Interactions and Oligomerization of Hantavirus Glycoproteins

In this report the basis for the structural architecture of the envelope of hantaviruses, family Bunyaviridae, is systematically studied by the interactions of two glycoproteins N and C (Gn and Gc, respectively) and their respective disulfide bridge-mediated homo- and heteromeric oligomerizations. In virion extracts Gn and Gc associated in both homo- and hetero-oligomers which were, at least partially, thiol bridge mediated. Due to strong homo-oligomerization, the hetero-oligomers of Gn and Gc are likely to be mediated by homo-oligomeric subunits. A reversible pH-induced disappearance of a neutralizing epitope in Gc and dissociation of the Gn-Gc complex at pH values below 6.2 provide proteochemical evidence for the fusogenicity of Gc. Incomplete inactivation of virions at acidic pH indicates that additional factors are required for hantavirus fusion, as in the case of pestiviruses of the Flaviviridae. Based on similarities to class II fusion proteins, a structure model was created of hantavirus Gc using the Semliki Forest virus E1 protein as a template. In total, 10 binding regions for Gn were found by peptide scanning, of which five represent homotypic (Gn_I to Gn_V) and five represent heterotypic (Gc_I to Gc_V) interaction sites that we assign as intra- and interspike connections, respectively. In conclusion, the glycoprotein associations were compiled to a model wherein the surface of hantaviruses is formed of homotetrameric Gn complexes interconnected with Gc homodimers. This organization would create the grid-like surface pattern described earlier for hantaviruses in negatively stained electron microscopy specimens.Hantaviruses, a genus in the family Bunyaviridae, are rodent- and insectivore-borne zoonotic viruses that are seemingly apathogenic to the carrier rodents (39, 57). A number of hantaviruses are human pathogens that in areas of endemicity are responsible for two diseases: hemorrhagic fever with renal syndrome in Eurasia and hantavirus cardiopulmonary syndrome in the Americas (49, 57, 61). Hantaviruses are enveloped viruses and have a trisegmented, single-stranded, negative-sense RNA genome that encodes an RNA-dependent RNA polymerase, two glycoproteins, and a nucleocapsid protein (22, 34, 49, 60). During assembly, the four proteins and the RNA genome are packed into a round or a pleomorphic particle enveloped with a lipid bilayer. The interactions among the structural components of hantavirus have not been described in sufficient detail to construct the basic architecture of the virus particle or to understand the mechanisms of its assembly and entry.The envelope glycoproteins are expressed as a precursor polypeptide, which is cotranslationally cleaved after a conserved pentapeptide WAASA into an N- and C-terminal portion prior to maturation of the envelope glycoproteins proteins N and C (Gn and Gc, respectively) (27). In the family Bunyaviridae the transport of newly synthesized glycoproteins from endoplasmic reticulum to the Golgi apparatus requires the presence of both Gn and Gc (36, 37, 50, 53). Recombinant coexpression of the hantavirus glycoproteins is sufficient to achieve proper folding and the expected cellular localization at the Golgi even when the glycoproteins are not expressed from a common precursor (6, 36, 50). This suggests that the expression of the precursor is not a prerequisite for interactions between Gn and Gc during maturation in which the formation of a Gn-Gc complex results in exposure of a conformational Golgi apparatus-targeting signal, present only in the heterodimeric Gn-Gc complex (6, 50).Entry of enveloped viruses via recognition of the cell surface receptors and subsequent fusion of the virus and cell membranes are accomplished by viral glycoproteins which often appear in homo- and/or heteromeric complexes. For example, the E1 and E2 of Semliki Forest virus (SFV) form a trimer of heterodimers (45), and the E protein of tick-borne encephalitis virus (TBEV) forms a homodimer (41) while the hemagglutinin of influenza A virus (67) and the S protein of severe acute respiratory syndrome coronavirus associate in homotrimers (4, 5). The mature glycoproteins extracted from virions of Uukuniemi phlebovirus exist as homodimers (44), whereas glycoprotein complex formations of many other members of the Bunyaviridae have not been defined. The viral fusion proteins can be classified into class I, class II, and class III (25). Between classes I and II, a distinguishing property is the orientation of a fusion protein in the metastable state. The class I proteins are oriented perpendicular to the viral membrane, and the class II protein is parallel to the viral membrane (7). The class II viral fusion proteins assemble in virions as metastable homo- or heterodimeric complexes which, upon exposure to low pH, fuse the viral and target cellular membranes (7). This process begins with a conformational change in the fusion protein, leading to the revelation of its fusion loop, which binds to the cellular target membrane (7). Additionally, the formation of a homotrimeric fusion protein complex and structural changes that drive the fusion into completion occur (7).Understanding the multimeric status, protein-protein interactions, and pH-dependent conformational changes of glycoproteins is paramount to our understanding of selectivity in cell receptor binding and mechanisms of virus entry. It is unknown whether higher-order oligomeric complexes are found in hantavirus particles. Many neutralizing monoclonal antibodies (MAbs) have been isolated and by MAb escape mutants shown to recognize epitopes in both Gn and Gc, typically localized at discontinuous sites (15). Different neutralization mechanisms for hantavirus MAbs have been elucidated. These range from inhibiting receptor binding to inhibition of virus fusion (2, 23, 28, 30, 65). It is known that hantaviral glycoproteins possess fusogenic activity. Glycoproteins of hantaviruses that cause hemorrhagic fever with renal syndrome can induce syncytia when subjected to low pH (32, 35), and infection by Hantaan virus was shown to use low-pH-dependent clathrin-mediated endocytosis (19). Hantavirus Gc is suggested to be a class II fusion protein (13, 55), and the N-linked glycosylation of Gc is essential for cell fusion activity (70); but no clear understanding exists of the fusion mechanism or conformational changes that mediate uncoating of virions after entry.Our study supports the hypothesis that the Gc of hantaviruses is a class II fusion protein. We show the interaction between Gn and Gc to be pH sensitive and dissociation to start at a pH below 6.4. The low-pH-induced Gc dissociation from Gn was reversible, suggesting that the conformational changes in Gc are also reversible. Both glycoproteins were found to form homodimeric and hetero-oligomeric complexes in virion extracts through thiol bridging. Interaction studies further suggested that the protruding part of the spike complex in the hantavirus virion consists of four Gn subunits and that the spike complexes interconnect with homodimeric Gc subunits. Finally, we mapped and compiled the interaction sites of Gn and Gc proteins in a class II fusion protein three-dimensional (3D) model of Gc. The identified Gn-Gn, Gn-Gc, and Gc-Gc interaction sites may play an important role in glycoprotein folding and maturation, spike assembly, virus fusion, and neutralization of infection.     

