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Interferons control viral replication and the growth of some malignant tumors. Since systemic application may cause severe adverse effects, tissue-specific expression is an attractive alternative. Liver-directed interferon gene therapy offers promising applications such as chronic viral hepatitis B or C or hepatocellular carcinoma and thus needs testing in vivo in suitable animal models. We therefore used the Tet-On system to regulate gene expression in adenoviral vectors, and studied the effect of liver-specific and regulated interferon?expression in a mouse model of chronic hepatitis B virus (HBV) infection. In a first generation adenoviral vector, genes encoding for firefly luciferase and interferonsa,ßor?, respectively, were coexpressed under control of the bidirectional tetracycline-regulated promoter Ptetbi. Liver-specific promoters driving expression of the reverse tetracycline controlled transactivator ensured local expression in the livers of HBV transgenic mice. Following gene transfer, we demonstrated low background, tight regulation and a 1000-fold induction of gene expression by doxycycline. Both genes within the bidirectional transcription unit were expressed simultaneously, and in a liver-specific fashion in cell culture and in living mice. Doxycycline-dependent interferon?expression effectively controlled HBV replication in mice, but did not eliminate HBV transcripts. This system will help to study the effects of local cytokine expression in mouse disease models in detail.Gene Therapy (2005) 12, 668-677. doi:10.1038/sj.gt.3302449 Published online 13 January 2005 [ABSTRACT FROM AUTHOR]

Nasal xenobiotic metabolizing enzymes (XMEs) are important for the sense of smell because they influence odorant availability and quality. Since the major part of the human nasal cavity is lined by a respiratory mucosa, we hypothesized that this tissue contributed to nasal odorant metabolism through XME activity. Thus, we built human respiratory tissue models and characterized the XME profiles using single-cell RNA sequencing. We focused on the XMEs dicarbonyl and l-xylulose reductase, aldehyde dehydrogenase (ALDH) 1A1, and ALDH3A1, which play a role in food odorant metabolism. We demonstrated protein abundance and localization in the tissue models and showed the metabolic activity of the corresponding enzyme families by exposing the models to the odorants 3,4-hexandione and benzaldehyde. Using gas chromatography coupled with mass spectrometry, we observed, for example, a significantly higher formation of the corresponding metabolites 4-hydroxy-3-hexanone (39.03 ± 1.5%, p = 0.0022), benzyl alcohol (10.05 ± 0.88%, p = 0.0008), and benzoic acid (8.49 ± 0.57%, p = 0.0004) in odorant-treated tissue models compared to untreated controls (0 ± 0, 0.12 ± 0.12, and 0.18 ± 0.18%, respectively). This is the first study that reveals the XME profile of tissue-engineered human respiratory mucosa models and demonstrates their suitability to study nasal odorant metabolism.

Aspirin, with its active compound acetylsalicylic acid (ASA), shows antiviral activity against rhino- and influenza viruses at high concentrations. We sought to investigate whether ASA and its metabolite salicylic acid (SA) inhibit SARS-CoV-2 since it might use similar pathways to influenza viruses. The compound-treated cells were infected with SARS-CoV-2. Viral replication was analysed by RTqPCR. The compounds suppressed SARS-CoV-2 replication in cell culture cells and a patient-near replication system using human precision-cut lung slices by two orders of magnitude. While the compounds did not interfere with viral entry, it led to lower viral RNA expression after 24 h, indicating that post-entry pathways were inhibited by the compounds.

