Adherent and suspension baby hamster kidney cells have a different cytoskeleton and surface receptor repertoire

Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is a mainstay of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell lines and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.

Adherent and suspension baby hamster kidney cells have a different cytoskeleton and surface receptor repertoire

Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is a mainstay of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell clones and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.

Mutant huntingtin reduces vesicular zinc level by inhibiting the binding of Sp1 to ZnT3 promoter

Background Synaptic dysfunction caused by mutant huntingtin greatly contributes to Huntington’s disease (HD) pathogenesis. HD patients show cognitive impairment as well as uncontrolled movements. Vesicular zinc is closely linked to modulating synaptic transmission and maintaining cognitive ability. However, whether does mutant huntingtin affect zinc homeostasis in the brain or not? This will be of great significance for further revealing the pathogenesis of HD. Methods N171-HD82Q transgenic mice and cultured BHK cells expressing N-terminal mutant huntingtin fragment containing 160 glutamines (160Q BHK cells) were used to investigate the effect of mutant huntingtin on zinc homeostasis and its molecular mechanisms. Results Herein, we have demonstrated that the density of synaptic vesicular zinc decreases in the cortex, striatum and hippocampus of N171-82Q mice. Given that vesicular zinc concentration depends on the abundance of zinc transporter 3 (ZnT3) on the membrane of synaptic vesicles, ZnT3 expression is detected in the brain of N171-82Q mice and 160Q BHK cells. Mutant huntingtin leads to a dramatical decrease in ZnT3 mRNA and protein levels in the three brain regions of these mice aged from 14 to 20 weeks. Significantly, Sp1 activates ZnT3 transcription via its binding to the GC boxes in ZnT3 promoter. Nevertheless, mutant huntingtin inhibits the binding of Sp1 to the promoter of ZnT3 gene and down-regulates ZnT3 expression. Furthermore, the overexpression of Sp1 ameliorates inhibition of ZnT3 gene transcription by mutant huntingtin. Conclusions Collectively, this first study to reveal a significant loss of synaptic vesicular zinc and ZnT3 expression caused by mutant huntingtin in the early stage of HD. Our findings have revealed the molecular mechanism underlying this change. Mutant huntingtin inhibits the binding of Sp1 to ZnT3 gene promoter to reduce ZnT3 expression. The imbalance of vesicular zinc homeostasis may be closely associated with synaptic dysfunction and cognitive deficits in HD. This work sheds novel mechanistic insights into the pathogenesis of HD and promises a potential therapeutic strategy for HD.

Genetic and Biological Diversity of Porcine Sapeloviruses Prevailing in Zambia

Porcine sapelovirus (PSV) has been detected worldwide in pig populations. Although PSV causes various symptoms such as encephalomyelitis, diarrhea, and pneumonia in pigs, the economic impact of PSV infection remains to be determined. However, information on the distribution and genetic diversity of PSV is quite limited, particularly in Africa. In this study, we investigated the prevalence of PSV infection in Zambia and characterized the isolated PSVs genetically and biologically. We screened 147 fecal samples collected in 2018 and found that the prevalences of PSV infection in suckling pigs and fattening pigs were high (36.2% and 94.0%, respectively). Phylogenetic analyses revealed that the Zambian PSVs were divided into three different lineages (Lineages 1–3) in the clade consisting of Chinese strains. The Zambian PSVs belonging to Lineages 2 and 3 replicated more efficiently than those belonging to Lineage 1 in Vero E6 and BHK cells. Bioinformatic analyses revealed that genetic recombination events had occurred and the recombination breakpoints were located in the L and 2A genes. Our results indicated that at least two biologically distinct PSVs could be circulating in the Zambian pig population and that genetic recombination played a role in the evolution of PSVs.

FVIII Protein Is Not Detectable in Human PBMCs or Livers from Dogs with an Intron-22 Inversion Mutation: Implications for FVIII Immunogenicity and Tolerance

