain hexameric short regularly spaced repeats (SRSR), which have been implicated in plasmid maintenance, and each plasmid carries multiple recombination motifs, concentrated in the variable regions, which likely provide sites for genomic rearrangements.

Base Sequence↗
PubMed2002

Efficient transduction of primary human B lymphocytes and nondividing myeloma B cells with HIV-1-derived lentiviral vectors.

We studied the transduction of primary human B lymphocytes and myeloma cells with lentiviral vectors. In peripheral blood B cells that had been activated with helper T cells (murine thymoma EL-4 B5) and cytokines, multiply attenuated HIV-1-derived vectors pseudotyped with vesicular stomatitis virus (VSV) G-envelope protein achieved the expression of green fluorescence protein (GFP) in 27% +/- 12% (mean +/- 1 SD; median, 27%) of B cells in different experiments. When compared in parallel cultures, the transducibility of B cells from different donors exhibited little variation. The human cytomegalovirus (CMV) promoter gave 4- to 6-fold higher GFP expression than did the human elongation factor-1alpha promoter. A murine retroviral vector pseudotyped with VSV G protein proved inefficient even in mitotically active primary B cells. B cells freshly stimulated with Epstein-Barr virus were also transducible by HIV vectors (24% +/- 9%), but B cells activated with CD40 ligand and cytokines resisted transduction. Thus, different culture systems gave different results. Freshly isolated, nondividing myeloma cells were efficiently transduced by HIV vectors; for 6 myelomas the range was 14% to 77% (median, 28%) GFP(+) cells. HIV vectors with a mutant integrase led to no significant GFP signal in primary B or myeloma cells, suggesting that vector integration was required for high transduction. In conclusion, HIV vectors are promising tools for studies of gene functions in primary human B cells and myeloma cells for the purposes of research and the development of gene therapies.

B-Lymphocytes↗
PubMed2006

Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio.

BACKGROUND: Integrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be). Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such cassette arrays are widespread and can range from 30 kb to 150 kb. Besides those found in completely sequenced genomes, no such array has yet been recovered in its entirety. We describe an approach to systematically isolate, sequence and annotate large integron gene cassette arrays from bacterial strains. RESULTS: The complete Vibrio sp. DAT722 integron cassette array was determined through the streamlined approach described here. To place it in an evolutionary context, we compare the DAT722 array to known vibrio arrays and performed phylogenetic analyses for all of its components (integrase, 59-be sites, gene cassette encoded genes). It differs extensively in terms of genomic context as well as gene cassette content and organization. The phylogenetic tree of the 59-be sites collectively found in the Vibrio gene cassette pool suggests frequent transfer of cassettes within and between Vibrio species, with slower transfer rates between more phylogenetically distant relatives. We also identify multiple cases where non-integron chromosomal genes seem to have been assembled into gene cassettes and others where cassettes have been inserted into chromosomal locations outside integrons. CONCLUSION: Our systematic approach greatly facilitates the isolation and annotation of large integrons gene cassette arrays. Comparative analysis of the Vibrio sp. DAT722 integron obtained through this approach to those found in other vibrios confirms the role of this genetic element in promoting lateral gene transfer and suggests a high rate of gene gain/loss relative to most other loci on vibrio chromosomes. We identify a relationship between the phylogenetic distance separating two species and the rate at which they exchange gene cassettes, interactions between the non-mobile portion of bacterial genomes and the vibrio gene cassette pool as well as intragenomic translocation events of integrons in vibrios.

DNA, Bacterial↗
PubMed2006

Modes of transmission and genetic diversity of foamy viruses in a Macaca tonkeana colony.

BACKGROUND: Foamy viruses are exogenous complex retroviruses that are highly endemic in several animal species, including monkeys and apes, where they cause persistent infection. Simian foamy viral (SFV) infection has been reported in few persons occupationally exposed to non-human primates (NHP) in zoos, primate centers and laboratories, and recently in few hunters from central Africa. Most of the epidemiological works performed among NHP populations concern cross-sectional studies without long-term follow-up. Therefore, the exact timing and the modes of transmission of SFVs remain not well known, although sexual and oral transmissions have been suspected. We have conducted a longitudinal study in a free-breeding colony of Macaca tonkeana in order (1) to determine the prevalence of the infection by foamy viruses, (2) to characterize molecularly the viruses infecting such animals, (3) to study their genetic variability overtime by long-term follow-up of several DNA samples in a series of specific animals, and (4) to get new insights concerning the timing and the modes of SFVs primary infection in these monkeys by combining serology and molecular means, as well as studies of familial structures and long-term behavioral observations. RESULTS/CONCLUSION: We first demonstrated that this colony was highly endemic for SFVs, with a clear increase of seroprevalence with age. Only 4.7% of immatures, and 43,7% of sub-adults were found seropositive, while 89.5% of adults exhibited antibodies directed against SFV. We further showed that 6 different strains of foamy viruses (exhibiting a very low intra-strain and overtime genetic variability in the integrase gene) are circulating within this group. This suggests a possible infection by different strains within an animal. Lastly, we provide strong evidence that foamy viruses are mostly acquired through severe bites, mainly in sub-adults or young adults. Most cases of seroconversion occur after 7 years of age; from this age individuals competed for access to sexual partners, thus increasing the likelihood of being wounded. Furthermore, all the serological and molecular data, obtained in this free-breeding colony, argue against a significant transmission of SFVs from mother or father to infants as well as between siblings.