Background: Anthocyanin-containing plant extracts and carotenoids, such as astaxanthin, have been well-known for their antiviral and anti-inflammatory activity, respectively. We hypothesised that a mixture of Ribes nigrum L. (Grossulariaceae) (common name black currant (BC)) and Vaccinium myrtillus L. (Ericaceae) (common name bilberry (BL)) extracts (BC/BL) with standardised anthocyanin content as well as single plant extracts interfered with the replication of Measles virus and Herpesviruses in vitro., Methods: We treated cell cultures with BC/BL or defined single plant extracts, purified anthocyanins and astaxanthin in different concentrations and subsequently infected the cultures with the Measles virus (wild-type or vaccine strain Edmonston), Herpesvirus 1 or 8, or murine Cytomegalovirus. Then, we analysed the number of infected cells and viral infectivity and compared the data to non-treated controls., Results: The BC/BL extract inhibited wild-type Measles virus replication, syncytia formation and cell-to-cell spread. This suppression was dependent on the wild-type virus-receptor-interaction since the Measles vaccine strain was unaffected by BC/BL treatment. Furthermore, the evidence was provided that the delphinidin-3-rutinoside chloride, a component of BC/BL, and purified astaxanthin, were effective anti-Measles virus compounds. Human Herpesvirus 1 and murine Cytomegalovirus replication was inhibited by BC/BL, single bilberry or black currant extracts, and the BC/BL component delphinidin-3-glucoside chloride. Additionally, we observed that BC/BL seemed to act synergistically with aciclovir. Moreover, BC/BL, the single bilberry and black currant extracts, and the BC/BL components delphinidin-3-glucoside chloride, cyanidin-3-glucoside, delphinidin-3-rutinoside chloride, and petunidin-3-galactoside inhibited human Herpesvirus 8 replication., Conclusions: Our data indicate that Measles viruses and Herpesviruses are differentially susceptible to a specific BC/BL mixture, single plant extracts, purified anthocyanins and astaxanthin. These compounds might be used in the prevention of viral diseases and in addition to direct-acting antivirals, such as aciclovir., (© 2022. The Author(s).)

For the treatment of large bone defects, the commonly used technique of autologous bone grafting presents several drawbacks and limitations. With the discovery of the bone-inducing capabilities of bone morphogenetic protein 2 (BMP2), several delivery techniques were developed and translated to clinical applications. Implantation of scaffolds containing adsorbed BMP2 showed promising results. However, off-label use of this protein-scaffold combination caused severe complications due to an uncontrolled release of the growth factor, which has to be applied in supraphysiological doses in order to induce bone formation. Here, we propose an alternative strategy that focuses on the covalent immobilization of an engineered BMP2 variant to biocompatible scaffolds. The new BMP2 variant harbors an artificial amino acid with a specific functional group, allowing a site-directed covalent scaffold functionalization. The introduced artificial amino acid does not alter BMP2's bioactivity in vitro. When applied in vivo, the covalently coupled BMP2 variant induces the formation of bone tissue characterized by a structurally different morphology compared to that induced by the same scaffold containing ab-/adsorbed wild-type BMP2. Our results clearly show that this innovative technique comprises translational potential for the development of novel osteoinductive materials, improving safety for patients and reducing costs.

To study the interaction of human pathogens with their host target structures, human tissue models based on primary cells are considered suitable. Complex tissue models of the human airways have been used as infection models for various viral and bacterial pathogens. The Gram-negative bacterium Bordetella pertussis is of relevant clinical interest since whooping cough has developed into a resurgent infectious disease. In the present study, we created three-dimensional tissue models of the human ciliated nasal and tracheo-bronchial mucosa. We compared the innate immune response of these models towards the B. pertussis virulence factor adenylate cyclase toxin (CyaA) and its enzymatically inactive but fully pore-forming toxoid CyaA-AC - . Applying molecular biological, histological, and microbiological assays, we found that 1 µg/ml CyaA elevated the intracellular cAMP level but did not disturb the epithelial barrier integrity of nasal and tracheo-bronchial airway mucosa tissue models. Interestingly, CyaA significantly increased interleukin 6, interleukin 8, and human beta defensin 2 secretion in nasal tissue models, whereas tracheo-bronchial tissue models were not significantly affected compared to the controls. Subsequently, we investigated the interaction of B. pertussis with both differentiated primary nasal and tracheo-bronchial tissue models and demonstrated bacterial adherence and invasion without observing host cell type-specific significant differences. Even though the nasal and the tracheo-bronchial mucosa appear similar from a histological perspective, they are differentially susceptible to B. pertussis CyaA in vitro . Our finding that nasal tissue models showed an increased innate immune response towards the B. pertussis virulence factor CyaA compared to tracheo-bronchial tissue models may reflect the key role of the nasal airway mucosa as the first line of defense against airborne pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sivarajan, Kessie, Oberwinkler, Pallmann, Walles, Scherzad, Hackenberg and Steinke.)

