Methanotrophs and methanotrophic activity in engineered landfill biocovers.

The dynamics and changes in the potential activity and community structure of methanotrophs in landfill covers, as a function of time and depth were investigated. A passive methane oxidation biocover (PMOB-1) was constructed in St-Nicéphore MSW Landfill (Quebec, Canada). The most probable number (MPN) method was used for methanotroph counts, methanotrophic diversity was assessed using denaturing gradient gel electrophoresis (DGGE) fingerprinting of the pmoA gene and the potential CH(4) oxidation rate was determined using soil microcosms. Results of the PMOB-1 were compared with those obtained for the existing landfill cover (silty clay) or a reference soil (RS). During the monitoring period, changes in the number of methanotrophic bacteria in the PMOB-1 exhibited different developmental phases and significant variations with depth. In comparison, no observable changes over time occurred in the number of methanotrophs in the RS. The maximum counts measured in the uppermost layer was 1.5x10(9) cells g dw(-1) for the PMOB-1 and 1.6x10(8) cells g dw(-1) for the RS. No distinct difference was observed in the methanotroph diversity in the PMOB-1 or RS. As expected, the potential methane oxidation rate was higher in the PMOB-1 than in the RS. The maximum potential rates were 441.1 and 76.0 microg CH(4) h(-1) g dw(-1) in the PMOB and RS, respectively. From these results, the PMOB was found to be a good technology to enhance methane oxidation, as its performance was clearly better than the starting soil that was present in the landfill site.

[Effect of regulator protein Nef on the viral activity of human immunodeficiency virus]. / Effet de la protéine régulatrice Nef sur le pouvoir infectieux du virus de l'immunodéficience humaine.

Despite 25 years of research on human immunodeficiency virus (HIV), the functions of some viral proteins are not fully understood. The role of Nef in the evolution of HIV-1 infection towards immunodeficiency is undeniable; however, the mechanisms involved in this function of Nef remain elusive. The interaction of Nef with a large number of cellular partners disrupts the metabolism of infected cells, including the endocytic pathway, in favor of viral spread. Down-regulation of cell-surface major histocompatibility complex (MHC) molecules by Nef enables infected cells to escape immune surveillance. Nef-induced down-regulation of CD4 increases the infectivity of virions by increasing the incorporation of the envelope glycoproteins into the viral membrane. In addition, Nef increases viral infectivity through a mechanism that is independent of MHC and CD4 down-regulation that nonetheless requires the ability of Nef to interfere with the endocytic process. Overall, these properties promote viral spread in the infected host.

[Nuclear import of the genome from the human immunodeficiency virus type 1]. / Import nucléaire du matériel génétique du virus de l'immunodéficience humaine de type 1.

HIV and other lentiviruses have the ability to replicate in non-dividing cells, such as macrophages and quiescent T lymphocytes, which represent major target cells during the course of infection. After virus entry, the viral genomic RNA is reverse transcribed into a linear double-strand DNA. This viral DNA associates with viral and host cell proteins into the so-called pre-integration complex (PIC). In contrast to oncoretroviruses which require nuclear envelope disintegration during mitosis to integrate their genome into host chromosomes, lentiviruses, such as HIV, have evolved an active strategy to import their own genome through the envelope of the interphasic nucleus. In this review, we will discuss on the most recent developments reported in the literature regarding the cellular and molecular bases that govern the intra-cytoplasmic routing and the translocation of the HIV-1 genome into the nuclear compartment, two crucial steps of the viral life cycle that are still poorly understood.

Putative homeodomain transcription factor 1 interacts with the feminization factor homolog fem1b in male germ cells.

The Phtf1 gene encodes a membrane protein abundantly expressed in male germinal cells. Using a two-hybrid screening procedure we have identified FEM1B, an ortholog of the C. elegans feminization factor 1 (FEM-1), as a binding partner for PHTF1. We studied FEM1B expression in the rodent testis and found that Fem1b mRNA is present at high levels during meiosis and after, during spermiogenesis, in a similar manner to Phtf1 mRNA. Accordingly, Western blot and immunofluorescence revealed the presence of PHTF1 and FEM1B in the same cell types, and by coimmunoprecipitation we demonstrated the association between these proteins. We characterized some aspects of this interaction and showed that the ANK domain of FEM1B is necessary for the interaction with the amino extremity of PHTF1. Next, we found that FEM1B can bind several intracellular organelles and demonstrated that PHTF1 would recruit FEM1B to the endoplasmic reticulum membrane. Previous in vitro experiments had suggested that the human FEM1B was involved in apoptosis. After comparing expression profiles of FEM1B and PHTF1 with apoptotic events occurring in the normal seminiferous tubules, we suggest that neither FEM1B nor PHTF1 are directly implicated in apoptosis in this tissue.

