Extension of human GCSF serum half-life by the fusion of albumin binding domain

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

Mammalian orthoreovirus reassortment proceeds with little constraint on segment mixing

Segmentation of viral genomes gives the potential for genetic exchange within co-infected cells. However, for this potential to be realized, co-infecting genomes must mix during the viral lifecycle. The efficiency of reassortment in turn dictates its potential to drive evolution. The opportunity for mixing within co-infected cells may vary greatly across virus families, such that the evolutionary implications of genome segmentation differ as a result of core features of the viral lifecycle. To investigate the relationship between viral replication compartments and genetic exchange, we quantified reassortment in mammalian orthoreovirus (reovirus). Reoviruses carry a 10-segmented, double-stranded RNA genome, which is replicated within proteinaceous structures termed inclusion bodies. We hypothesized that inclusions impose a barrier to reassortment. We quantified reassortment between wild-type ( wt ) and variant ( var ) reoviruses that differ by one nucleotide per segment. Wt/var systems in both T1L and T3D backgrounds revealed frequent reassortment without bias towards particular genotypes. However, reassortment was more efficient in the T3D serotype. Since T1L and T3D viruses exhibit different inclusion body morphologies, we tested the impact of this phenotype on reassortment. In both serotypes, reassortment levels did not differ by inclusion morphology. Reasoning that the merging of viral inclusions may be critical for genome mixing, we then tested the effect of blocking merging. Reassortment proceeded efficiently even under these conditions. Our findings indicate that reovirus reassortment is highly efficient despite the localization of many viral processes to inclusion bodies, and that the robustness of this genetic exchange is independent of inclusion body structure and fusion. Importance Quantification of reassortment in diverse viral systems is critical to elucidate the implications of genome segmentation for viral evolution. In principle, genome segmentation offers a facile means of genetic exchange between coinfecting viruses. In practice, there may be physical barriers within the cell that limit mixing of viral genomes. Here, we tested the hypothesis that localization of the various stages of the mammalian orthoreovirus lifecycle within cytoplasmic inclusion bodies compartmentalizes viral replication and limits genetic exchange. Contrary to this hypothesis, our data indicate that reovirus reassortment occurs readily within co-infected cells and is not strongly affected by the structure or dynamics of viral inclusion bodies. We conclude that the potential for reassortment to contribute to reovirus evolution is high.

Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing

Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

TDP-43 Pathology in Alzheimer’s Disease

Transactive response DNA binding protein of 43 kDa (TDP-43) is an intranuclear protein encoded by the TARDBP gene that is involved in RNA splicing, trafficking, stabilization, and thus, the regulation of gene expression. Cytoplasmic inclusion bodies containing phosphorylated and truncated forms of TDP-43 are hallmarks of amyotrophic lateral sclerosis (ALS) and a subset of frontotemporal lobar degeneration (FTLD). Additionally, TDP-43 inclusions have been found in up to 57% of Alzheimer’s disease (AD) cases, most often in a limbic distribution, with or without hippocampal sclerosis. In some cases, TDP-43 deposits are also found in neurons with neurofibrillary tangles. AD patients with TDP-43 pathology have increased severity of cognitive impairment compared to those without TDP-43 pathology. Furthermore, the most common genetic risk factor for AD, apolipoprotein E4 (APOE4), is associated with increased frequency of TDP-43 pathology. These findings provide strong evidence that TDP-43 pathology is an integral part of multiple neurodegenerative conditions, including AD. Here, we review the biology and pathobiology of TDP-43 with a focus on its role in AD. We emphasize the need for studies on the mechanisms that lead to TDP-43 pathology, especially in the setting of age-related disorders such as AD.

Characterization of the interaction domains between the phosphoprotein and the nucleoprotein of human Metapneumovirus

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein (N), forming an RNA-N complex (N Nuc ), which serves as template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to N Nuc and L, allowing the attachment of the L polymerase to the N Nuc template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between HMPV P and N Nuc , which has not been demonstrated yet. Here, we finely characterized the binding P- N Nuc interaction domains by using recombinant proteins, combined with a functional assay for the polymerase complex activity, and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to N Nuc , that P binds to the N-terminal domain of N (N NTD ), and identified conserved N residues critical for the interaction. Our results allowed to propose a structural model for the HMPV P-N Nuc interaction. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Nowadays, no vaccine or efficient antiviral treatments are available for this pneumovirus. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction, and identified a pocket on the surface of the N protein, a potential target of choice for the design of compounds interfering with N-P complexes and inhibiting viral replication.

A subpopulation of arenavirus nucleoprotein localizes to mitochondria

Viruses need cells for their replication and, therefore, ways to hijack cellular functions. Mitochondria play fundamental roles within the cell in metabolism, immunity and regulation of homeostasis due to which some viruses aim to alter mitochondrial functions. Herein we show that the nucleoprotein (NP) of arenaviruses enters the mitochondria of infected cells, affecting the mitochondrial morphology. Reptarenaviruses cause boid inclusion body disease (BIBD) that is characterized, especially in boas, by the formation of cytoplasmic inclusion bodies (IBs) comprising reptarenavirus NP within the infected cells. We initiated this study after observing electron-dense material reminiscent of IBs within the mitochondria of reptarenavirus infected boid cell cultures in an ultrastructural study. We employed immuno-electron microscopy to confirm that the mitochondrial inclusions indeed contain reptarenavirus NP. Mutations to a putative N-terminal mitochondrial targeting signal (MTS), identified via software predictions in both mamm- and reptarenavirus NPs, did not affect the mitochondrial localization of NP, suggesting that it occurs independently of MTS. In support of MTS-independent translocation, we did not detect cleavage of the putative MTSs of arenavirus NPs in reptilian or mammalian cells. Furthermore, in vitro translated NPs could not enter isolated mitochondria, suggesting that the translocation requires cellular factors or conditions. Our findings suggest that MTS-independent mitochondrial translocation of NP is a shared feature among arenaviruses. We speculate that by targeting the mitochondria arenaviruses aim to alter mitochondrial metabolism and homeostasis or affect the cellular defense.