To circumvent time-consuming clinical trials, testing whether existing drugs are effective inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this path and screened approved medications "off-label" against SARS-CoV-2. Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8 µg/ml significantly in these screenings, and the EC50 was determined with 387 ng/ml. Furthermore, Fluoxetine reduced viral infectivity in precision-cut human lung slices showing its activity in relevant human tissue targeted in severe infections. Fluoxetine treatment resulted in a decrease in viral protein expression. Fluoxetine is a racemate consisting of both stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that both isomers show similar activity on the virus, indicating that the R-form might specifically be used for SARS-CoV-2 treatment. Fluoxetine inhibited neither Rabies virus, human respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1 gene expression, indicating that it acts virus-specific. Moreover, since it is known that Fluoxetine inhibits cytokine release, we see the role of Fluoxetine in the treatment of SARS-CoV-2 infected patients of risk groups.

Bordetella pertussis is a highly contagious pathogen which causes whooping cough in humans. A major pathophysiology of infection is the extrusion of ciliated cells and subsequent disruption of the respiratory mucosa. Tracheal cytotoxin (TCT) is the only virulence factor produced by B. pertussis that has been able to recapitulate this pathology in animal models. This pathophysiology is well characterized in a hamster tracheal model, but human data are lacking due to scarcity of donor material. We assessed the impact of TCT and lipopolysaccharide (LPS) on the functional integrity of the human airway mucosa by using in vitro airway mucosa models developed by co-culturing human tracheobronchial epithelial cells and human tracheobronchial fibroblasts on porcine small intestinal submucosa scaffold under airlift conditions. TCT and LPS either alone and in combination induced blebbing and necrosis of the ciliated epithelia. TCT and LPS induced loss of ciliated epithelial cells and hyper-mucus production which interfered with mucociliary clearance. In addition, the toxins had a disruptive effect on the tight junction organization, significantly reduced transepithelial electrical resistance and increased FITC-Dextran permeability after toxin incubation. In summary, the results indicate that TCT collaborates with LPS to induce the disruption of the human airway mucosa as reported for the hamster tracheal model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kessie, Lodes, Oberwinkler, Goldman, Walles, Steinke and Gross.)

Three-dimensional respiratory tissue models have been generated using, for example, human primary airway epithelial cells (hAEC) or respective cell lines. To investigate ciliopathies, such as primary ciliary dyskinesia, the presence of functional kinocilia in vitro is an essential prerequisite. Since access to hAEC of healthy donors is limited, we aimed to identify a respiratory epithelial cell line that is capable to display functional kinocilia on at least 60% of the apical surface. Thus, we cultured four different human respiratory cell lines with human primary airway fibroblasts under airlift conditions, characterized the morphology, and analyzed ciliary function. Only one of the tested cell lines showed beating kinocilia; however, <10% of the whole surface was covered and ciliary beating was undirected. Positive control tissue models using hAEC and fibroblasts displayed expected directed ciliary beating pattern around 11 Hz. Our data show that the available cell lines are not suitable for basic and applied research questions whenever functional kinocilia are required and that, rather, hAEC- or human induced pluripotent stem cell-derived tissue models need to be generated. Impact Statement To study ciliopathies or Bordetella pertussis infection in vitro , three-dimensional respiratory tissue models with functional kinocilia covering at least 60% of the model's surface are mandatory. We cultured four respiratory cell lines on a fibroblast-loaded biological scaffold and showed that none of them met this requirement. In contrast, primary airway cell-derived models sufficiently reflected the mucociliary phenotype. To further search for an alternative to primary respiratory cells, investigations on other cell lines should be conducted or even new cell lines have to be generated.