A clinically relevant question is whether partial FVIII proteins are expressed in tissues from patients with severe hemophilia A (HA) due to an intron-22 inversion mutation. If so, this could in principle confer central immune tolerance to the expressed FVIII regions, thereby lowering the risk of anti-FVIII immune responses. In 2013, Pandey et al. reported detection of FVIII proteins in peripheral blood mononuclear cells (PBMCs), B cells and liver tissues from an intron-22 inversion subject and non-HA controls. They concluded that partial FVIII proteins were translated from inverted mRNA encoding F8 exons 1-22, and also from the F8B transcript, which contains F8 exons 23-26 (Nature Medicine 19(10), 1318-24). In 2014, the Montgomery and Ginsburg labs reported that FVIII protein is expressed principally, and possibly exclusively, in endothelial cells (ECs). These studies utilized mouse models with EC-directed deletion of F8 exons 17-18, or of the FVIII transporter protein LMAN1, respectively. The F8-EC-knockout mice had a severe hemophilia A (HA) phenotype. Earlier studies from several labs, including Pandey et al., relying primarily on antibody staining, had reported FVIII expression in additional cell types, including PBMCs and hepatocytes. The present study tests the hypothesis that partial FVIII proteins are expressed from inverted mRNA encoding F8 exons 1-22 and/or from F8B mRNA. A panel of FVIII-specific polyclonal and monoclonal antibodies (mAbs) was tested for FVIII specificity by staining permeabilized PBMCs, monocytes, T cells, blood outgrowth ECs, monocyte-derived macrophages and dendritic cells, HUVECs and B-cell lines. Positive and negative controls included FVIII-expressing BHK cells and non-engineered BHK cells. Specificity was further tested by immunoprecipitation (IP) of cell lysates followed by SDS-PAGE, Western blots and mass spectrometry to define proteins pulled down by the anti-FVIII antibodies. In addition, immunohistochemistry (IHC) staining was carried out for liver sections from HA dogs with an intron-22 inversion mutation and from otherwise normal (non-HA) control dogs. IP followed by SDS-PAGE, Westerns and/or mass spec revealed that a significant fraction of the anti-FVIII antibodies bound to other proteins besides FVIII in the tissues and cells examined. A cocktail of 4 anti-FVIII mAbs that were validated using (+) and (-) control samples was used to stain isolated and cultured cells and tissues. Cross-recognition of canine FVIII was confirmed by IP + Western blots. IHC using the mAb cocktail followed by HRP-conjugated secondary antibodies produced variable staining of liver tissues from HA and non-HA dogs, regardless of tissue preparation methods. However, use of fluorescent-labeled secondary mAbs produced signals well above background in ECs of control dog livers but not in the HA livers. Multiple confocal images were selected randomly, and average MFI values of 10 non-HA liver sections were well above those of 10 HA sections (p = 0.006). A second staining method, the Duolink Proximity Ligation Assay, was employed as an independent test to detect interactions between FVIII and VWF. Again, only normal control liver sections showed fluorescence above background (p =0.0004). F8B mRNA was not detected in canine liver and was not expected based on the canine intron-22 DNA sequence. We conclude that if protein is expressed from inverted mRNA encoding F8 exons 1-22, it is below the detection limit of these assays. Permeabilized human cells were tested for FVIII expression by staining using the mAb cocktail, or validated anti-FVIII-C2 mAbs to detect the putative protein encoded by F8B, followed by fluorescent detection. Cells were also stimulated with histamine followed by a chromogenic FVIII activity assay of supernatants. Results indicated that if FVIII or F8B protein are expressed in non-ECs, they are below the detection limit of these assays. We also note that if the putative F8B-encoded protein confers tolerance, immune responses of HA patients to the FVIII C2 domain would be quite rare, which is not the case. Although neonatal thymic expression of partial FVIII proteins may occur, immune tolerance to self-antigens requires repeated exposures of the immune system to the antigens. We conclude it is unlikely that HA patients with an intron-22 inversion mutation have universally acquired central tolerance to partial proteins encoded by inverted F8 mRNA or F8B. Disclosures Ragni: Shire/Takeda: Consultancy, Other: Study drug; Sangamo: Research Funding; OPKO: Research Funding; Bioverativ/Sanofi: Consultancy, Research Funding; Bayer: Consultancy; ICER: Consultancy; Biomarin: Consultancy, Research Funding; Alnylam/Sanofi: Consultancy, Research Funding; Spark Therapeutics: Consultancy, Research Funding. Pratt:Bloodworks NW: Patents & Royalties: inventor on patents related to FVIII immunogenicity; Grifols, Inc: Research Funding.

Single-Site Glycoprotein Mutants Inhibit a Late Event in Sindbis Virus Assembly

ABSTRACTA panel of Sindbis virus mutants that were suspected to have deficiencies in one or more aspects of their replication cycles was examined in baby hamster kidney (BHK) cells. These included an amino acid deletion (ΔH230) and substitution (H230A) in the Sindbis glycoprotein E1_H230 and similar mutants in E2_G209 (G209A, G209D, and ΔG209). Neither H230 mutation produced a measurable titer, but repeated passaging of the H230A mutant in BHK cells produced a second-site compensatory mutant (V231I) that partially rescued both H230 mutants. Electron micrograph (EM) images of these mutants showed assembled viral nucleocapsids but no completed, mature virions. EM of the compensatory mutant strains showed complete virus particles, but these now formed paracrystalline arrays. None of the E2_G209 substitution mutants had any effect on virus production; however, the deletion mutant (ΔG209) showed a very low titer when grown at 37°C and no titer when grown at 28°C. When the deletion mutant grown at 28°C was examined by EM, partially budded virions were observed at the cell surface.35S labeling of this mutant confirmed the presence of mutant virus protein in the transfected BHK cell lysate. We conclude that H230 is essential for the assembly of complete infectious Sindbis virus virions and that the presence of an amino acid at E2 position 209 is required for complete budding of Sindbis virus particles although several different amino acids can be at this location without affecting the titer.IMPORTANCEOur data show the importance of single-site mutations at E1_H230 and E2_G209 in Sindbis virus glycoproteins. These sites have been shown to affect assembly and antibody binding in previous studies. Our data indicate that mutation of one histidine residue in E1 is detrimental to the assembly of Sindbis virus particles in baby hamster kidney cells. Repeated passaging leads to a second-site substitution that partially restores the titer although EM still shows an altered phenotype. Substitutions at position G209 in E2 have no effect on titer, but deletion of this residue greatly reduces titer and again prevents assembly. When this mutant is grown at a lower temperature, virus particles bud from the host cell, but budding arrests before the progeny virus escapes. These results allow us to conclude that these sites have essential roles in assembly, and E2_G209 shows us a new viral egress phenotype.