Characterization of the interaction domains between the phosphoprotein and the nucleocapsid of human Metapneumovirus

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein, forming an RNA-N complex (NNuc), which serves as template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to NNuc and L, allowing the attachment of the L polymerase to the nucleocapsid template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between P and NNuc. However, the HMPV P-NNuc interaction still remains to characterize. Here, we finely characterized the binding domains involved in HMPV P and NNuc interaction by studying binding between recombinant proteins, combined with the use of a functional assay of the polymerase complex activity and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to NNuc, that P binds the N-terminal domain of N (NNTD), and identified conserved N residues critical for the interaction. Our results allowed to propose a structural model of the HMPV P-NNuc interaction.

Multinucleate parietal cells and cytoplasmic inclusion bodies in the gastric epithelium of callitrichids

Inclusion bodies (IBs) and multinucleate cells can be associated with viral infections; however, IBs and multinucleate cells have been described in normal tissue and with non-viral disease processes in multiple species. We examined fundic stomach from 50 callitrichids histologically for bi- and multinucleate parietal cells and cytoplasmic IBs in gastric epithelial cells. Callitrichids represented included 6 genera: Saguinus (4 spp.), Leontopithecus (1 sp.), Mico (3 spp.), Cebuella (1 sp.), Callithrix (1 sp.), Callimico (1 sp.), and 13 unspecified marmosets. Gastric epithelial IBs were present in 46 of 47 (98%) of the callitrichids from which the stomach was sufficiently well preserved to identify IBs. Cytoplasmic IBs were identified in gastric surface pit epithelial cells (43 of 44, 98%), mucous neck cells (43 of 44, 98%), parietal cells (43 of 44, 98%), and chief cells (43 of 44, 98%). The IBs were eosinophilic, ovoid, round, elongate, or variably indented, sometimes slightly refractile, and 1–6 × 1–13 µm. IBs were sometimes perinuclear and molded around the nucleus. Electron microscopy of the gastric epithelium of one marmoset indicated that IBs were composed of intermediate filaments. The IBs did not stain with immunohistochemical markers for cytokeratin AE1/AE3 or vimentin. Binucleate parietal cells were found in 49 of 50 (98%) callitrichids, and multinucleate parietal cells were observed in 40 of 49 (82%) callitrichids. Gastric epithelial cytoplasmic IBs and bi- and multinucleate parietal cells are likely a normal finding in callitrichids, and, to our knowledge, have not been reported previously.

Experimental Reptarenavirus Infection of Boa constrictor and Python regius

Boid inclusion body disease (BIBD) causes losses in captive snake populations globally. BIBD is associated with the formation of cytoplasmic inclusion bodies (IBs), which mainly comprise reptarenavirus nucleoprotein (NP). In 2017, BIBD was reproduced by cardiac injection of boas and pythons with reptarenaviruses, thus demonstrating a causative link between reptarenavirus infection and the disease. Here, we report experimental infections of Python regius (n = 16) and Boa constrictor (n = 16) with three reptarenavirus isolates. First, we used pythons (n = 8) to test two virus delivery routes: intraperitoneal injection and tracheal instillation. Viral RNAs but no IBs were detected in brains and lungs at 2 weeks postinoculation. Next, we inoculated pythons (n = 8) via the trachea. During the 4 months following infection, snakes showed transient central nervous system (CNS) signs but lacked detectable IBs at the time of euthanasia. One of the snakes developed severe CNS signs; we succeeded in reisolating the virus from the brain of this individual and could demonstrate viral antigen in neurons. In a third attempt, we tested cohousing, vaccination, and sequential infection with multiple reptarenavirus isolates on boas (n = 16). At 10 months postinoculation, all but one snake tested positive for viral RNA in lung, brain, and/or blood, but none exhibited the characteristic IBs. Three of the four vaccinated snakes seemed to sustain challenge with the same reptarenavirus; however, neither of the two snakes rechallenged with different reptarenaviruses remained uninfected. Comparison of the antibody responses in experimentally versus naturally reptarenavirus-infected animals indicated differences in the responses. IMPORTANCE In the present study, we experimentally infected pythons and boas with reptarenavirus via either intraperitoneal injection or tracheal instillation. The aims were to experimentally induce boid inclusion body disease (BIBD) and to develop an animal model for studying disease transmission and pathogenesis. Both virus delivery routes resulted in infection, and infection via the trachea could reflect the natural route of infection. In the experimentally infected snakes, we did not find evidence of inclusion body (IB) formation, characteristic of BIBD, in pythons or boas. Most of the boas (11/12) remained reptarenavirus infected after 10 months, which suggests that they developed a persistent infection that could eventually have led to BIBD. We demonstrated that vaccination using recombinant protein or an inactivated virus preparation prevented infection by a homologous virus in three of four snakes. Comparison of the antibody responses of experimentally and naturally reptarenavirus-infected snakes revealed differences that merit further studies.