Exploring the relationship between α-actinin-3 deficiency and obesity in mice and humans.

Obesity is a worldwide health crisis, and the identification of genetic modifiers of weight gain is crucial in understanding this complex disorder. A common null polymorphism in the fast fiber-specific gene ACTN3 (R577X) is known to influence skeletal muscle function and metabolism. α-Actinin-3 deficiency occurs in an estimated 1.5 billion people worldwide, and results in reduced muscle strength and a shift towards a more efficient oxidative metabolism. The X-allele has undergone strong positive selection during recent human evolution, and in this study, we sought to determine whether ACTN3 genotype influences weight gain and obesity in mice and humans. An Actn3 KO mouse has been generated on two genetic backgrounds (129X1/SvJ and C57BL/6J) and fed a high-fat diet (HFD, 45% calories from fat). Anthropomorphic features (including body weight) were examined and show that Actn3 KO 129X1/SvJ mice gained less weight compared to WT. In addition, six independent human cohorts were genotyped for ACTN3 R577X (Rs1815739) and body mass index (BMI), waist-to-hip ratio-adjusted BMI (WHRadjBMI) and obesity-related traits were assessed. In humans, ACTN3 genotype alone does not contribute to alterations in BMI or obesity.

α-Actinin-3 deficiency alters muscle adaptation in response to denervation and immobilization.

Homozygosity for a common null polymorphism (R577X) in the ACTN3 gene results in the absence of the fast fibre-specific protein, α-actinin-3 in ∼16% of humans worldwide. α-Actinin-3 deficiency is detrimental to optimal sprint performance and benefits endurance performance in elite athletes. In the general population, α-actinin-3 deficiency is associated with reduced muscle mass, strength and fast muscle fibre area, and poorer muscle function with age. The Actn3 knock-out (KO) mouse model mimics the human phenotype, with fast fibres showing a shift towards slow/oxidative metabolism without a change in myosin heavy chain (MyHC) isoform. We have recently shown that these changes are attributable to increased activity of the calcineurin-dependent signalling pathway in α-actinin-3 deficient muscle, resulting in enhanced response to exercise training. This led us to hypothesize that the Actn3 genotype influences muscle adaptation to disuse, irrespective of neural innervation. Separate cohorts of KO and wild-type mice underwent 2 weeks immobilization and 2 and 8 weeks of denervation. Absence of α-actinin-3 resulted in reduced atrophic response and altered adaptation to disuse, as measured by a change in MyHC isoform. KO mice had a lower threshold to switch from the predominantly fast to a slower muscle phenotype (in response to immobilization) and a higher threshold to switch to a faster muscle phenotype (in response to denervation). We propose that this change is mediated through baseline alterations in the calcineurin signalling pathway of Actn3 KO muscle. Our findings have important implications for understanding individual responses to muscle disuse/disease and training in the general population.

Determinants of organ tropism of Sendai virus.

Wild-type Sendai virus is exclusively pneumotropic in mice. Protease activation mutants, ts-f1 and F1-R, were isolated from persistently infected tissue culture cells. Additional mutants were isolated from wild-type Sendai virus with phenotypes similar to the pantropic mutant, F1-R. The genome of the mutants was sequenced and mutations were revealed in several proteins encoded by the genes. Three of the six mutations in the fusion (F) proteins were considered prime candidates for the determinant of pantropism. Characterization of the mutants led to the finding that the exchange (Ser to Pro) residue 115 next to the cleavage site of the F protein was the primary determinant that resulted in the enhanced cleavability of the F protein. Another important finding was bipolar budding of F1-R in polarized epithelial cells and mouse bronchial epithelium. This has been attributed to two mutations in the matrix (M) protein, at residues 128 (Asp to Gly) and 210 (Ile to Thr). Thus, the determinants of pantropism of F1-R are protease activation of the F protein and biopolar budding attributed to the mutated M protein.

Determinants of organ tropism of sendai virus.

Wild-type Sendai virus is exclusively pneumotropic in mice. Protease activation mutants, ts-f1 and F1-R, were isolated from persistently infected tissue culture cells. Additional mutants were isolated from wild-type Sendai virus with phenotypes similar to the pantropic mutant, F1-R. The genome of the mutants was sequenced and mutations were revealed in several proteins encoded by the genes. Three of the six mutations in the fusion (F) proteins were considered prime candidates for the determinant of pantropism. Characterization of the mutants led to the finding that the exchange (Ser to Pro) residue 115 next to the cleavage site of the F protein was the primary determinant that resulted in the enhanced cleavability of the F protein. Another important finding was bipolar budding of F1-R in polarized epithelial cells and mouse bronchial epithelium. This has been attributed to two mutations in the matrix (M) protein, at residues 128 (Asp to Gly) and 210 (Ile to Thr). Thus the determinants of pantropism of F1-R are protease activation of the F protein and bipolar budding attributed to the mutated M protein and enhanced disruption of microtubules.

