Late Domain-Independent Rescue of a Release-Deficient Moloney Murine Leukemia Virus by the Ubiquitin Ligase Itch

Moloney murine leukemia virus (MoMLV) Gag utilizes its late (L) domain motif PPPY to bind members of the Nedd4-like ubiquitin ligase family. These interactions recruit components of the cell''s budding machinery that are critical for virus release. MoMLV Gag contains two additional L domains, PSAP and LYPAL, that are believed to drive residual MoMLV release via interactions with cellular proteins Tsg101 and Alix, respectively. We found that overexpression of Tsg101 or Alix failed to rescue the release of PPPY-deficient MoMLV via these other L domains. However, low-level expression of the ubiquitin ligase Itch potently rescued the release and infectivity of MoMLV lacking PPPY function. In contrast, other ubiquitin ligases such as WWP1, Nedd4.1, Nedd4.2, and Nedd4.2s did not rescue this release-deficient virus. Efficient rescue required the ubiquitin ligase activity of Itch and an intact C2 domain but not presence of the endophilin-binding site. Additionally, we found Itch to immunoprecipitate with MoMLV Gag lacking the PPPY motif and to be incorporated into rescued MoMLV particles. The PSAP and LYPAL motifs were dispensable for Itch-mediated virus rescue, and their absence did not affect the incorporation of Itch into the rescued particles. Itch-mediated rescue of release-defective MoMLV was sensitive to inhibition by dominant-negative versions of ESCRT-III components and the VPS4 AAA ATPase, indicating that Itch-mediated correction of MoMLV release defects requires the integrity of the host vacuolar sorting protein pathway. RNA interference knockdown of Itch suppressed the residual release of the MoMLV lacking the PPPY motif. Interestingly, Itch stimulation of the PPPY-deficient MoMLV release was accompanied by the enhancement of Gag ubiquitination and the appearance of new ubiquitinated Gag proteins in virions. Together, these results suggest that Itch can facilitate MoMLV release in an L domain-independent manner via a mechanism that requires the host budding machinery and involves Gag ubiquitination.Retroviruses require access to the host budding machinery to exit the cell (5, 13, 40). To this end, retroviral Gag polyproteins use short sequences called late (L) domains to promote virus release by recruiting members of the host vacuolar protein sorting (vps) machinery. In the cell, vps proteins are involved in membrane dynamics that facilitate the separation of daughter cells at the completion of cytokinesis (9, 39) and the budding of vesicles into endosomal compartments or multivesicular bodies (MVB) (2, 23), a process topologically similar to virus budding (57). Class E vps proteins are organized into three heteromeric endosomal complexes (called endosomal sorting complexes) required for transport, namely, ESCRT-I, -II, and -III (2). In the current model for budding, sequential recruitment of ESCRT components on the cytoplasmic face of the membrane facilitates vesicle invagination into MVB compartments and viral egress from the cell (2). The disassembly of ESCRT-III components is catalyzed by the activity of VPS4 AAA-type ATPase, which in turn is presumed to trigger membrane fission events (3, 50). Any disruption in this sequence, such as mutations in L domain motifs or dominant-negative interference with the function of ESCRT-III members or the VPS4 ATPase, adversely affects virus release. This indicates that Gag interactions with the ESCRT machinery are necessary for virus budding and separation from the cell (19, 21, 34, 49, 57).Currently, three types of L domain motifs have been identified: PT/SAP, LYPX_nL, and PPPY. All retroviral Gag molecules contain at least one of these motifs, as multiple L domains are believed to synergistically function to ensure efficient viral release. Moloney murine leukemia virus (MoMLV) Gag carries all three L domain motifs, PSAP, LYPAL, and PPPY, which bind the vps protein Tsg101, the ESCRT-associated protein Alix (46), and members of the Nedd4-ubiquitin ligase family (33), respectively. In HIV-1, the PTAP motif in the p6 region of Gag binds Tsg101 (16, 56), which functions in viral budding (16, 35) as a member of ESCRT-I (16, 36, 57). The LYPX_nL motif is also located in p6 and is the binding site for Alix (49, 57), a protein that also interacts with the nucleocapsid domain of HIV-1 Gag (14, 43) and links Gag to components of ESCRT-III (14). Similarly, the human T-cell leukemia virus (HTLV-I) Gag carries PPPY and PTAP L domains, which both contribute to efficient HTLV-1 release (6, 7, 21). The PPPY L domain motif, which is found in numerous retroviral Gag polyproteins (6, 7, 19, 21, 27, 28, 61, 62), plays a critical role in MoMLV release, as mutations disrupting its sequence lead to significant decreases in virus budding and release (33, 62). PSAP and LYPAL, the additional L domain motifs, are believed to serve little to no role in the release of MoMLV Gag virus-like particles (45, 46).The role of Nedd4-like ubiquitin ligases in budding events was initially established by data obtained with the yeast Nedd4-like ligase Rsp5, an enzyme that ubiquitinates surface proteins, thus signaling their incorporation into the MVB pathway (26). From retroviral budding studies, multiple findings support the notion that Nedd4-like ubiquitin ligases link PPPY-containing Gag proteins to the host ESCRT machinery. For example, mutations in the PPPY motif or expression of dominant-negative versions of Nedd4-like ligases resulted in budding defects similar to those seen upon interference with the function of ESCRT-III members (7, 21, 27, 28, 33, 62). Overexpression of Nedd4-like ligases WWP1 and Itch corrected the budding defects of a MoMLV PPPY mutant that retained residual binding to both ligases (33). Also, when transplanted to a heterologous retroviral Gag, the PPPY L domain creates a requirement for Nedd4-like ubiqutin ligase activity to facilitate viral release that is dependent on the presence of a functional ESCRT pathway (63). Collectively, these observations support the notion that Nedd4-like ubiquitin ligases link retroviral Gag polyproteins to components of the ESCRT pathway necessary for budding.Both endosomal and viral budding require the ubiquitin conjugation properties of Nedd4-like ligases, indicating that ubiquitin transfer to a key protein(s) is necessary to promote budding. A role for Gag ubiquitination in viral budding has been suggested (8, 20, 22, 48). In fact, ubiquitin attachment to equine infectious anemia virus (EIAV) Gag can substitute for the lack of L domains and rescue viral budding (25), suggesting that ubiquitin molecules conjugated to Gag can signal the recruitment of the host ESCRT machinery. For feline immunodeficiency virus, efficient budding seems to require L domain-dependent ubiquitination of Gag proteins (8) that is independent of the L domain ability to directly recruit Nedd4-like ubiquitin ligases (i.e., by means of the PT/SAP L domain motif) (8). Similarly, ubiquitination of HTLV-1 Gag was also shown to play a significant role in viral release (22). Conversely, data arguing in favor of a role for the ubiquitination of transacting factors, but not Gag, in the facilitation of viral budding have also been reported (10, 63). Thus Gag polyproteins recruit, in a PPPY-dependent or -independent manner, enzymatically active Nedd4-like ubiquitin ligases that conjugate ubiquitin molecules to Gag or to Gag-binding host factors. Such interactions, whether direct or indirect, are believed to link the viral protein to the host ESCRT pathway and facilitate release.In addition to the well-characterized cellular proteins that bind primary L domain motifs, retroviral Gag can recruit other host factors, either via secondary L domains or independently of L domains (10, 24, 29, 55, 59). These cellular factors are believed to promote virus production by facilitating Gag protein trafficking to the plasma membrane and/or providing additional L domain-independent links to the host vps pathway. Examples of these parallel pathways are illustrated in the rescue of a budding-defective HIV-1 lacking the PTAP domain by overexpression of Alix (15, 54) and in the remarkably potent rescue of HIV-1 lacking all known L domains by the overexpression of Nedd4.2s, a Nedd4.2 isoform that belongs to the Nedd4-like ubiquitin ligase family (10, 55). In this study, we sought to identify host cell factors that rescue budding defects of the MoMLV mutant lacking the PPPY motif (MoMLV AAAY mutant). Our studies provide evidence that Itch overexpression rescued budding and infectivity defects of the MoMLV AAAY mutant virus, indicating that Gag can recruit the ubiquitin ligase Itch in an L domain-independent manner to facilitate MoMLV release via a mechanism that involves Gag ubiquitination.     