Animals↗
PubMed2006

Retroviral E-DNA: persistence and gene expression in nondividing immune cells.

Following retroviral infection of cells, not only is the proviral DNA integrated into the host genome, but there is also an accumulation of unintegrated extrachromosomal DNA (E-DNA), both linear and circular. Although the integrated DNA is responsible for the production of viral proteins and new viral progeny, the role of E-DNA has remained uncertain. Several reports have shown that E-DNA is transcriptionally active producing both RNA, as well as viral proteins and that circular E-DNA can persist in nondividing cells, raising questions regarding the potential consequences of this reservoir. Furthermore, integrase inhibitors, presently in clinical trials, shifts the balance of proviral DNA to the E-DNA form. This review is focused on recent work in this field with an emphasis on exploring the potential role of E-DNA in both pathogenesis of retroviral infections, especially HIV-1, and as a tool to deliver and express genes.

Animals↗
PubMed2005

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.

CD4-Positive T-Lymphocytes↗
PubMed2006

Retroviral DNA integration: viral and cellular determinants of target-site selection.

Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV) integrates preferentially within active transcription units, whereas murine leukemia virus (MLV) integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN) coding region into HIV (to make HIVmIN) caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN) further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag) displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I-hypersensitive sites (i.e., +/- 1 kb), and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins.

Attachment Sites, Microbiological↗
PubMed2003

The next generation of HIV/AIDS drugs: novel and developmental antiHIV drugs and targets.

There are presently 42 million people worldwide living with HIV/AIDS, the majority of which have limited access to antiretrovirals. Even if worldwide penetration was possible, our current chemotherapeutic strategies still suffer from issues of cost, patient compliance, deleterious acute and chronic side effects, emerging single and multidrug resistance, and generalized treatment and economic issues. Even our best antiretroviral therapeutic strategy, highly active antiretroviral therapy (HAART), falls short of completely suppressing HIV replication. Therefore, expansion of current therapeutic options by discovering new antiretrovirals and targets will be critical in the coming years. This review addresses the current status of reverse transcriptase and protease inhibitor development, and summarizes the progress in emerging classes of HIV inhibitors, including entry (T-20, T-1249), coreceptor (SCH-C, SCH-D), integrase (beta-Diketos) and p7 nucleocapsid Zn finger inhibitors (thioesters and PATEs). In addition, the processes of virus entry, PIC transport to the nucleus, HIV interaction with nuclear pores, Tat function, Rev function and virus budding (Tsg101 and ubiquitination) are examined, and proof of concept inhibitors and potential antiviral targets discussed.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Highly active antiretroviral therapy: current state of the art, new agents and their pharmacological interactions useful for improving therapeutic outcome.