Polarized trafficking and surface expression of the AQP4 water channel are coordinated by serial and regulated interactions with different clathrin-adaptor complexes.

Aquaporin 4 (AQP4) is the predominant water channel in the brain. It is targeted to specific membrane domains of astrocytes and plays a crucial role in cerebral water balance in response to brain edema formation. AQP4 is also specifically expressed in the basolateral membranes of epithelial cells. However, the molecular mechanisms involved in its polarized targeting and membrane trafficking remain largely unknown. Here, we show that two independent C-terminal signals determine AQP4 basolateral membrane targeting in epithelial MDCK cells. One signal involves a tyrosine-based motif; the other is encoded by a di-leucine-like motif. We found that the tyrosine-based basolateral sorting signal also determines AQP4 clathrin-dependent endocytosis through direct interaction with the mu subunit of AP2 adaptor complex. Once endocytosed, a regulated switch in mu subunit interaction changes AP2 adaptor association to AP3. We found that the stress-induced kinase casein kinase (CK)II phosphorylates the Ser276 immediately preceding the tyrosine motif, increasing AQP4-mu 3A interaction and enhancing AQP4-lysosomal targeting and degradation. AQP4 phosphorylation by CKII may thus provide a mechanism that regulates AQP4 cell surface expression.

Interaction of human immunodeficiency virus type 1 Vpr with the HHR23A DNA repair protein does not correlate with multiple biological functions of Vpr.

The virion-associated Vpr protein of human immunodeficiency virus type 1 (HIV-1) alters cell cycle progression from the G2 phase, influences the virus in vivo mutation rate, and participates in the nuclear translocation of viral DNA. While many Vpr-interacting proteins have been identified, the functional relevance of these interactions remains to be thoroughly documented. We have explored the contribution of the interaction of HIV-1 Vpr with HHR23A, a cellular protein implicated in DNA repair, to the known phenotypes of Vpr. The association of Vpr with HHR23A required the core region of Vpr, which encompasses the two alpha-helical structures of the protein. No binding of HHR23A was detected with the Vpr and Vpx proteins of other primate lentiviruses. HIV-1 Vpr variants containing single amino acid substitutions in each alpha-helix and deficient for binding to HHR23A were isolated. The functional characterization of these Vpr variants indicated that binding to HHR23A did not correlate with the ability of Vpr to induce cell cycle arrest, even though it was previously proposed that HHR23A is a mediator of the Vpr-induced G2 arrest. Also, the Vpr-HHR23A interaction did not influence the HIV-1 in vivo mutation rate. Finally, Vpr and HHR23A are both localized in the nucleus, but no correlation was observed between the nuclear targeting of Vpr and the interaction with HHR23A. Further analysis is needed to determine the functional role(s) of the Vpr-HHR23A association during the HIV-1 life cycle.

Nef-induced CD4 downregulation: a diacidic sequence in human immunodeficiency virus type 1 Nef does not function as a protein sorting motif through direct binding to beta-COP.

The Nef protein from the human immunodeficiency virus (HIV) induces CD4 cell surface downregulation by interfering with the endocytic machinery. It has been recently proposed that binding of HIV type 1 Nef to the beta subunit of COPI coatomers participated in the Nef-induced CD4 downregulation through recognition of a novel diacidic motif found in the C-terminal disordered loop of Nef (V. Piguet, F. Gu, M. Foti, N. Demaurex, J. Gruenberg, J. L. Carpentier, and D. Trono, Cell 97:63-73, 1999). We have mutated the glutamate residues which formed this motif in order to document this observation. Surprisingly, mutation of the diacidic sequence of Nef did not significantly affect its ability (i) to interact with beta-COP, (ii) to downregulate CD4 cell surface expression, and (iii) to address an integral resident membrane protein containing Nef as the cytoplasmic domain to the endocytic pathway. Our results indicate that these acidic residues are not involved in the connection of Nef with the endocytic machinery through binding to beta-COP. Additional studies are thus required to characterize the residues of Nef involved in the binding to beta-COP and to evaluate the contribution of this interaction to the Nef-induced perturbations of membrane trafficking.

Characterization of the nuclear import pathway for HIV-1 integrase.