Construction, database integration, and application of an Oenothera EST library

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.     

The lantibiotic mersacidin is an autoinducing peptide

The lantibiotic (lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide consisting of 20 amino acids and is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene (mrsA) and the genes for producer self-protection, modification enzymes, transport proteins, and regulator proteins are organized in a 12.3-kb biosynthetic gene cluster on the chromosome of the producer strain. Mersacidin is produced in stationary phase in a synthetic medium (K. Altena, A. Guder, C. Cramer, and G. Bierbaum, Appl. Environ. Microbiol. 66:2565-2571, 2000). To investigate the influence of the alternative sigma factor H on mersacidin biosynthesis, a SigH knockout was constructed. The knockout mutant was asporogenous, and a comparison to the wild-type strain indicated no significant differences concerning mersacidin production and immunity. Characterization of the mrsA promoter showed that the gene is transcribed by the housekeeping sigma factor A. The biosynthesis of some lantibiotic peptides like nisin or subtilin is regulated in a cell-density-dependent manner (M. Kleerebezem, Peptides 25:1405-1414, 2004). When mersacidin was added at a concentration of 2 mg/liter to an exponentially growing culture, an earlier production of antibacterial activity against Micrococcus luteus ATCC 4698 in comparison to that of the control culture was observed, suggesting that mersacidin itself functions as an autoinducer. In real-time PCR experiments, the expression of mrsA was remarkably increased in the induced culture compared to the control. In conclusion, mersacidin is yet another lantibiotic peptide whose biosynthesis can be regulated by an autoinducing mechanism.     

Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions: IMPLICATIONS FOR BINDING SPECIFICITY*

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K_D values varying between 38 nm (FGF-18) and 620 nm (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 m⁻¹ s⁻¹ and FGF-9, 130,000 m⁻¹ s⁻¹). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.     

Budding of retroviruses utilizing divergent L domains requires nucleocapsid

We recently reported that human immunodeficiency virus type 1 (HIV-1) carrying PTAP and LYPX(n)L L domains ceased budding when the nucleocapsid (NC) domain was mutated, suggesting a role for NC in HIV-1 release. Here we investigated whether NC involvement in virus release is a property specific to HIV-1 or a general requirement of retroviruses. Specifically, we examined a possible role for NC in the budding of retroviruses relying on divergent L domains and structurally homologous NC domains that harbor diverse protein sequences. We found that NC is critical for the release of viruses utilizing the PTAP motif whether it functions within its native Gag in simian immunodeficiency virus cpzGAB2 (SIVcpzGAB2) or SIVsmmE543 or when it is transplanted into the heterologous Gag protein of equine infectious anemia virus (EIAV). In both cases, virus release was severely diminished even though NC mutant Gag proteins retained the ability to assemble spherical particles. Moreover, budding-defective NC mutants, which displayed particles tethered to the plasma membrane, were triggered to release virus when access to the cell endocytic sorting complex required for transport pathway was restored (i.e., in trans expression of Nedd4.2s). We also examined the role of NC in the budding of EIAV, a retrovirus relying exclusively on the (L)YPX(n)L-type L domain. We found that EIAV late budding defects were rescued by overexpression of the isolated Alix Bro1 domain (Bro1). Bro1-mediated rescue of EIAV release required the wild-type NC. EIAV NC mutants lost interactions with Bro1 and failed to produce viruses despite retaining the ability to self-assemble. Together, our studies establish a role for NC in the budding of retroviruses harboring divergent L domains and evolutionarily diverse NC sequences, suggesting the utilization of a common conserved mechanism and/or cellular factor rather than a specific motif.     

The alteration of 5-HT(2A) and 5-HT(2C) receptors is involved in neuronal apoptosis of goldfish cerebellum following traumatic experience

5-HT receptor changes remain controversial in posttraumatic stress disorder (PTSD) models. This study looks at the relationship between traumatic injuries and the alterations in 5-HT(2A) and 5-HT(2C) receptors in the goldfish brain. The effect of treatment with doxepin and fluoxetine, known to be selective serotonin reuptake inhibitor (SSRI) antidepressants, on 5-HT receptor expression in goldfish with fin ablation was also investigated. We demonstrated that fin ablation induced anxiety-like behavioural alterations and significant up-regulation of c-fos expression in goldfish cerebellum. The behavioural alterations correlated well with an increased expression of 5-HT(2A) receptors in the cerebellum of the fish with traumatic injury. An increase in the number of apoptotic cells and a higher caspase-8 protein level was present in the brains of goldfish with fin ablation compared to the control. Our findings suggest that neuronal apoptosis occured in the cerebellum as a result of fin ablation and may be related to the alterations of 5-HT(2A) and 5-HT(2C) levels and that the beneficial clinical effects of doxepin/fluoxetine treatment are due to the down-regulation of 5-HT(2A) and up-regulation of 5-HT(2C) receptors in the brain.     

Differential proteomic analysis of a polymicrobial biofilm

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia exist in a polymicrobial biofilm associated with chronic periodontitis. The aim of this study was to culture these three species as a polymicrobial biofilm and to determine proteins important for bacterial interactions. In a flow cell all three species attached and grew as a biofilm; however, after 90 h of culture P. gingivalis and T. denticola were closely associated and dominated the polymicrobial biofilm. For comparison, planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. Whole cell lysates were subjected to SDS-PAGE, followed by in-gel proteolytic H(2)(16)O/H(2)(18)O labeling. From two replicates, 135 and 174 P. gingivalis proteins and 134 and 194 T. denticola proteins were quantified by LC-MALDI TOF/TOF MS. The results suggest a change of strategy in iron acquisition by P. gingivalis due to large increases in the abundance of HusA and HusB in the polymicrobial biofilm while HmuY and other iron/haem transport systems decreased. Significant changes in the abundance of peptidases and enzymes involved in glutamate and glycine catabolism suggest syntrophy. These data indicate an intimate association between P. gingivalis and T. denticola in a biofilm that may play a role in disease pathogenesis.