Autophagic clearance of Sin Nombre hantavirus glycoprotein Gn promotes virus replication in cells

Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells.     

汉滩病毒包膜糖蛋白糖基化位点突变体重组假病毒的构建及初步鉴定

目的：为进一步研究汉坦病毒包膜糖蛋白的糖基化与病毒的感染性和免疫原性等的关系,构建含有汉滩病毒（HTNV）囊膜糖蛋白（GP）糖基化位点突变体的重组假病毒。方法：利用定点突变的方法,分别突变了HTNV 76-118株的5个N-糖基化位点并克隆入慢病毒表达载体,与包装质粒共转染293T细胞,构建5株重组假病毒。感染HEK293细胞后,进行RT-PCR鉴定及免疫荧光检测。结果：经测序显示构建的含有N-糖基化位点突变体的5个重组假病毒原序列中的天冬酰胺（N）均被置换为谷氨酰胺（Q）。RT-PCR结果显示5个重组假病毒均有HTNV GP基因的表达。免疫荧光检测5个重组假病毒均可表达HTNV的Gn和Gc蛋白。结论：成功构建了含有HTNV包膜糖蛋白糖基化位点突变体的5个重组假病毒,分别命名为rLV-M1、rLV-M2、rLV-M3、rLV-M4和rLV-M5。本研究为明确N-糖基化对汉坦病毒生物学活性的影响提供了有利的研究工具,并为汉坦病毒疫苗及致病机理的进一步研究打下了一定的基础。     

Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.     

Host mTORC1 Signaling Regulates Andes Virus Replication

Hantavirus pulmonary syndrome (HPS) is a severe respiratory disease characterized by pulmonary edema, with fatality rates of 35 to 45%. Disease occurs following infection with pathogenic New World hantaviruses, such as Andes virus (ANDV), which targets lung microvascular endothelial cells. During replication, the virus scavenges 5′-m⁷G caps from cellular mRNA to ensure efficient translation of viral proteins by the host cell cap-dependent translation machinery. In cells, the mammalian target of rapamycin (mTOR) regulates the activity of host cap-dependent translation by integrating amino acid, energy, and oxygen availability signals. Since there is no approved pharmacological treatment for HPS, we investigated whether inhibitors of the mTOR pathway could reduce hantavirus infection. Here, we demonstrate that treatment with the FDA-approved rapamycin analogue temsirolimus (CCI-779) blocks ANDV protein expression and virion release but not entry into primary human microvascular endothelial cells. This effect was specific to viral proteins, as temsirolimus treatment did not block host protein synthesis. We confirmed that temsirolimus targeted host mTOR complex 1 (mTORC1) and not a viral protein, as knockdown of mTORC1 and mTORC1 activators but not mTOR complex 2 components reduced ANDV replication. Additionally, primary fibroblasts from a patient with tuberous sclerosis exhibited increased mTORC1 activity and increased ANDV protein expression, which were blocked following temsirolimus treatment. Finally, we show that ANDV glycoprotein Gn colocalized with mTOR and lysosomes in infected cells. Together, these data demonstrate that mTORC1 signaling regulates ANDV replication and suggest that the hantavirus Gn protein may modulate mTOR and lysosomal signaling during infection, thus bypassing the cellular regulation of translation.     

Characterization of monoclonal antibodies against Gnathostoma nipponicum

Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (>42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P < 0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.     

Dual-gradient high-performance liquid chromatography for identification of cytosolic high-mannose-type free glycans

It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N′-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells.     

Detection of 23-27 kDa GTP-binding proteins in platelets and other cells.