Scanning electron microscope examination of epithelial cells infected with enveloped viruses.

The apical surface structure of virus infected epithelial cells (MDBK and MDCK) was examined in the SEM. Cells were infected with related enveloped viruses (influenza, Sendai, and vesicular stomatitis) under conditions of productive, nonproductive, and persistent infections. Fewer microvilli were seen in persistent infections with Sendai virus and the standard virus infected cells appeared to be normal. Extensive morphological changes, stunting of microvilli or budding effects, were noted in productive infections with influenza virus (WSN) and a reduction in the relative number of microvilli in a nonproductive infection with the PR8 strain. Although budding occurs on the basal lateral surface of VSV infected cells, morphological alterations were evident on the apical surface unlike that seen in influenza and Sendai virus infections.

Immunoelectron microscopic study of the detection of the glycoproteins of influenza and Sendai viruses in infected cells by the immunoperoxidase method.

The envelope glycoproteins of influenza virus (HA and NA) and paramyxovirus (HN and F) were visualized on the surface of infected cells by immunoelectron microscopy using the indirect immunoperoxidase technique. In X7 influenza virus-infected fibroblasts, the hemagglutinin (HA) and the neuraminidase (NA) were observed on the cell membrane respectively 2 and 3--4 h after infection. The antigens were initially seen as discrete patches and later evenly distributed along the plasma membrane prior to budding. Antibody induction of HA and NA was observed as cytoplasmic inclusions, with peroxidase-positive activity, attributed to endocytosis. The redistribution of HA and NA supports the hypothesis of lateral mobility of the viral glycoproteins in cellular membranes as visualized by the immunoperoxidase method. The glycoproteins of Sendai virus, in infected Madin--Darby bovine kidney cells, were found to be evenly distributed along the plasma membrane and endoplasmic reticulum, the latter by the indirect microperoxidase method. The immunoperoxidase methods may be useful for investigating the polarized distribution of envelope glycoproteins.

A gene for speed: contractile properties of isolated whole EDL muscle from an alpha-actinin-3 knockout mouse.

The actin-binding protein alpha-actinin-3 is one of the two isoforms of alpha-actinin that are found in the Z-discs of skeletal muscle. alpha-Actinin-3 is exclusively expressed in fast glycolytic muscle fibers. Homozygosity for a common polymorphism in the ACTN3 gene results in complete deficiency of alpha-actinin-3 in about 1 billion individuals worldwide. Recent genetic studies suggest that the absence of alpha-actinin-3 is detrimental to sprint and power performance in elite athletes and in the general population. In contrast, alpha-actinin-3 deficiency appears to be beneficial for endurance athletes. To determine the effect of alpha-actinin-3 deficiency on the contractile properties of skeletal muscle, we studied isolated extensor digitorum longus (fast-twitch) muscles from a specially developed alpha-actinin-3 knockout (KO) mouse. alpha-Actinin-3-deficient muscles showed similar levels of damage to wild-type (WT) muscles following lengthening contractions of 20% strain, suggesting that the presence or absence of alpha-actinin-3 does not significantly influence the mechanical stability of the sarcomere in the mouse. alpha-Actinin-3 deficiency does not result in any change in myosin heavy chain expression. However, compared with alpha-actinin-3-positive muscles, alpha-actinin-3-deficient muscles displayed longer twitch half-relaxation times, better recovery from fatigue, smaller cross-sectional areas, and lower twitch-to-tetanus ratios. We conclude that alpha-actinin-3 deficiency results in fast-twitch, glycolytic fibers developing slower-twitch, more oxidative properties. These changes in the contractile properties of fast-twitch skeletal muscle from alpha-actinin-3-deficient individuals would be detrimental to optimal sprint and power performance, but beneficial for endurance performance.

Molecular biology of the pathogenesis of Sendai viruses.