Highly active antiretroviral therapy (HAART) dramatically changed the course of HIV infection. Currently, this therapy involves the use of agents from at least two distinct classes of antivirals: a protease inhibitor (PI) in combination with two nucleoside/nucleotide reverse transcriptase inhibitors (N(t)RTIs), or a non-nucleoside reverse transcriptase inhibitor (NNRTI) in combination with NRTIs. Recently, the third family of antivirals started to be used clinically, with the advent of enfuvirtide, the first fusion inhibitor (FI). Several pharmacological agents are available form these classes of antivirals, NRTIs, NNRTIs, PIs and FIs, which will be briefly reviewed here. Some more agents are in advanced clinical evaluation or have recently been approved (such as tenofovir, a NtRTI; atazanavir, a PI; tipranavir, another PI), mainly against drug-resistant viruses. Compounds inhibiting HIV integrase, the third enzyme of HIV, are also available ultimately, with several such derivatives in clinical trials (L-731, 988 and S-1360). Another approach to inhibit the growth of retroviruses, including HIV, targets the ejection of zinc ions from critical zinc finger viral proteins, which has as a consequence the inhibition of viral replication in the absence of mutations leading to drug resistance phenotypes. All steps in the process of HIV entry into the cell may be targeted by specific compounds that might be developed as novel types of antiretrovirals. Thus, inhibitors of the gp120-CD4 interaction have been detected (zintevir, FP-21399 and BMS-378806 in clinical trials). Small molecule chemokine antagonists acting as HIV entry inhibitors also were described in the last period, which interact both with the CXCR4 coreceptor (such as AMD3100; AMD3465; ALX40-4C; T22, T134 and T140), or which are antagonist of the CCR5 coreceptor (TAK-779, TAK-220, SCH-C, SCH-D, E913, AK-602 and NSC 651016 in clinical trials), together with new types of fusion inhibitors possessing the same mechanism of action as enfuvirtide (such as T1249). Compounds interacting with Tat/Tar have also been detected which inhibit HIV replication in low micromolar range (EM2487, tamacrazine, CGP 64222 or CGA 137053 among others). Unexploited viral and cellular targets (such as the maturation process-with a first potent compound available, PA-457; the cellular proteins Tsg101, APOBEC3G, or the viral ones Vif, Rev or RNase H) are also presented, together with recently emerged approaches for eradication of HIV reservoirs. A review on the pharmacology and interactions of these agents with other drugs is presented here, with emphasis on how these pharmacological interferences may improve the clinical use of antivirals, or how side effects due to these drugs may be managed better by taking them into account.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Anti-HIV therapy: Current and future directions.

Although combinations of drugs that target the HIV reverse transcriptase and protease enzymes have clearly revolutionized the treatment of HIV/AIDS, problems with these agents, such as viral escape mutants, persistence of viral reservoirs, poor patient compliance due to complicated regimens, and toxic side effects, have emphasized the need for development of new drugs with novel mechanisms of action, as well as an HIV vaccine. Recently two new classes of drugs have been identified that interfere with the membrane fusion reaction required for HIV entry of target cells. Two such agents, T-20 (enfuvirtide) and T-1249, which have been approved by the Food and Drug Administration (FDA), block the action of the fusogenic envelope glycoprotein gp41. Others target the HIV coreceptors CCR5 and CXCR4, and are now in clinical trials. Also under development are novel agents that target the HIV integrase and HIV regulatory gene products as well as immunomodulators such as IL-12 and IL-2. This article will focus on these and other novel approaches to HIV therapeutics.

Anti-HIV Agents↗
PubMed2004

Probability issues in molecular design: predictive and modeling ability in 3D-QSAR schemes.

In the current work we investigated 3D-QSAR data by the use of the coupled leave-several-out (LSO) and leave-one-out (LOO) cross-validation (CV) procedures. We verified the above mentioned scheme using both simulated data and real 3D QSAR data describing a series of CoMFA steroids, heterocyclic azo dyes and styrylquinoline HIV integrase inhibitors. Unlike in standard analyses, this technique characterizes individual method not by a single performance metrics but screens a whole possible modeling space by sampling different molecules into the training and test sets, respectively. This allowed us for the discussion of the information included in the estimators validating cross-validation procedures, as well as the comparison of the efficiency of several 3D QSAR schemes, in particular, Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Surface Analysis (CoMSA). Moreover, it allows one to acquire some general knowledge about predictive and modeling ability in 3D QSAR method.

Drug Design↗
PubMed2006

Comparative molecular surface analysis (CoMSA) for virtual combinatorial library screening of styrylquinoline HIV-1 blocking agents.

We used comparative molecular surface analysis to design molecules for the synthesis as part of the search for new HIV-1 integrase inhibitors. We analyzed the virtual combinatorial library (VCL) constituted from various moieties of styrylquinoline and styrylquinazoline inhibitors. Since imines can be applied in a strategy of dynamic combinatorial chemistry (DCC), we also tested similar compounds in which the -C=N- or -N=C- linker connected the heteroaromatic and aromatic moieties. We then used principal component analysis (PCA) or self-organizing maps (SOM), namely, the Kohonen neural networks to obtain a clustering plot analyzing the diversity of the VCL formed. Previously synthesized compounds of known activity, used as molecular probes, were projected onto this plot, which provided a set of promising virtual drugs. Moreover, we further modified the above mentioned VCL to include the single bond linker -C-N- or -N-C-. This allowed increasing compound stability but expanded also the diversity between the available molecular probes and virtual targets. The application of the CoMSA with SOM indicated important differences between such compounds and active molecular probes. We synthesized such compounds to verify the computational predictions.