Membrane proteins from rabbit and human platelets were separated by SDS/polyacrylamide-gel electrophoresis and the resolved polypeptides blotted on nitrocellulose. A family of GTP-binding proteins, termed Gn proteins, was detected by incubation of these blots with [alpha-32P]GTP in the presence of Mg2+. A major Gn protein with a molecular mass of 27 kDa (Gn27) and lesser amounts of 23, 24 and 25 kDa Gn proteins were observed in platelet membranes; much smaller amounts were in the platelet soluble fraction. Binding of [alpha-32P]GTP by platelet Gn proteins was blocked by GDP, GTP or guanosine 5'-[gamma-thio]triphosphate, but not by GMP or adenosine 5'-[beta gamma-imido]triphosphate. Rabbit and human red-cell membranes contained only Gn27. When rat tissues were analysed for Gn proteins, the largest amounts were found in brain, which contained two membrane-bound forms (Gn27 and Gn26) and a soluble form (Gn26).     

Carbohydrate engineering of the recognition motifs in streptococcal co-aggregation receptor polysaccharides

The cell wall polysaccharides of certain oral streptococci function as receptors for the lectin-like surface adhesins on other members of the oral biofilm community. Recognition of these receptor polysaccharides (RPS) depends on the presence of a host-like motif, either GalNAcbeta1-3Gal (Gn) or Galbeta1-3GalNAc (G), within the oligosaccharide repeating units of different RPS structural types. Type 2Gn RPS of Streptococcus gordonii 38 and type 2G RPS of Streptococcus oralis J22 are composed of heptasaccharide repeats that are identical except for their host-like motifs. In the current investigation, the genes for the glycosyltransferases that synthesize these motifs were identified by high-resolution nuclear magnetic resonance (NMR) analysis of genetically altered polysaccharides. RPS production was switched from type 2Gn to 2G by replacing wefC and wefD in the type 2Gn gene cluster of S. gordonii 38 with wefF and wefG from the type 2G cluster of S. oralis J22. Disruption of either wefC or wefF abolished cell surface RPS production. In contrast, disruption of wefD in the type 2Gn cluster or wefG in the type 2G cluster eliminated beta-GalNAc from the Gn motif or beta-Gal from the G motif, resulting in mutant polysaccharides with hexa- rather than heptasaccharide subunits. The mutant polysaccharides reacted like wild-type RPS with rabbit antibodies against type 2Gn or 2G RPS but were inactive as co-aggregation receptors. Additional mutant polysaccharides with GalNAcbeta1-3GalNAc or Galbeta1-3Gal recognition motifs were engineered by replacing wefC in the type 2Gn cluster with wefF or wefF in the type 2G cluster with wefC respectively. The reactions of these genetically modified polysaccharides as antigens and receptors provide further insight into the structural basis of RPS function.     

Sexual behavior of men with isolated hypogonadotropic hypogonadism or prepubertal anterior panhypopituitarism

Sexual behavior of men with secondary hypogonadism was studied. Seven of the thirteen subjects presented with hypogonadism secondary to isolated gonadotropin (Gn) deficit, whereas the other six had idiopathic prepubertal anterior panhypopituitarism. Testosterone (T) levels were low and did not differ between the two groups. All subjects were evaluated both during replacement therapy (Gn in the first group; Gn plus cortisone and thyroxine in the second group) and 2 months after withdrawal of Gn therapy. During and after withdrawal of Gn administration, men with isolated deficit of Gn retained sexual activity and nocturnal penile tumescence, although they were partially compromised compared with a control group; on the other hand, panhypopituitarics reported compromised sexual function during Gn treatment and no sexual function when Gn therapy was not given. We conclude that different lesions of the hypothalamus-pituitary axis were accompanied by varying degrees of sexual impairment in the two groups of men presenting both secondary hypogonadism and very low T levels.     

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.     

Overexpression of the Golgi-localized enzyme alpha-mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl-N-acetylchitobiose to tetramannosyl-N-acetylchitobiose in the N-glycan-processing pathway.