Protease activation mutant (ts-f1) was isolated from persistently infected cells, and a pantropic mutant, F1-R, was derived from ts-f1. The mutants have been found to be extremely useful for investigations on the molecular biology of paramyxoviruses. The genome of the mutants has been sequenced and mutations were revealed in several proteins encoded by the genes. Three of the six mutations in the fusion (F) proteins were considered prime candidates for the determinants of pantropism. Characterization of the revertants, that are no longer pantropic and derived from F1-R, revealed that the mutation at amino acid residue (115 Arg to Pro) of the F protein is responsible for pantropism. Another important finding was bipolar budding of F1-R in polarized epithelial cells and mouse bronchial epithelium. It has been postulated that mutation(s) in the matrix (M) protein may be associated with bipolar budding since the revertants retained this phenotype of F1-R.

Functional significance of sialidase during influenza virus multiplication: an electron microscope study.

Morphological evidence has been obtained by electron microscopy in support of previous findings that one of the most important functions of sialidase is associated with the release of virus from infected host cells. Highly specific antiserum against fowl plague virus enzyme and specific antiserum against X7 recombinant influenza virus enzyme were shown to influence the morphology of cells infected with their homologous virus. In the presence of enzyme antiserum, an accumulation and aggregation of virus particles were evident on the cell surface and in the extracellular space of infected host cells. The aggregation of virus particles was interpreted to result from the inhibition of the release of virus.

Glycoproteins of Sendai virus: purification and antigenic analysis.

Solubilized envelope antigens (glycoproteins) of Sendai virus were purified in a DEAE-Bio-Gel A column. Monospecific antisera were prepared against the antigens, designated HN and F glycoproteins. These glycoproteins are antigenically distinct. Immunodiffusion analyses of the envelope antigens of three representative paramyxoviruses (Sendai, Yucaipa, and Newcastle disease virus)did not reveal any cross-reactions among them with antisera specific for Sendai virus glycoproteins.

Electron microscope study of ribonucleic acid of myxoviruses.

Intact ribonucleic acid (RNA) molecules in an extended form were extracted from purified influenza virus and observed in the electron microscope. For this study, the RNA extraction procedure and the Kleinschmidt protein monolayer technique were modified. The mean lengths of RNA from X7, X7-F1, and WSN strains of influenza virus were found to be 2.69, 2.55, and 2.37 mum, respectively. From these measurements, the corresponding estimated molecular weights would be 2.9, 2.8, and 2.5 x 10(6) daltons. X7 and WSN RNA preparations were exposed to pH 3 to disrupt intact molecules. Histograms of length measurements showed five peaks, which were interpreted to represent the five pieces of RNA reported to exist in the influenza virion. X7 RNA appeared to be more stable than WSN RNA when stored at 4 C. The profiles of histograms of incomplete virus RNA suggest that the high molecular-weight component is missing. In preliminary experiments on Newcastle disease virus RNA, molecules of various lengths were observed.

Electron microscope study of cultured cells of the chorioallantoic membrane infected with representative paramyxoviruses.

Chorioallantoic membrane (CAM) tissue cultures were found to be permissive for representative paramyxoviruses. The CAM cells can be used for plaque assay without the presence of trypsin. Ultrastructures of CAM cells infected with paramyxovirus Yucaipa (PMY), Sendai virus, and NDV were different. Nucleocapsids were readily seen in budding structures of cells infected with PMY, and in Sendai virus infected cells, large clusters of nucleocapsids were clearly evident in the cytoplasm. The site of glycoprotein cleavage does not have any effect on the nature of budding. It appears that a factor or factors in addition to the nature of the plasma membrane influences the morphology of cells infected with paramyxoviruses.

Persistent infections with Sendai virus and Newcastle disease viruses.

Persistent infections (Pi) were established in two host-cell systems [Madin-Darby bovine kidney (MDBK) and Madin-Darby canine kidney (MDCK)] with Sendai virus and three strains of NDV, to test the influence of different viruses and host-cell systems. Virus was recovered from the persistently infected cells. An RNA- ts mutant was recovered from a Pi of MDBK cells, but no Pi could be established in MDCK cells with the three strains of NDV. Additionally, the Pi was established exclusively by a virulent strain, NDV-Milano. On the other hand, Sendai virus could establish Pi in MDBK and MDCK cell-systems. Several ts mutants were recovered from "late" passages of Pi, and from an accidental infection, a ts mutant with an altered P polypeptide. Ten other ts mutants were tested, however, the specific ts lesion could not be identified. From three Pi in MDCK cells, host range mutants (ts-f1, ts-f2, and ts-f3) were recovered. One of the mutants (ts-f1) has an altered M (matrix) protein. The host range mutants undergo a productive infection in MDBK and MDCK cells, which are nonpermissive for wild type Sendai virus. The possible significance of the results are discussed.

Comparison of protective effects of serum antibody on respiratory and systemic infection of Sendai virus in mice.