Anti-HIV Agents↗
PubMed2003

Nuclear import of the pre-integration complex (PIC): the Achilles heel of HIV?

Current treatments against the Aquired immune deficiency syndrome (AIDS) are reasonably effective in reducing the amount of human immunodeficiency virus (HIV) present in infected patients, but their side-effects, and the emergence of drug-resistant HIV strains have intensified the renewed search for novel anti-HIV therapies. An essential step in HIV infection is the integration of the viral genome into the host cell chromosomes within the nucleus. Unlike other retroviruses, HIV can transport its genetic material, in the form of the large nucleoprotein pre-integration complex (PIC), into the nucleus through the intact nuclear envelope (NE). This enables HIV to infect non-dividing cells such as macrophages and microglial cells. Detailed knowledge of the signal-dependent pathways by which cellular proteins and RNAs cross the NE has accumulated in the past decade, but although several different components of the PIC have been implicated in its nuclear import, the mechanism of nuclear entry remains unclear. Since specifically inhibiting PIC nuclear import would undoubtedly block HIV infection in non-dividing cells, this critical step of HIV replication is of great interest as a drug target. This review examines the complex and controversial literature regarding three PIC components--the HIV proteins matrix, integrase and Vpr--proposed to facilitate PIC nuclear import, and existing models of HIV PIC nuclear import. It also suggests approaches to move towards a better understanding of PIC nuclear import, through examining the role of individual PIC components in the context of the intact PIC by direct visualisation, in order to develop new anti-HIV therapeutics.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Nonviral vectors for cancer gene therapy: prospects for integrating vectors and combination therapies.

Gene therapy has the potential to improve the clinical outcome of many cancers by transferring therapeutic genes into tumor cells or normal host tissue. Gene transfer into tumor cells or tumor-associated stroma is being employed to induce tumor cell death, stimulate anti-tumor immune response, inhibit angiogenesis, and control tumor cell growth. Viral vectors have been used to achieve this proof of principle in animal models and, in select cases, in human clinical trials. Nevertheless, there has been considerable interest in developing nonviral vectors for cancer gene therapy. Nonviral vectors are simpler, more amenable to large-scale manufacture, and potentially safer for clinical use. Nonviral vectors were once limited by low gene transfer efficiency and transient or steadily declining gene expression. However, recent improvements in plasmid-based vectors and delivery methods are showing promise in circumventing these obstacles. This article reviews the current status of nonviral cancer gene therapy, with an emphasis on combination strategies, long-term gene transfer using transposons and bacteriophage integrases, and future directions.

Bacteriophages↗
PubMed2004

[Recent progress in anti-HIM-1 research].

Establishment of highly active antiretroviral therapy (HAART) has brought about dramatic improvement of the prognosis of HIV-1 infection. On the other hand, several drawbacks associated with long-term HARRT have been demonstrated. Among them, emergence of drug-resistant viruses is a serious problem; therefore compounds with novel mechanisms of action have been investigated to overcome the problem. Novel reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs) recently approved for clinical use are effective against existing resistant strains by once daily administration. In addition, the virus entry (fusion) inhibitor has also been licensed. Since this compound targets a molecule other than reverse transcriptase and protease for inhibition of HIV-1 replication, it is active against HIV-1 highly resistant to RTIs and PIs. Furthermore, clinical trials with CCR 5 (coreceptor of HIV-1) antagonists are in progress, and inhibitors of integrase, HIV-1 gene expression, and virion assembly have been identified.

Anti-HIV Agents↗
PubMed2004

Human cell proteins and human immunodeficiency virus DNA integration.

Integration, catalyzed by the viral integrase (IN) protein, is a crucial step in the life cycle of all retroviruses including human immunodeficiency virus type 1 (HIV-1). Although purified HIV-1 IN protein is sufficient to catalyze the DNA breakage and joining steps of integration in the absence of any other protein factor, a number of studies indicate that cellular proteins participate in the integration process in cells. These host cell proteins have been proposed to act through binding the pre-integrated viral cDNA substrate, by directly interacting with the IN protein, and/or by repairing the single-stranded DNA gaps that occur at viral/chromosomal DNA junctions during integration. In this paper we summarize the identification and potential roles of specific cell factors in HIV-1 integration. We also present experimental results of human cell proteins that coimmunoprecipitated with HIV-1 IN following its expression in HeLa cells and discuss these results in light of the previously-identified integration cofactors.

Animals↗