Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.

NC1-Peptide From Collagen α3 (IV) Chains in the Basement Membrane of Testes Regulates Spermatogenesis via p-FAK-Y407

The blood–testis barrier (BTB) in the testis is an important ultrastructure to support spermatogenesis. This blood-tissue barrier undergoes remodeling at late stage VII to early stage IX of the epithelial cycle to support the transport of preleptotene spermatocytes across the BTB to prepare for meiosis I/II at the apical compartment through a mechanism that remains to be delineated. Studies have shown that NC1-peptide-derived collagen α3 (IV) chain in the basement membrane is a bioactive peptide that induces BTB remodeling. It also promotes the release of fully developed spermatids into the tubule lumen. Thus, this endogenously produced peptide coordinates these 2 cellular events across the seminiferous epithelium. Using an NC1-peptide complementary deoxyribonucleic acid (cDNA) construct to transfect adult rat testes for overexpression, NC1-peptide was found to effectively induce germ cell exfoliation and BTB remodeling, which was associated with a surge and activation of p-rpS6, the downstream signaling protein of mTORC1 and the concomitant downregulation of p-FAK-Y407 in the testis. In order to define the functional relationship between p-rpS6 and p-FAK-Y407 signaling to confer the ability of NC1-peptide to regulate testis function, a phosphomimetic (and thus constitutively active) mutant of p-FAK-Y407 (p-FAK-Y407E-MT) was used for its co-transfection, utilizing Sertoli cells cultured in vitro with a functional tight junction (TJ) barrier that mimicked the BTB in vivo. Overexpression of p-FAK-Y407E-MT blocked the effects of NC1-peptide to perturb Sertoli cell BTB function by promoting F-actin and microtubule cytoskeleton function, and downregulated the NC1-peptide-mediated induction of p-rpS6 activation. In brief, NC1-peptide is an important endogenously produced biomolecule that regulates BTB dynamics.

A gene-specific T2A-GAL4 library for Drosophila

We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3’ of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.

Presenilin 2 overexpression is associated with apoptosis in Neuro2a cells

Presenilin 1 (PS1) and PS2 are evolutionarily conserved transmembrane proteins of the aspartyl protease family. Initially, they were reported to be associated with the early onset of familial, early-onset Alzheimer’s disease. PS1 has been implicated in several crucial brain functions including developmental processes, synaptic plasticity, and processing of various molecules, while PS2 has been poorly studied and is considered to be a compensatory partner of PS1. Certain controversial reports have suggested that PS2 has a role in apoptosis, though the underlying mechanism is not clear. To ascertain the role of PS2 in apoptosis, mouse neuroblastoma cells (Neuro2a) were transfected with a cDNA construct encoding full length mouse PS2 and analyzed for viability, expression of PS1, PS2, Bax and p53, Bax protein, and status of chromatin condensation. Our results showed reduced viability, condensed chromatin and higher expression of Bax at mRNA and protein levels, but no change in the expression of p53 and PS1 in PS2-overexpressing Neuro2a cells. Thus, it is evident that PS2, independent of PS1, is associated with apoptosis via a Bax-mediated pathway. These findings might help in the understanding of the involvement of PS2 in apoptosis and its associated brain disorders.

Micropuncture gene delivery and intravital two-photon visualization of protein expression in rat kidney

Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the virally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any fluorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology.

B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with Sp1 and CBF factors in scleroderma fibroblasts

We investigated the role of B-Myb, a cell-cycle-regulated transcription factor, in the expression of the α1 (I) pro-collagen gene (COL1A1) in scleroderma fibroblasts. Scleroderma or SSc (systemic sclerosis) is a fibrotic disease characterized by excessive production of extracellular matrix components, especially type I collagen. Northern-blot analysis showed an inverse relationship between COL1A1 mRNA expression and that of B-Myb during exponential cell growth and during quiescence in human SSc fibroblasts. Overexpression of B-Myb in SSc fibroblasts was correlated with decreased COL1A1 mRNA expression. Transient transfections localized the down-regulatory effect of B-Myb to a region containing the proximal 174 bp of the COL1A1 promoter that does not contain B-Myb consensus binding sites. Gel-shift analysis, using nuclear extracts from normal and SSc fibroblasts transfected with B-Myb, showed no differences in DNA–protein complex formation when compared with the nuclear extracts from mock-transfected cells. However, we found that B-Myb decreases Sp1 (specificity protein 1) and CBF (CCAAT-binding factor) binding for their specific sites localized in the 174 bp COL1A1 proximal promoter. These results were also confirmed using B-Myb-immunodepleted nuclear extracts. Furthermore, immunoprecipitation assays using SSc nuclear extracts demonstrated a physical interaction of B-Myb with Sp1 and CBF transcription factors, and also an interaction between Sp1 and CBF. In addition, by employing full-length or deleted B-Myb cDNA construct, we found that B-Myb down-regulates the COL1A1 proximal promoter through its C-terminal domain. Thus these results suggest that B-Myb may be an important factor in the pathway(s) regulating collagen production in SSc fibroblasts.