The protective effects of the passive administration of convalescent serum from mice infected with Sendai virus were evaluated in mice challenged intranasally with wild-type and a pantropic variant (F1-R) of Sendai virus. Adoptive transfer of the serum efficiently prevented F1-R from infecting the systemic organs, but it failed to protect the mice from infections of the respiratory tracts by either virus. Virus replication in nasal turbinates was not diminished while infection in the lung was suppressed sufficiently for the infected mice to survive the infection. These findings suggest that serum antibody is less effective for the protection against viral infections on the surface of the respiratory tract, but it is effective for inhibition of spread of the virus into the systemic organs.

The site of cleavage in infected cells and polypeptides of representative paramyxoviruses grown in cultured cells of the chorioallantoic membrane.

Cultured cells of the chorioallantoic membrane (CAM) fulfilled the need of using the same cell system that was permissive for representative paramyxoviruses to carry out studies on the biosynthesis of their glycoproteins in infected cells. The polypeptides composition of the respective paramyxoviruses [Newcastle disease virus (NDV), paramyxovirus Yucaipa (PMY), and Sendai virus], grown in eggs and CAM-cells, was essentially identical. In egg-grown PMY a large glycoprotein (LGP) was present but only in some CAM-grown preparations of virus labeled with [3H]-glucosamine and rarely in [35S]-methionine or [3H]-amino acids (valine, leucine, and tyrosine) labeled viruses. The site of cleavage of precursor F0 to F1,2 was not the same. In contrast to the cleavage of Sendai virus glycoprotein, cleavage was intracellular in NDV and PMY infected cells. Homologous antisera against the glycoproteins failed to inhibit cleavage of HN0 or F0 in cells infected with the representative paramyxoviruses.

Organ tropism of Sendai virus in mice: proteolytic activation of the fusion glycoprotein in mouse organs and budding site at the bronchial epithelium.

Wild-type Sendai virus is exclusively pneumotropic in mice, while a host range mutant, F1-R, is pantropic. The latter was attributed to structural changes in the fusion (F) glycoprotein, which was cleaved by ubiquitous proteases present in many organs (M. Tashiro, E. Pritzer, M. A. Khoshnan, M. Yamakawa, K. Kuroda, H.-D. Klenk, R. Rott, and J. T. Seto, Virology 165:577-583, 1988). These studies were extended by investigating, by use of an organ block culture system of mice, whether differences exist in the susceptibility of the lung and the other organs to the viruses and in proteolytic activation of the F protein of the viruses. Block cultures of mouse organs were shown to synthesize the viral polypeptides and to support productive infections by the viruses. These findings ruled out the possibility that pneumotropism of wild-type virus results because only the respiratory organs are susceptible to the virus. Progeny virus of F1-R was produced in the activated form as shown by infectivity assays and proteolytic cleavage of the F protein in the infected organ cultures. On the other hand, much of wild-type virus produced in cultures of organs other than lung remained nonactivated. The findings indicate that the F protein of wild-type virus was poorly activated by ubiquitous proteases which efficiently activated the F protein of F1-R. Thus, the activating protease for wild-type F protein is present only in the respiratory organs. These results, taken together with a comparison of the predicted amino acid substitutions between the viruses, strongly suggest that the different efficiencies among mouse organs in the proteolytic activation of F protein must be the primary determinant for organ tropism of Sendai virus. Additionally, immunoelectron microscopic examination of the mouse bronchus indicated that the budding site of wild-type virus was restricted to the apical domain of the epithelium, whereas budding by F1-R occurred at the apical and basal domains. Bipolar budding was also observed in MDCK monolayers infected with F1-R. The differential budding site at the primary target of infection may be an additional determinant for organ tropism of Sendai virus in mice.

Altered budding site of a pantropic mutant of Sendai virus, F1-R, in polarized epithelial cells.

A protease activation mutant of Sendai virus, F1-R, causes a systemic infection in mice, whereas wild-type virus is exclusively pneumotropic (M. Tashiro, E. Pritzer, M. A. Khoshnan, M. Yamakawa, K. Kuroda, H.-D. Klenk, R. Rott, and J. T. Seto, Virology 165:577-583, 1988). Budding of F1-R has been observed bidirectionally at the apical and basolateral surfaces of the bronchial epithelium of mice and of MDCK cells, whereas wild-type virus buds apically (M. Tashiro, M. Yamakawa, K. Tobita, H.-D. Klenk, R. Rott, and J. T. Seto, J. Virol. 64:3627-3634, 1990). In this study, wild-type virus was shown to be produced primarily from the apical site of polarized MDCK cells grown on permeable membrane filters. Surface immunofluorescence and immunoprecipitation analyses revealed that transmembrane glycoproteins HN and F were expressed predominantly at the apical domain of the plasma membrane. On the other hand, infectious progeny of F1-R was released from the apical and basolateral surfaces, and HN and F were expressed at both regions of the cells. Since F1-R has amino acid substitutions in F and M proteins but none in HN, the altered budding of the virus and transport of the envelope glycoproteins might be attributed to interactions by F and M proteins. These findings suggest that in addition to proteolytic activation of the F glycoprotein, the differential site of budding, at the primary target of infection, is a determinant for organ tropism of Sendai virus in mice.