Coronary flow reserve and heart failure in experimental coxsackievirus myocarditis. A transthoracic Doppler echocardiography study

The objective of this study was to apply transthoracic Doppler echocardiography (TTDE) in mice to study coronary flow reserve (CFR), an index of coronary microvascular function, in mild and severe forms of experimental viral myocarditis. Regarding methodology, BALB/c mice were infected with cardiotropic coxsackieviruses causing either a mild (Nancy strain) or a severe (Woodruff strain) myocarditis. Left ventricular dimensions, fractional shortening, and CFR (ratio of left coronary artery flow velocity during maximal adenosine-induced vasodilatation to rest) were measured by TTDE before infection and again 1 or 2 wk after infection. As a result, the resting flow velocity did not change after infection. In contrast, CFR reduced significantly 1 wk after infection with either virus variant [from 2.5 (SD 0.3) to 1.4 (SD 0.1) in severe and from 2.4 (SD 0.4) to 2.1 (SD 0.3) in mild myocarditis], being significantly lower in the severe than mild myocarditis. CFR remained low in severe myocarditis 2 wk after infection. Fractional shortening decreased to the same levels 1 wk after infection with either virus variant [from 0.54 (SD 0.02) to 0.43 (SD 0.03) in severe and from 0.51 (SD 0.03) to 0.44 (SD 0.02) in mild myocarditis, P < 0.05]. However, 2 wk after infection, mice with severe myocarditis had enlarged left ventricles and lower fractional shortening [0.31 (SD 0.03)] than mice with mild myocarditis [0.47 (SD 0.02), P < 0.01]. In conclusion, CFR measured with TTDE is reduced in coxsackievirus myocarditis in mice. Low CFR is associated with progressive heart failure, indicating that dysfunction of coronary microcirculation is a determinant of poor outcome in viral myocarditis.     