Unlabelled: The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like., Importance: Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two- and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: The endocrine balance between atrial natriuretic peptide (ANP) and the renin-angiotensin-aldosterone system is critical for the maintenance of arterial blood pressure and volume homeostasis. This study investigated whether a cardiac imbalance between ANP and aldosterone, toward increased mineralocorticoid receptor (MR) signaling, contributes to adverse left ventricular remodeling in response to pressure overload., Methods and Results: We used the MR-selective antagonist eplerenone to test the role of MRs in mediating pressure overload-induced dilatative cardiomyopathy of mice with abolished local, cardiac ANP activity. In response to 21 days of transverse aortic constriction, mice with cardiomyocyte-restricted inactivation (knockout) of the ANP receptor (guanylyl cyclase [GC]-A) or the downstream cGMP-dependent protein kinase I developed enhanced left ventricular hypertrophy and fibrosis together with contractile dysfunction. Treatment with eplerenone (100 mg/kg/d) attenuated left ventricular hypertrophy and fully prevented fibrosis, dilatation, and failure. Transverse aortic constriction induced the cardiac expression of profibrotic connective tissue growth factor and attenuated the expression of SERCA2a (sarcoplasmic reticulum Ca(2+)-ATPase) in knockout mice, but not in controls. These genotype-dependent molecular changes were similarly prevented by eplerenone. ANP attenuated the aldosterone-induced nuclear translocation of MRs via GC-A/cGMP-dependent protein kinase I in transfected HEK 293 (human embryonic kidney) cells. Coimmunoprecipitation and fluorescence resonance energy transfer experiments demonstrated that a population of MRs were membrane associated in close interaction with GC-A and cGMP-dependent protein kinase I and, moreover, that aldosterone caused a conformational change of this membrane MR/GC-A protein complex which was prevented by ANP., Conclusions: ANP counter-regulates cardiac MR activation in hypertensive heart disease. An imbalance in cardiac ANP/GC-A (inhibition) and aldosterone/MR signaling (augmentation) favors adverse cardiac remodeling in chronic pressure overload., (© 2014 American Heart Association, Inc.)

Actin and actin-binding proteins are incorporated into HIV-1 particles, and F-actin has been suggested to bind the NC domain in HIV-1 Gag. Furthermore, F-actin has been frequently observed in the vicinity of HIV-1 budding sites by cryo-electron tomography (cET). Filamentous structures emanating from viral buds and suggested to correspond to actin filaments have been observed by atomic force microscopy. To determine whether the NC domain of Gag is required for actin association with viral buds and for actin incorporation into HIV-1, we performed comparative analyses of virus-like particles (VLPs) obtained by expression of wild-type HIV-1 Gag or a Gag variant where the entire NC domain had been replaced by a dimerizing leucine zipper [Gag(LZ)]. The latter protein yielded efficient production of VLPs with near-wild-type assembly kinetics and size and exhibited a regular immature Gag lattice. Typical HIV-1 budding sites were detected by using cET in cells expressing either Gag or Gag(LZ), and no difference was observed regarding the association of buds with the F-actin network. Furthermore, actin was equally incorporated into wild-type HIV-1 and Gag- or Gag(LZ)-derived VLPs, with less actin per particle observed than had been reported previously. Incorporation appeared to correlate with the relative intracellular actin concentration, suggesting an uptake of cytosol rather than a specific recruitment of actin. Thus, the NC domain in HIV-1 Gag does not appear to have a role in actin recruitment or actin incorporation into HIV-1 particles. Importance: HIV-1 particles bud from the plasma membrane, which is lined by a network of actin filaments. Actin was found to interact with the nucleocapsid domain of the viral structural protein Gag and is incorporated in significant amounts into HIV-1 particles, suggesting that it may play an active role in virus release. Using electron microscopy techniques, we previously observed bundles of actin filaments near HIV-1 buds, often seemingly in contact with the Gag layer. Here, we show that this spatial association is observed independently of the proposed actin-binding domain of HIV-1. The absence of this domain also did not affect actin incorporation and had a minor effect on the viral assembly rate. Furthermore, actin was not enriched in the virus compared to the average levels in the respective producing cell. Our data argue against a specific recruitment of actin to HIV-1 budding sites by the nucleocapsid domain of Gag., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: C-type natriuretic peptide (CNP)/natriuretic peptide receptor 2 (NPR2) signaling is essential for long bone growth. Enhanced CNP production caused by chromosomal translocations results in tall stature, a Marfanoid phenotype, and skeletal abnormalities. A similar phenotype was described in a family with an activating NPR2 mutation within the guanylyl cyclase domain., Case: Here we describe an extremely tall male without skeletal deformities, with a novel NPR2 mutation (p.Arg655Cys) located in the kinase homology domain., Objectives: The objective of the study was to investigate the functional and structural effects of the NPR2 mutation., Methods: Guanylyl cyclase activities of wild-type vs mutant NPR2 were analyzed in transfected human embryonic kidney 293 cells and in skin fibroblasts. The former were also used to study possible interactions between both isoforms. Homology modeling was performed to understand the molecular impact of the mutation., Results: CNP-stimulated cGMP production by the mutant NPR2 was markedly increased in patient skin fibroblasts and transfected human embryonic kidney 293 cells. The stimulatory effects of ATP on CNP-dependent guanylyl cyclase activity were augmented, suggesting that this novel mutation enhances both the responsiveness of NPR2 to CNP and its allosteric modulation/stabilization by ATP. Coimmunoprecipitation showed that wild-type and mutant NPR2 can form stable heterodimers, suggesting a dominant-positive effect. In accordance with augmented endogenous receptor activity, plasma N-terminal pro-CNP (a marker of CNP production in tissues) was reduced in the proband., Conclusions: We report the first activating mutation within the kinase homology domain of NPR2, resulting in extremely tall stature. Our observations emphasize the important role of this domain in the regulation of guanylyl cyclase activity and bone growth in response to CNP.