The karyophilic properties of the human immunodeficiency virus, type I (HIV-1) pre-integration complex (PIC) allow the virus to infect non-dividing cells. To better understand the mechanisms responsible for nuclear translocation of the PIC, we investigated nuclear import of HIV-1 integrase (IN), a PIC-associated viral enzyme involved in the integration of the viral genome in the host cell DNA. Accumulation of HIV-1 IN into nuclei of digitonin-permeabilized cells does not result from passive diffusion but rather from an active transport that occurs through the nuclear pore complexes. HIV-1 IN is imported by a saturable mechanism, implying that a limiting cellular factor is responsible for this process. Although IN has been previously proposed to contain classical basic nuclear localization signals, we found that nuclear accumulation of IN does not involve karyopherins alpha, beta1, and beta2-mediated pathways. Neither the non-hydrolyzable GTP analog, guanosine 5'-O-(thiotriphosphate), nor the GTP hydrolysis-deficient Ran mutant, RanQ69L, significantly affects nuclear import of IN, which depends instead on ATP hydrolysis. Therefore these results support the idea that IN import is not mediated by members of the karyopherin beta family. More generally, in vitro nuclear import of IN does not require addition of cytosolic factors, suggesting that cellular factor(s) involved in this active but atypical pathway process probably remain associated with the nuclear compartment or the nuclear pore complexes from permeabilized cells.

Cellular distribution and karyophilic properties of matrix, integrase, and Vpr proteins from the human and simian immunodeficiency viruses.

Infections by human and simian immunodeficiency viruses (HIV and SIV) are independent of host cell division since the preintegration complex (PIC), containing the viral DNA, is able to undergo active nuclear import after viral entry. In order to clarify the mechanisms responsible for nuclear import of the PIC, we have analyzed the subcellular distribution and the karyophilic properties of its viral components, matrix protein (MA), integrase (IN), Vpr, and Vpx. Although MA has been reported to contain a nuclear localization signal, the MA/GFP fusions are excluded from the nucleus and associated with cellular membranes. In contrast, both HIV-1 and SIV IN and Vpr localize in the nucleus of transfected cells. Interestingly, only Vpx from SIVsm virus accumulate in the nucleus while SIVsm Vpr is uniformly distributed throughout nucleus and cytoplasm. Coexpression of MA, Vpr, and IN does not induce any change in their respective intracellular localizations. Finally, we confirm the karyophilic properties of HIV-1 IN and Vpr using an in vitro nuclear import assay. These results indicate that the viral proteins IN and Vpr, which are strongly associated with the viral DNA within PIC, may participate in the nuclear import of the HIV PIC.

The interaction of vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 In vivo mutation rate.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) influences the in vivo mutation rate of the virus. Since Vpr interacts with a cellular protein implicated in the DNA repair process, uracil DNA glycosylase (UNG), we have explored the contribution of this interaction to the mutation rate of HIV-1. Single-amino-acid variants of Vpr were characterized for their differential UNG-binding properties and used to trans complement vpr null mutant HIV-1. A striking correlation was established between the abilities of Vpr to interact with UNG and to influence the HIV-1 mutation rate. We demonstrate that Vpr incorporation into virus particles is required to influence the in vivo mutation rate and to mediate virion packaging of the nuclear form of UNG. The recruitment of UNG into virions indicates a mechanism for how Vpr can influence reverse transcription accuracy. Our data suggest that distinct mechanisms evolved in primate and nonprimate lentiviruses to reconcile uracil misincorporation into lentiviral DNA.

Mutation of a conserved residue (D123) required for oligomerization of human immunodeficiency virus type 1 Nef protein abolishes interaction with human thioesterase and results in impairment of Nef biological functions.

Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.

HIV-2 and SIV nef proteins target different Src family SH3 domains than does HIV-1 Nef because of a triple amino acid substitution.

The nef gene is required for optimal viral spread of human and simian immunodeficiency viruses. However, the molecular mechanisms underlying the action of the Nef proteins may not be identical for all viral families. Here we investigate the interaction between the Nef protein of human and simian immunodeficiency viruses and SH3 domains from Src family kinases. Using the yeast two-hybrid system and immunoblotting we show that, in contrast to HIV-1 Nef, SIV and HIV-2 Nef poorly interact with Hck SH3 but bind to Src and Fyn SH3 domains. The molecular basis of these differences in SH3 targeting was revealed by sequence analysis and homology modeling of the putative SH3-Nef structures. Three amino acids (Trp-113, Thr-117, and Gln-118) that localize in a "hydrophobic pocket" implicated in SH3 binding of HIV-1 Nef, are systematically substituted in SIV/HIV-2 alleles (by Tyr, Glu, and Glu, respectively). We demonstrate that site-directed mutagenesis of these residues in SIV(mac239) Nef suffices to restore Hck SH3 binding and co-immunoprecipitation with full-length Hck from transfected cells. Our findings identify fundamental mechanistic differences in targeting of Src family kinases by HIV and SIV Nef. The herein described mechanism of SH3 selection by Nef via a "pocket" proximal to the canonical proline-rich motif may be a common feature for SH3 recognition by their natural ligands.