Cathepsin B is a differentiation-resistant target for nitroxyl (HNO) in THP-1 monocyte/macrophages

We previously showed that the one-electron reduction product of nitric oxide (NO), nitroxyl (HNO), irreversibly inhibits the proteolytic activity of the model cysteine protease papain. This result led us to investigate the differential effects of the nitrogen oxides, such as nitroxyl (HNO), NO, and in situ-generated peroxynitrite on cysteine modification-sensitive cellular proteolytic enzymes. We used Angeli's salt, diethylaminenonoate (DEA/NO), and 3-morpholinosydnoniminehydrochloride (SIN-1), as donors of HNO, NO, and peroxynitrite, respectively. In this study we evaluated their inhibitory activities on the lysosomal mammalian papain homologue cathepsin B and on the cytosolic 26S proteasome in THP-1 monocyte/macrophages after LPS activation or TPA differentiation. HNO-generating Angeli's salt caused a concentration-dependent (62 +/- 4% at 316 muM) inhibition of the 26S proteasome activity, resulting in accumulation of protein-bound polyubiquitinylated proteins in LPS-activated cells, whereas neither DEA/NO nor SIN-1 showed any effect. Angeli's salt, but not DEA/NO or SIN-1, also caused (94 +/- 2% at 316 muM) inhibition of lysosomal cathepsin B activity in LPS-activated cells. Induction of macrophage differentiation did not significantly alter the inhibitory effect of HNO on lysosomal cathepsin B activity, but protected the proteasome from HNO-induced inhibition. The protection awarded by macrophage differentiation was associated with induction of the GSH synthesis rate-limiting enzyme gamma-glutamylcysteine synthetase, as well as with increased intracellular GSH. In conclusion, HNO abrogates both lysosomal and cytosolic proteolysis in THP-1 cells. Macrophage differentiation, associated with upregulation of antioxidant defenses such as increased cellular GSH, does not protect the lysosomal cysteine protease cathepsin B from inhibition.     

Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling

Lysyl hydroxylase 3 (LH3), the multifunctional enzyme associated with collagen biosynthesis that possesses lysyl hydroxylase and collagen glycosyltransferase activities, has been characterized in the extracellular space in this study. Lysine modifications are known to occur in the endoplasmic reticulum (ER) prior to collagen triple-helix formation, but in this study we show that LH3 is also present and active in the extracellular space. Studies with in vitro cultured cells indicate that LH3, in addition to being an ER resident, is secreted from the cells and is found both in the medium and on the cell surface associated with collagens or other proteins with collagenous sequences. Furthermore, in vivo, LH3 is present in serum. LH3 protein levels correlate with the galactosylhydroxylysine glucosyltransferase (GGT) activity of mouse tissues. This, together with other data, indicates that LH3 is responsible for GGT activity in the tissues and that GGT activity assays can be used to quantify LH3 in tissues. LH3 in vivo is located in two compartments, in the ER and in the extracellular space, and the partitioning varies with tissue type. In mouse kidney the enzyme is located mainly intracellularly, whereas in mouse liver it is located solely in the extracellular space. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, nondenaturated conformation reveals a new dynamic in extracellular matrix remodeling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine-linked carbohydrates in collagenous proteins.     

MASQOT-GUI: spot quality assessment for the two-channel microarray platform

MASQOT-GUI provides an open-source, platform-independent software pipeline for two-channel microarray spot quality control. This includes gridding, segmentation, quantification, quality assessment and data visualization. It hosts a set of independent applications, with interactions between the tools as well as import and export support for external software. The implementation of automated multivariate quality control assessment, which is a unique feature of MASQOT-GUI, is based on the previously documented and evaluated MASQOT methodology. Further abilities of the application are outlined and illustrated. AVAILABILITY: MASQOT-GUI is Java-based and licensed under the GNU LGPL. Source code and installation files are available for download at http://masqot-gui.sourceforge.net/     

Conformation study of 2-arylbenzimidazoles as inhibitors of Chlamydia pneumoniae growth

Chlamydia pneumoniae is a worldwide cause of various respiratory track diseases ranging from asymptomatic pharyngeal infection to severe, sometimes fatal pneumonia. We have previously identified 2-arylbenzimidazoles as highly active antichlamydial compounds. In this work the importance of conformational effects on the structure-activity relationship of these compounds was studied. To simplify calculations, properly substituted N-phenylbenzamides, or the corresponding heterocyclic compounds, and 2-arylbenzimidazoles were used as model compounds. They were energy minimized and the energy differences between certain conformations were calculated. The main finding was that the compounds which can more easily adopt a non-planar conformation show higher bioactivity. This finding can be utilized in designing new derivatives or in constructing a pharmacophore model.     