Objective: Histamine increases microvascular endothelial leakage by activation of complex calcium-dependent and -independent signaling pathways. Atrial natriuretic peptide (ANP) via its cGMP-forming guanylyl cyclase-A (GC-A) receptor counteracts this response. Here, we characterized the molecular mechanisms underlying this interaction, especially the role of cGMP-dependent protein kinase I (cGKI)., Approach and Results: We combined intravital microscopy studies of the mouse cremaster microcirculation with experiments in cultured microvascular human dermal endothelial cells. In wild-type mice, ANP had no direct effect on the extravasation of fluorescent dextran from postcapillary venules, but strongly reduced the histamine-provoked vascular leakage. This anti-inflammatory effect of ANP was abolished in mice with endothelial-restricted inactivation of GC-A or cGKI. Histamine-induced increases in endothelial [Ca(2+)]i in vitro and of vascular leakage in vivo were markedly attenuated by the Ca(2+)-entry inhibitor SKF96365 and in mice with ablated transient receptor potential canonical (TRPC) 6 channels. Conversely, direct activation of TRPC6 with hyperforin replicated the hyperpermeability responses to histamine. ANP, via cGKI, stimulated the inhibitory phosphorylation of TRPC6 at position Thr69 and prevented the hyperpermeability responses to hyperforin. Moreover, inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil prevented the edematic actions of histamine in wild types but not in mice with endothelial GC-A or cGKI deletion., Conclusions: ANP attenuates the inflammatory actions of histamine via endothelial GC-A/cGMP/cGKI signaling and inhibitory phosphorylation of TRPC6 channels. The therapeutic potential of this novel regulatory pathway is indicated by the observation that sildenafil improves systemic endothelial barrier functions by enhancing the endothelial effects of endogenous ANP.

Aims: Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism., Methods and Results: To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to β-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca(2+)]i-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte Ca(2+)i homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca(2+)i-handling, and contractility via cGKI., Conclusion: These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca(2+)i handling and contractility.

HIV protease is a primary target for the design of virostatics. Screening of libraries of non-peptide low molecular weight compounds led to the identification of several new compounds that inhibit HIV PR in the low micromolar range. X-ray structure of the complex of one of them, a dibenzo[b,e][1,4]diazepinone derivative, showed that two molecules of the inhibitor bind to the PR active site. Covalent linkage of two molecules of such a compound by a two-carbon linker led to a decrease of the inhibition constant of the resulting compound by 3 orders of magnitude. Molecular modeling shows that these dimeric inhibitors form two crucial hydrogen bonds to the catalytic aspartates that are responsible for their improved activity compared to the monomeric parental building blocks. Dibenzo[b,e][1,4]diazepinone analogues might represent a potential new class of HIV PIs.