Golgi alpha-mannosidase II is an enzyme that processes the intermediate oligosaccharide Gn(1)M(5)Gn(2) to Gn(1)M(3)Gn(2) during biosynthesis of N-glycans. Previously, we isolated a cDNA encoding a protein homologous to alpha-mannosidase II and designated it alpha-mannosidase IIx. Here, we show by immunocytochemistry that alpha-mannosidase IIx resides in the Golgi in HeLa cells. When coexpressed with alpha-mannosidase II, alpha-mannosidase IIx colocalizes with alpha-mannosidase II in COS cells. A protein A fusion of the catalytic domain of alpha-mannosidase IIx hydrolyzes a synthetic substrate, 4-umbelliferyl-alpha-D-mannoside, and this activity is inhibited by swainsonine. [(3)H]glucosamine-labeled Chinese hamster ovary cells overexpressing alpha-mannosidase IIx show a reduction of M(6)Gn(2) and an accumulation of M(4)Gn(2). Structural analysis identified M(4)Gn(2) to be Man alpha 1-->6(Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc. The results suggest that alpha-mannosidase IIx hydrolyzes two peripheral Man alpha 1-->6 and Man alpha 1-->3 residues from [(Man alpha 1-->6)(Man alpha 1-->3)Man alpha 1-->6](Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc, during N-glycan processing.     

Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts

High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.     

Secretory activity of gonadotropin and the responsiveness of gonadotrophs to gonadotropin-releasing hormone during the annual reproductive cycle of male bats, Rhinolophus ferrumequinum: analysis by cell immunoblot assay

The purpose of this study was to examine secretory activity of gonadotropin (Gn) and the responsiveness of Gn secretion to Gn-releasing hormone (GnRH) in male horseshoe bats, Rhinolophus ferrumequinum, during the annual reproductive cycle. Anterior pituitary cells were monodispersed and subjected to cell immunoblot assay for Gn. Cell blots specific for follicle stimulating hormone (FSH) or luteinizing hormone (LH) were quantified using a microscopic image analyzer. The percentages of LH- or FSH-secreting cells detected as immunoreactive cell blots were markedly increased in the spermatogenic period (summer) and decreased in the hibernation period (winter). The mean Gn secretion from individual cells and total Gn secretion per unit area of the transfer membrane also showed similar changes. The responsiveness of Gn secretion to GnRH was greater in the spermatogenic period than in other seasons. On the other hand, although the secretory activity of Gn was markedly decreased during hibernation, a stimulatory effect of GnRH on Gn secretion was observed. These findings suggest that seasonal changes in the release of Gn required for gametogenesis and gonadal steroidogenesis varied depending on the reproductive activity and seasonal changes in Gn sensitivity to stimulatory effects of GnRH due to alterations in GnRH receptor numbers and/or in postreceptor events of gonadotrophs.     

细胞诱导分化剂对HL60细胞β-1,4-半乳糖基转移酶活性的影响

用荧光标记的受体底物（Ｇｎβ１－２Ｍα１－６（Ｇｎβ１－２Ｍα１－３）Ｍβ１－４Ｇｎβ１－４（Ｆｕｃα１－６）Ｇｎ－ＰＡ）,结合高效液相层析（ＨＰＬＣ）建立了细胞β－１,４－半乳糖基转移酶的活性检测方法．研究了ＨＬ６０细胞在体外低血清培养后不同的时间其酶活性的变化,发现１２至２４ｈ酶活性达到一个高峰,为５０．１４ｐｍｏｌ／ｍｉｎ（１０６ｃｅｌ）,此时细胞处在分裂间期,其它各测定时间变化不大．用ＰＭＡ,ＲＡ等细胞诱导分化剂处理ＨＬ６０细胞株时,发现其活性发生了较明显的变化,ＰＭＡ诱导的细胞其酶活性在２４ｈ变化最大,升高到对照的１．３２倍;而ＲＡ处理的细胞其酶活性在７２ｈ变化最大,升高到对照的２．１５倍．     

Biochemical discrimination of the predominant pertussis toxin substrate of rabbit neutrophils from brain Gi and Go: isoelectric focusing improves resolution of pertussis toxin substrates