Two independent regions of HIV-1 Nef are required for connection with the endocytic pathway through binding to the mu 1 chain of AP1 complex.

The Nef protein from the human immunodeficiency virus (HIV) induces down-regulation of the CD4 and major histocompatibility complex class I molecules from the cell surface by interfering with the endocytic machinery. This work focuses on the interaction of HIV-1 Nef with the mu 1 chain of adaptor protein type 1 (AP1) complex and its contribution to the Nef-induced alterations of membrane trafficking. Two independent regions surrounding a disordered loop located in the C-terminal part of Nef are involved in mu 1 binding. Each region can separately interact with mu 1, and simultaneous point mutations within both regions are needed to abolish binding. We used CD8 chimeras in which the cytoplasmic tail was replaced by Nef mutants to show that these mu 1-binding sites contain determinants required to induce CD4 down-regulation and to target the chimera to the endocytic pathway by promoting AP1 complex recruitment. Ultrastructural analysis revealed that the CD8-Nef chimera provokes morphological alterations of the endosomal compartments and co-localizes with AP1 complexes. These data indicate that the recruitment by Nef of AP1 via binding to mu 1 participates in the connection of Nef with the endocytic pathway.

HEED, the product of the human homolog of the murine eed gene, binds to the matrix protein of HIV-1.

heed, the human homolog of mouse eed and Drosophila esc, two members of the trithorax (trx) and Polycomb group (Pc-G) of genes, was isolated by screening an activated lymphocyte cDNA library versus the immunodeficiency virus type 1 (HIV-1) MA protein used as a bait in a two-hybrid system in yeast. The human EED protein (HEED) had 99. 5% identity with the mouse EED protein and contained seven WD repeats. Two heed gene transcripts were identified, with a putative 407-nucleotide-long intron, giving rise to two HEED protein isoforms of 535 and 494 residues in length, respectively. The shorter HEED isoform, originated from the unspliced message, lacked the seventh WD repeat. HEED was found to bind to MA protein in vitro, as efficiently as in vivo in yeast cells. Site-directed mutagenesis and phage biopanning suggested that the interaction between HEED and MA involved the N-terminal region of the MA protein, including the first polybasic signal, in a MA conformation-dependent manner. In the HEED protein, however, two discrete linear MA-binding motifs were identified within residues 388-403, overlapping the origin of the fifth WD repeat. Deletion of the C-terminal 41 residues of HEED, spanning the seventh WD repeat, as in the 494-residue HEED protein, was detrimental to HEED-MA interaction in vivo, suggesting the existence of another C-terminal binding site and/or a conformational role of the HEED C-terminal domain in the MA-HEED interaction. MA and HEED proteins co-localized within the nucleus of co-transfected human cells and of recombinant baculovirus co-infected insect cells. This and the failure of HEED to bind to uncleaved GAG precursor suggested a role of HEED at the early stages of virus infection, rather than late in the virus life cycle.

Interaction with the p6 domain of the gag precursor mediates incorporation into virions of Vpr and Vpx proteins from primate lentiviruses.

Vpr and Vpx proteins from human and simian immunodeficiency viruses (HIV and SIV) are incorporated into virions in quantities equivalent to those of the viral Gag proteins. We demonstrate here that Vpr and Vpx proteins from distinct lineages of primate lentiviruses were able to bind to their respective Gag precursors. The capacity of HIV type 1 (HIV-1) Vpr mutants to bind to Pr55(Gag) was correlated with their incorporation into virions. Molecular analysis of these interactions revealed that they required the C-terminal p6 domain of the Gag precursors. While the signal for HIV-1 Vpr binding lies in the leucine triplet repeat region of the p6 domain reported to be essential for incorporation, SIVsm Gag lacking the equivalent region still bound to SIVsm Vpr and Vpx, indicating that the determinants for Gag binding are located upstream of this region of the p6 domain. Binding to Gag cleavage products showed that HIV-1 Vpr interacted directly with the nucleocapsid protein (NC), whereas SIVsm Vpr and Vpx did not interact with NC but with the p6 protein. These results (i) reveal differences between HIV-1 and SIVsm for the p6 determinants required for Vpr and Vpx binding to Gag and (ii) suggest that HIV-1 Vpr and SIVsm Vpr and Vpx interact with distinct cleavage products of the precursor following proteolytic processing in the virions.