Pairs of amino acid positions that evolve in a correlated manner are proposed to play important roles in protein structure or function. Methods to detect them might fare better with families for which sequences of thousands of closely related homologs are available than families with only a few distant relatives. We applied co-evolution analysis to thousands of sequences of HIV Gag, finding that the most significantly co-evolving positions are proximal in the quaternary structures of the viral capsid. A reduction in infectivity caused by mutating one member of a significant pair could be rescued by a compensatory mutation of the other.

Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein driving assembly and release of virions from infected cells. Gag alone is capable of self-assembly in vitro, but host factors have been shown to play a role in efficient viral replication and particle morphogenesis within the living cell. In a series of affinity purification experiments, we identified the cellular protein Lyric to be an HIV-1 Gag-interacting protein. Lyric was previously described to be an HIV-inducible gene and is involved in various signaling pathways. Gag interacts with endogenous Lyric via its matrix (MA) and nucleocapsid (NC) domains. This interaction requires Gag multimerization and Lyric amino acids 101 to 289. Endogenous Lyric is incorporated into HIV-1 virions and is cleaved by the viral protease. Gag-Lyric interaction was also observed for murine leukemia virus and equine infectious anemia virus, suggesting that it represents a conserved feature among retroviruses. Expression of the Gag binding domain of Lyric increased Gag expression levels and viral infectivity, whereas expression of a Lyric mutant lacking the Gag binding site resulted in lower Gag expression and decreased viral infectivity. The results of the current study identify Lyric to be a cellular interaction partner of HIV-1 Gag and hint at a potential role in regulating infectivity. Further experiments are needed to elucidate the precise role of this interaction.

Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A, to exert its diverse functions. This process involves a cGMP-dependent signaling pathway preventing pathological [Ca(2+)](i) increases in myocytes. In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization. Here we show that, in this situation, ANP binding to GC-A stimulates a unique cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca(2+) levels. This pathway involves the activation of Ca(2+)-permeable transient receptor potential canonical 3/6 (TRPC3/C6) cation channels by GC-A, which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting cation influx activates voltage-dependent L-type Ca(2+) channels and ultimately increases myocyte Ca(2)(+)(i) levels. These observations reveal a dual role of the ANP/GC-A-signaling pathway in the regulation of cardiac myocyte Ca(2+)(i) homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca(2+)(i)-enhancing action of hypertrophic factors such as angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca(2+)](i) might increase the propensity to cardiac hypertrophy and arrhythmias.

The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.

HIV protease (HIV PR) is a primary target for anti-HIV drug design. We have previously identified and characterized substituted metallacarboranes as a new class of HIV protease inhibitors. In a structure-guided drug design effort, we connected the two cobalt bis(dicarbollide) clusters with a linker to substituted ammonium group and obtained a set of compounds based on a lead formula [H(2)N-(8-(C(2)H(4)O)(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))-3,3'-Co)(2)]Na. We explored inhibition properties of these compounds with various substitutions, determined the HIV PR:inhibitor crystal structure, and computationally explored the conformational space of the linker. Our results prove the capacity of linker-substituted dual-cage cobalt bis(dicarbollides) as lead compounds for design of more potent inhibitors of HIV PR.

The expression of a membrane-anchored gp41-derived peptide (M87) has been shown to confer protection from infection through human immunodeficiency virus type 1 (HIV-1) (Hildinger et al., J. Virol. 75:3038-3042, 2001). In an effort to characterize the mechanism of action of this membrane-anchored peptide in comparison to the soluble peptide T-20, we selected resistant variants of HIV-1(NL4-3) and HIV-1(BaL) by serial virus passage using PM1 cells stably expressing peptide M87. Sequence analysis of the resistant isolates showed different patterns of selected point mutations in heptad repeat regions 1 and 2 (HR1 and HR2, respectively) for the two viruses analyzed. For HIV-1(NL4-3) a single amino acid change at position 33 in HR1 (L33S) was selected, whereas for HIV-1(BaL) the majority of the sequences obtained showed two amino acid changes, one in HR1 and one in HR2 (I48V/N126K). In both selections the most important contiguous 3-amino-acid sequence, GIV, within HR1, associated with resistance to soluble T-20, was not changed. Site-directed mutagenesis studies confirmed the importance of the characterized point mutations to confer resistance to M87 as well as to soluble T-20 and T-649. Replication capacity and dual-color competition assays revealed that the double mutation I48V/N126K in HIV-1(BaL) results in a strong reduction of viral fitness, whereas the L33S mutation in HIV-1(NL4-3) did enhance viral fitness compared to the respective parental viruses. However, the selected point mutations did not confer resistance to the more recently described optimized membrane-anchored fusion inhibitor M87o (Egelhofer et al., J. Virol. 78:568-575, 2004), strengthening the importance of this novel antiviral concept for gene therapy approaches.