In the present study, the predominant pertussis toxin substrate in rabbit neutrophils, Gn, was biochemically compared to Gi and Go purified from brain, after being [32P]ADP-ribosylated by activated pertussis toxin and [32P]NAD. On SDS-polyacrylamide gels, a poorly resolved doublet from neutrophil membranes was observed; the upper band, corresponding to approximately equal to 25% labeling, comigrated with Gi-alpha and the predominant lower band, Gn, migrated intermediately between Gi-alpha and Go-alpha. Peptide maps generated by limited-digestion of the labeled Gn, Gi and Go with S. aureus V8 protease were slightly, but definitively and reproducibly different. Isoelectric focusing clearly distinguished Gn from the other two pertussis toxin substrates. The pI value of Gn, 5.60, was distinctly different from those of Gi, 5.75 and 5.80. Although the pI values for Go and Gn were similar (5.60), the patterns of the two proteins were qualitatively different, with Go being resolved into an equal doublet (pI = 5.55 and 5.60) while Gn appeared predominantly as a single band. Thus, Gn is biochemically distinguishable from Gi and Go of brain and these structural differences are most clearly evident following isoelectric focusing.     

G alpha(q) signal in osteoblasts is inhibitory to the osteoanabolic action of parathyroid hormone

This study examined the role of the Gα(q) signal constituted by Gα(q) and Gα(11) (encoded by Gnα(q) and Gnα(11), respectively), a major intracellular pathway of parathyroid hormone (PTH), in the PTH osteoanabolic action by the gain- and loss-of-function analyses. Transgenic mice with osteoblast-specific overexpression of the constitutively active Gnα(q) gene under the control of 2.3-kb type I collagen α1 chain (Col1a1) promoter exhibited osteopenia with decreased bone formation parameters and did not respond to the daily PTH treatment. We then established osteoblast-specific Gnα(q) and Gnα(11) double-knock-out (cDKO) mice by crossing the 2.3-kb Col1a1 promoter-Cre recombinase transgenic mice and those with Gnα(q) gene flanked with loxP and global ablation of Gnα(11) (Col1a1-Cre(+/-);Gna(q)(fl/fl);Gna(11)(-/-)) and found that the cDKO and single knock-out littermates of Gnα(q) or Gnα(11) exhibited normal bone volume and turnover under physiological conditions. With a daily injection of PTH, however, the cDKO mice, but not the single knock-out mice, showed higher bone volume and turnover than the wild-type littermates. Cultures of primary osteoblasts derived from cDKO and wild-type littermates confirmed enhancement of the PTH osteoanabolic action by the Gα(q) signal deficiency in a cell-autonomous mechanism, in association with the membrane translocation of protein kinase Cδ. This enhancement was reproduced by overexpression of regulator of G protein signaling-2, a Gα(q) signal inhibitor, in osteoblastic MC3T3-E1 cells. Hence, the Gα(q) signal plays an inhibitory role in the PTH osteoanabolic action, suggesting that its suppression may lead to a novel treatment in combination with PTH against osteoporosis.     

Structural studies of Hantaan virus

Hantaan virus is the prototypic member of the Hantavirus genus within the family Bunyaviridae and is a causative agent of the potentially fatal hemorrhagic fever with renal syndrome. The Bunyaviridae are a family of negative-sense RNA viruses with three-part segmented genomes. Virions are enveloped and decorated with spikes derived from a pair of glycoproteins (Gn and Gc). Here, we present cryo-electron tomography and single-particle cryo-electron microscopy studies of Hantaan virus virions. We have determined the structure of the tetrameric Gn-Gc spike complex to a resolution of 2.5 nm and show that spikes are ordered in lattices on the virion surface. Large cytoplasmic extensions associated with each Gn-Gc spike also form a lattice on the inner surface of the viral membrane. Rod-shaped ribonucleoprotein complexes are arranged into nearly parallel pairs and triplets within virions. Our results differ from the T=12 icosahedral organization found for some bunyaviruses. However, a comparison of our results with the previous tomographic studies of the nonpathogenic Tula hantavirus indicates a common structural organization for hantaviruses.