A novel human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif.

HIV-1 Vpu interacts with CD4 in the endoplasmic reticulum and triggers CD4 degradation, presumably by proteasomes. Human beta TrCP identified by interaction with Vpu connects CD4 to this proteolytic machinery, and CD4-Vpu-beta TrCP ternary complexes have been detected by coimmunoprecipitation. beta TrCP binding to Vpu and its recruitment to membranes require two phosphoserine residues in Vpu essential for CD4 degradation. In beta TrCP, WD repeats at the C terminus mediate binding to Vpu, and an F box near the N terminus is involved in interaction with Skp1p, a targeting factor for ubiquitin-mediated proteolysis. An F-box deletion mutant of beta TrCP had a dominant-negative effect on Vpu-mediated CD4 degradation. These data suggest that beta TrCP and Skp1p represent components of a novel ER-associated protein degradation pathway that mediates CD4 proteolysis.

Nef interacts with the mu subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC I molecules.

The surface expression of MHC I is reduced in HIV-infected cells. We show that the Nef protein affects the intracellular sorting of HLA-A and -B molecules. In the presence of Nef, these proteins accumulate in the Golgi and colocalize with clathrin-coated vesicles. MHC I modulation relies on a tyrosine-based sorting signal located in the cytoplasmic domain of HLA-A and -B heavy chains. This cryptic sorting signal becomes operative only in the presence of Nef. Nef interacts with the medium (mu) subunit of AP adaptor complexes involved in the recognition of tyrosine-based sorting signals, likely facilitating the connection between MHC I and the clathrin-dependent sorting machinery.

The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling.

BACKGROUND: Human immunodeficiency virus (HIV) Nef protein accelerates virulent progression of acquired immunodeficiency syndrome (AIDS) by its interaction with specific cellular proteins involved in signal transduction and host cell activation. Nef has been shown to bind specifically to a subset of the Src family of kinases. The structures of free Nef and Nef bound to Src homology region 3 (SH3) domain are important for the elucidation of how the affinity and specificity for the Src kinase family SH3 domains are achieved, and also for the development of potential drugs and vaccines against AIDS. RESULTS: We have determined the crystal structures of the conserved core of HIV-1 Nef protein alone and in complex with the wild-type SH3 domain of the p59fyn protein tyrosine kinase (Fyn), at 3.0 A resolution. Comparison of the bound and unbound Nef structures revealed that a proline-rich motif (Pro-x-x-Pro), which is implicated in SH3 binding, is partially disordered in the absence of the binding partner; this motif only fully adopts a left-handed polyproline type II helix conformation upon complex formation with the Fyn SH3 domain. In addition, the structures show how an arginine residue (Arg77) of Nef interacts with Asp 100 of the so-called RT loop within the Fyn SH3 domain, and triggers a hydrogen-bond rearrangement which allows the loop to adapt to complement the Nef surface. The Arg96 residue of the Fyn SH3 domain is specifically accommodated in the same hydrophobic pocket of Nef as the isoleucine residue of a previously described Fyn SH3 (Arg96-->lle) mutant that binds to Nef with higher affinity than the wild type. CONCLUSIONS: The three-dimensional structures support evidence that the Nef-Fyn complex forms in vivo and may have a crucial role in the T cell perturbating action of Nef by altering T cell receptor signaling. The structures of bound and unbound Nef reveal that the multivalency of SH3 binding may be achieved by a ligand induced flexibility in the RT loop. The structures suggest possible targets for the design of inhibitors which specifically block Nef-SH3 interactions.

Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest.

The Vpr protein encoded by human immunodeficiency virus type 1 (HIV-1) is important for growth of virus in macrophages and prevents infected cells from passing into mitosis (G2 arrest). The cellular target for these functions is not known, but Vpr of HIV-1 and the related Vpr from simian immunodeficiency virus of sooty mangabeys (SIV(SM)) bind the DNA repair enzyme UNG, while the Vpx protein of SIV(SM) does not. Nonetheless, a mutational analysis of Vpr showed that binding to UNG is neither necessary nor sufficient for the effect of Vpr on the cell cycle.