Possible involvement of microtubule disruption in bipolar budding of a Sendai virus mutant, F1-R, in epithelial MDCK cells.

Envelope glycoproteins F and HN of wild-type Sendai virus are transported to the apical plasma membrane domain of polarized epithelial MDCK cells, where budding of progeny virus occurs. On the other hand, a pantropic mutant, F1-R, buds bipolarly at both the apical and basolateral domains, and the viral glycoproteins have also been shown to be transported to both of these domains (M. Tashiro, M. Yamakawa, K. Tobita, H.-D. Klenk, R. Rott, and J.T. Seto, J. Virol. 64:4672-4677, 1990). MDCK cells were infected with wild-type virus and treated with the microtubule-depolymerizing drugs colchicine and nocodazole. Budding of the virus and surface expression of the glycoproteins were found to occur in a nonpolarized fashion similar to that found in cells infected with F1-R. In uninfected cells, the drugs were shown to interfere with apical transport of a secretory cellular glycoprotein, gp80, and basolateral uptake of [35S]methionine as well as to disrupt microtubule structure, indicating that cellular polarity of MDCK cells depends on the presence of intact microtubules. Infection by the F1-R mutant partially affected the transport of gp80, uptake of [35S]methionine, and the microtubule network, whereas wild-type virus had a marginal effect. These results suggest that apical transport of the glycoproteins of wild-type Sendai virus in MDCK cells depends on intact microtubules and that bipolar budding by F1-R is possibly due, at least in part, to the disruption of microtubules. Nucleotide sequence analyses of the viral genes suggest that the mutated M protein of F1-R might be involved in the alteration of microtubules.

Significance of basolateral domain of polarized MDCK cells for Sendai virus-induced cell fusion.

Fusion (fusion from within) of polarized MDCK monolayer cells grown on porous membranes was examined after infection with Sendai viruses. Wild-type virus, that buds at the apical membrane domain, did not induce cell fusion even when the F glycoprotein expressed at the apical domain was activated with trypsin. On the other hand, a protease activation mutant, F1-R, with F protein in the activated form and that buds bipolarly at the apical and basolateral domains, caused syncytia formation in the absence of exogenous protease. Anti-Sendai virus antibodies added to the basolateral side, but not at the apical side, inhibited cell fusion induced by F1-R. In addition, T-9, a mutant with bipolar budding phenotype of F1-R but with an uncleavable F protein phenotype like wild-type virus, induced cell fusion exclusively when trypsin was added to the basolateral medium. By electron microscopy, cell-to-cell fusion was shown to occur at the lateral domain of the plasma membrane. These results indicate that in addition to proteolytic activation of the F protein, basolateral expression of Sendai virus envelope glycoproteins is required to induce cell fusion.

Tryptase Clara, an activating protease for Sendai virus in rat lungs, is involved in pneumopathogenicity.

Tryptase Clara is an arginine-specific serine protease localized exclusively in and secreted from Clara cells of the bronchial epithelium of rats (H. Kido, Y. Yokogoshi, K. Sakai, M. Tashiro, Y. Kishino, A. Fukutomi, and N. Katunuma, J. Biol. Chem. 267:13573-13579, 1992). The purified protease was shown in vitro to behave similarly to trypsin, cleaving the precursor glycoprotein F of Sendai virus at residue Arg-116 and activating viral infectivity in a dose-dependent manner. Anti-tryptase Clara antibody inhibited viral activation by the protease in vitro in lung block cultures and in vivo in infected rats. When the enzyme-specific antibody was administered intranasally to rats that were also infected intranasally with Sendai virus, activation of progeny virus in the lungs was significantly inhibited. Thus, multiple cycles of viral replication were suppressed, resulting in a reduction in lung lesions and in the mortality rate. These findings indicate that tryptase Clara is an activating protease for Sendai virus in rat lungs and is therefore involved in pulmonary pathogenicity of the virus in rats.