Mutant hepatitis B viruses are useful tools to study the viral life cycle and viral pathogenesis. Furthermore, recombinant hepatitis B viruses are candidate vectors for liver-directed gene therapy. Because wild-type viruses present in recombinant or mutant virus stocks may falsify experimental results and are detrimental for a viral vector, we investigated whether and to what extent wild-type virus is present in recombinant virus stocks and where it originates from. We took advantage of the duck model of hepatitis B virus infection which allows very sensitive detection of replication-competent viruses by infection of primary duck hepatocytes or of ducklings in vivo. Recombinant hepatitis B virus stocks contained significant amounts of wild-type viruses, which were most probably generated by homologous recombination between plasmids containing homologous viral sequences. In addition, replication-competent viral genomes were reconstituted from plasmids which contained replication-deficient but redundant viral sequences. Using a stable cell line for packaging of deficient viral genomes, no wild-type virus was detected, neither by infection of primary hepatocytes nor in vivo.

For the study of hepatitis B virus infection, no permissive cell line or small animal is available. Stably transfected cell lines and transgenic mice which contain hepadnavirus genomes produce virus, but--unlike in natural infection--from an integrated viral transcription template. To transfer hepadnavirus genomes across the species barrier, we developed adenovirus vectors in which 1.3-fold-overlength human and duck hepatitis B virus genomes were inserted. The adenovirus-mediated genome transfer efficiently initiated hepadnavirus replication from an extrachromosomal template in established cell lines, in primary hepatocytes from various species, and in the livers of mice. Following the transfer, hepatitis B virus proteins, genomic RNA, and all replicative DNA intermediates were detected. Detection of covalently closed circular DNA in hepatoma cell lines and in primary hepatocytes indicated that an intracellular replication cycle independent from the transferred linear viral genome was established. High-titer hepatitis B virions were released into the culture medium of hepatoma cells and the various primary hepatocytes. In addition, infectious virions were secreted into the sera of mice. In conclusion, adenovirus-mediated genome transfer initiated efficient hepatitis B virus replication in cultured liver cells and in the experimental animals from an extrachromosomal template. This will allow development of small-animal systems of hepatitis B virus infection and will facilitate study of pathogenicity of wild-type and mutant viruses as well as of virus-host interaction and new therapeutic approaches.

Cyritestin is a membrane-anchored sperm protein belonging to the ADAM (f1.gif" BORDER="0"> f2.gif" BORDER="0">isintegrin and f1.gif" BORDER="0"> f3.gif" BORDER="0">etalloprotease) family of proteins, which are proposed to be involved in cell-cell adhesion through binding to integrin receptors. Several lines of evidence support a role of cyritestin and other members of this protein family in the fusion of sperm and the egg plasma membrane. In an effort to elucidate the physiological function of cyritestin, we have disrupted its locus by homologous recombination. Male homozygous null mutants are infertile, even though spermatogenesis, mating, and migration of sperm from the uterus into the oviduct are normal. In vitro experiments showed that infertility is due to the inability of the cyritestin-deficient sperm to bind to the zona pellucida. However, after removal of the zona pellucida, sperm-egg membrane fusion monitored by the presence of pronuclei and generation of 2- and 4-cell embryos did not reveal any differences from the wild-type situation. These results demonstrate that cyritestin is crucial in the fertilization process at the level of the sperm-zona pellucida interaction.