Factor VIII ectopically expressed in platelets: efficacy in hemophilia A treatment

Activated platelets release their granule content in a concentrated fashion at sites of injury. We examined whether ectopically expressed factor VIII in developing megakaryocytes would be stored in α-granules and whether its release from circulating platelets would effectively ameliorate bleeding in a factor VIIInull mice model. Using the proximal glycoprotein 1bα promoter to drive expression of a human factor VIII cDNA construct, transgenic lines were established. One line had detectable human factor VIII that colocalizes with von Willebrand factor in platelets. These animals had platelet factor VIII levels equivalent to 3% to 9% plasma levels, although there was no concurrent plasma human factor VIII detectable. When crossed onto a factor VIIInull background, whole blood clotting time was partially corrected, equivalent to a 3% correction level. In a cuticular bleeding time study, these animals also had only a partial correction, but in an FeCl3 carotid artery, thrombosis assay correction was equivalent to a 50% to 100% level. These studies show that factor VIII can be expressed and stored in platelet α-granules. Our studies also suggest that platelet-released factor VIII is at least as potent as an equivalent plasma level and perhaps even more potent in an arterial thrombosis model. (Blood. 2003;102:4006-4013)

Expression of Immunogenic S1 Glycoprotein of Infectious Bronchitis Virus in Transgenic Potatoes

ABSTRACT The expression of infectious bronchitis virus (IBV) S1 glycoprotein in potatoes and its immunogenicity in mice and chickens were investigated. Potato plants were genetically transformed with a cDNA construct encoding the IBV S1 glycoprotein with the Agrobacterium system. Genomic DNA and mRNA analyses of the transformed plantlets confirmed the integration of the foreign cDNA into the potato genome, as well as its transcription. Mice and chickens vaccinated with the expressed IBV S1 glycoprotein produced antibodies that neutralized IBV infectivity. After three immunizations, vaccinated chickens were completely protected from virulent IBV infection. These results demonstrate that transgenic potatoes expressing IBV S1 glycoprotein can be used as a source of recombinant antigen for vaccine production.

The peripheral cannabinoid receptor Cb2, a novel oncoprotein, induces a reversible block in neutrophilic differentiation

We previously identified a novel common virus integration site, Evi11, by means of retroviral insertional mutagenesis. We demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb2) is the potential target, suggesting that Cb2 is a proto-oncogene. To elucidate a role for this G protein–coupled receptor (GPCR) in leukemic transformation we generated a Cb2-EGFP cDNA construct that was introduced into 32D/G-CSF-R cells. These cells require interleukin 3 (IL-3) to proliferate in vitro, whereas in the presence of granulocyte–colony-stimulating factor (G-CSF) they differentiate toward mature neutrophils. We demonstrate that 32D/G-CSF-R/Cb2-EGFP cells migrate in a transwell assay in reponse to the Cb2 ligand 2-arachidonoylglycerol (2-AG), indicating that the fusion protein was functional. When cultured in the presence of G-CSF neutrophilic differentiation of Cb2-EGFP–expressing 32D/G-CSF-R cells was completely blocked. Moreover, a Cb2-specific antagonist fully recovered the G-CSF–induced neutrophilic differentiation of 32D/G-CSF-R/Cb2-EGFP cells. To investigate which signal transduction pathway(s) may be involved in the block of neutrophilic maturation, differentiation experiments were carried out using specific inhibitors of signaling routes. Interestingly, full rescue of G-CSF–induced neutrophilic differentiation was observed when cells were cultured with the mitogen-induced extracellular kinase (MEK) inhibitors, PD98059 or U0126, and partial recovery was detected with the phosphoinositide 3-kinase (PI3-K) inhibitor LY-294002. These studies demonstrate that the Cb2 receptor is an oncoprotein that blocks neutrophilic differentiation when overexpressed in myeloid precursor cells. Cb2 appears to mediate its activity through MEK/extracellular signal-related kinase (ERK) and PI3-K pathways.

Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59

ABSTRACT A novel method was developed to assemble a full-length infectious cDNA of the group II coronavirus mouse hepatitis virus strain A59 (MHV-A59). Seven contiguous cDNA clones that spanned the 31.5-kb MHV genome were isolated. The ends of the cDNAs were engineered with unique junctions and assembled with only the adjacent cDNA subclones, resulting in an intact MHV-A59 cDNA construct of ∼31.5 kb in length. The interconnecting restriction site junctions that are located at the ends of each cDNA are systematically removed during the assembly of the complete full-length cDNA product, allowing reassembly without the introduction of nucleotide changes. RNA transcripts derived from the full-length MHV-A59 construct were infectious, although transfection frequencies were enhanced 10- to 15-fold in the presence of transcripts encoding the nucleocapsid protein N. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar growth kinetics, plaque morphology, and cytopathology in murine cells as did wild-type MHV-A59. Molecularly cloned viruses recognized the MHV receptor (MHVR) for docking and entry, and pretreatment of cells with monoclonal antibodies against MHVR blocked virus entry and replication. Cells infected with molecularly cloned MHV-A59 virus expressed replicase (gene 1) proteins identical to those of laboratory MHV-A59. Importantly, the molecularly cloned viruses contained three marker mutations that had been derived from the engineered component clones. Full-length infectious constructs of MHV-A59 will permit genetic modifications of the entire coronavirus genome, particularly in the replicase gene. The method has the potential to be used to construct viral, microbial, or eukaryotic genomes approaching several million base pairs in length and used to insert restriction sites at any given nucleotide in a microbial genome.