Net atmospheric impacts of forest bioenergy production and utilization in Finnish boreal conditions

The net CO₂ exchange of forests was investigated to study net atmospheric impact of forest bioenergy production (BP) and utilization in Finnish boreal conditions. Net CO₂ exchange was simulated with a life cycle assessment tool over a 90‐year period and over the whole Finland based on National Forest Inventory data. The difference in the net exchanges between the traditional timber production (TP) and BP regime was considered the net atmospheric impact of forest bioenergy utilization. According to the results, forests became net sources of CO₂ after about 20 years of simulation, and the net exchange was higher in the BP regime than in the TP regime until the middle of the simulation period. From 2040 onwards, the net exchange started to decrease in both regimes and became higher in the TP regime, excluding the last decade of the simulation. The shift of forests to becoming a CO₂ source reflected the decrease in CO₂ sequestration due to the increasing share of recently harvested and seedling stands that are acting as sources of CO₂, and an increase of emissions from degradation of wood products. When expressed in terms of radiative forcing, the net atmospheric impact was on average 19% less for bioenergy compared with that for coal energy over the whole simulation period. The results show the importance of time dependence when considering dynamic forest ecosystems in BP and climate change mitigation. Furthermore, the results emphasize the dualistic role and possibilities of forest management in controlling the build and release of carbon into and from the stocks and in controlling the rate of the build speed, i.e. growth. This information is needed in identifying the capability and possibilities of ecosystems to produce biomass for energy, alongside other products and ecosystem services (e.g. pulp wood and timber), and simultaneously to mitigate climate change.     

New insights into Staphylococcus aureus stress tolerance and virulence regulation from an analysis of the role of the ClpP protease in the strains Newman, COL, and SA564

In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here reassessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, and arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.     

Seasonal movements and habitat use of river whitefish (Coregonus lavaretus) in the Koitajoki River (Finland), as determined by Carlin tagging and acoustic telemetry

In the Koitajoki River, Eastern Finland, there exists a self-sustaining population of river-spawning densely rakered whitefish (Coregonus lavaretus pallasi). The population has been classified as near-threatened due to overexploitation and possible hybridization with the lake-spawning sparsely rakered whitefish form. Thus, knowledge of habitat use, movements, and migrations is important for threat assessment and management decisions. We employed conventional tagging and acoustic telemetry to provide information on movements and home range of the Koitajoki River whitefish. We tagged mature individuals at their spawning areas and followed fish movements by tag recoveries and continuously recording fixed receiving stations. The maximum movement distances were 40–50?km both upstream and downstream from the releasing site. There were indications of a clear seasonal migration pattern and spawning-site fidelity; riverine habitats were mainly occupied for spawning and overwintering, while feeding and growth mainly took place in lacustrine environments within several lakes of the river system. Due to their migratory nature, whitefish is vulnerable to environmental disturbances and overfishing in a large geographic area.     

Genetics of microenvironmental sensitivity of body weight in rainbow trout (Oncorhynchus mykiss) selected for improved growth

Microenvironmental sensitivity of a genotype refers to the ability to buffer against non-specific environmental factors, and it can be quantified by the amount of residual variation in a trait expressed by the genotype's offspring within a (macro)environment. Due to the high degree of polymorphism in behavioral, growth and life-history traits, both farmed and wild salmonids are highly susceptible to microenvironmental variation, yet the heritable basis of this characteristic remains unknown. We estimated the genetic (co)variance of body weight and its residual variation in 2-year-old rainbow trout (Oncorhynchus mykiss) using a multigenerational data of 45,900 individuals from the Finnish national breeding programme. We also tested whether or not microenvironmental sensitivity has been changed as a correlated genetic response when genetic improvement for growth has been practiced over five generations. The animal model analysis revealed the presence of genetic heterogeneity both in body weight and its residual variation. Heritability of residual variation was remarkably lower (0.02) than that for body weight (0.35). However, genetic coefficient of variation was notable in both body weight (14%) and its residual variation (37%), suggesting a substantial potential for selection responses in both traits. Furthermore, a significant negative genetic correlation (-0.16) was found between body weight and its residual variation, i.e., rapidly growing genotypes are also more tolerant to perturbations in microenvironment. The genetic trends showed that fish growth was successfully increased by selective breeding (an average of 6% per generation), whereas no genetic change occurred in residual variation during the same period. The results imply that genetic improvement for body weight does not cause a concomitant increase in microenvironmental sensitivity. For commercial production, however, there may be high potential to simultaneously improve weight gain and increase its uniformity if both criteria are included in a selection index.