RESUMEN

Coxsackievirus A5 (CV-A5) is a re-emerging enterovirus that causes hand, foot, and mouth disease in children under five years of age. CV-A5-M14-611 is a mouse-adapted strain that can infect orally and lead to the death of 14-day-old mice. Here, recombinants based on CV-A5-M14-611 were constructed carrying three reporter genes in different lengths. Smaller fluorescent marker proteins, light, oxygen, voltage sensing (iLOV), and nano luciferase (Nluc) were proven to be able to express efficiently in vitro. However, the recombinant with the largest insertion of the red fluorescence protein gene (DsRed) was not rescued. The construction strategy of reporter viruses was to insert the foreign genes between the C-terminus of VP1 and the N-terminus of 2A genes and to add a 2A protease cleavage domain at both ends of the insertions. The iLOV-tagged or Nluc-tagged recombinants, CV-A5-iLOV or CV-A5-Nluc, exhibited a high capacity for viral replication, genetic stability in cells and pathogenicity in mice. They were used to establish a rapid, inexpensive and convenient neutralizing antibody assay and greatly facilitated virus neutralizing antibody titration. Living imaging was performed on mice with CV-A5-Nluc, which exhibited specific bioluminescence in virus-disseminated organs, while fluorescence induced by CV-A5-iLOV was weakly detected. The reporter-gene-tagged CV-A5 can be used to study the infection and mechanisms of CV-A5 pathogenicity in a mouse model. They can also be used to establish rapid and sensitive assays for detecting neutralizing antibodies.

Asunto(s)

RESUMEN

The mechanism of discontinuous transcription for the synthesis of a series of sub-genomic mRNAs to express the structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) potentially allows for the simultaneous expression of multiple foreign genes. This can occur by insertion of multiple novel independent transcription units between the ORF sequences of the PRRSV genome. Here, an expression cassette consisting of a red fluorescent protein (RFP) gene flanked at its 3' end by transcription-regulating sequences (TRS) and an expression cassette consisting of an iLOV gene flanked at its 5' end by TRS, was constructed. The resulting expression cassette containing a RFP and an iLOV gene were introduced between ORF1b and 2 as well as ORF7 and 3'UTR, respectively, in an infectious PRRSV cDNA clone. Transfection of the resulting clone (pGX-12RFP-73iLOV) into cells resulted in the recovery of a recombinant virus (rGX-12RFP-73iLOV). Simultaneous expression of RFP and iLOV was observed in MARC-145 cells infected with rGX-RFP-iLOV. To test the ability of the PRRSV genome to express all three reporter genes simultaneously, an expression cassette containing the Gluc gene and one containing the iLOV gene were also inserted in between ORF1b and 2 as well as ORF7 and 3'UTR, respectively. This was performed in a recently obtained infectious PRRSV cDNA clone carrying a RFP gene in nsp2. Transfection of the construct (pGX-R-Gluc-iLOV) carrying the three reporter genes into cells allowed the rescue of the recombinant reporter virus (rGX-R-Gluc-iLOV) which showed similar growth characteristics to the parental virus but yielded 100-fold less infectious viruses. Fluorescence microscopy of cells infected with rGX-R-Gluc-iLOV demonstrated the presence of both RFP and iLOV genes. Gluc activities in supernatants harvested at different time points from cells infected with recombinant viruses carrying Gluc showed increased levels of Gluc activity as the infection progressed. This indicated that Gluc gene as well as its activity were acceptable parameters to monitor viral propagation. Our results indicate that it is possible to introduce at least three foreign proteins simultaneously in a PRRSV-based vector and such studies will prove invaluable in our future understanding of these viruses.

Asunto(s)

RESUMEN

Porcine astrovirus (PAstV) is a common cause of diarrhea in swine farms. The current understanding of the molecular virology and pathogenesis of PAstV is incomplete, especially due to the limited functional tools available. Here, ten sites in the open reading frame 1b (ORF1b) of the PAstV genome were determined to tolerate random 15 nt insertions based on the infectious full-length cDNA clones of PAstV using transposon-based insertion-mediated mutagenesis of three selected regions of the PAstV genome. Insertion of the commonly used Flag tag into seven of the ten insertion sites allowed the production of infectious viruses and allowed their recognition by specifically labeled monoclonal antibodies. Indirect immunofluorescence showed that the Flag-tagged ORF1b protein partially overlapped with the coat protein within the cytoplasm. An improved light-oxygen-voltage (iLOV) gene was also introduced into these seven sites, and only one viable recombinant virus that expressed the iLOV reporter gene at the B2 site was recovered. Biological analysis of the reporter viruses showed that these exhibited similar growth characteristics to the parental virus, but they produced fewer infectious virus particles and replicated at a slower rate. The recombinant viruses containing iLOV fused to ORF1b protein, which maintained their stability and displayed green fluorescence for up to three generations after passaging in cell culture. The porcine astroviruses (PAstVs) expressing iLOV were then used to assess the in vitro antiviral activities of mefloquine hydrochloride and ribavirin. Altogether, the recombinant PAstVs expressing iLOV can be used as a reporter virus tool for the screening of anti-PAstV drugs as well as the investigation of PAstV replication and the functional activities of proteins in living cells.

Asunto(s)

RESUMEN

A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.

Asunto(s)

RESUMEN

The development of Drug Delivery Systems (DDS) has led to increasingly efficient therapies for the treatment and detection of various diseases. DDS use a range of nanoscale delivery platforms produced from polymeric of inorganic materials, such as micelles, and metal and polymeric nanoparticles, but their variant chemical composition make alterations to their size, shape, or structures inherently complex. Genetically encoded protein nanocages are highly promising DDS candidates because of their modular composition, ease of recombinant production in a range of hosts, control over assembly and loading of cargo molecules and biodegradability. One example of naturally occurring nanocompartments are encapsulins, recently discovered bacterial organelles that have been shown to be reprogrammable as nanobioreactors and vaccine candidates. Here we report the design and application of a targeted DDS platform based on the Thermotoga maritima encapsulin reprogrammed to display an antibody mimic protein called Designed Ankyrin repeat protein (DARPin) on the outer surface and to encapsulate a cytotoxic payload. The DARPin9.29 chosen in this study specifically binds to human epidermal growth factor receptor 2 (HER2) on breast cancer cells, as demonstrated in an in vitro cell culture model. The encapsulin-based DDS is assembled in one step in vivo by co-expressing the encapsulin-DARPin9.29 fusion protein with an engineered flavin-binding protein mini-singlet oxygen generator (MiniSOG), from a single plasmid in Escherichia coli. Purified encapsulin-DARPin_miniSOG nanocompartments bind specifically to HER2 positive breast cancer cells and trigger apoptosis, indicating that the system is functional and specific. The DDS is modular and has the potential to form the basis of a multi-receptor targeted system by utilising the DARPin screening libraries, allowing use of new DARPins of known specificities, and through the proven flexibility of the encapsulin cargo loading mechanism, allowing selection of cargo proteins of choice.

RESUMEN

Nanomaterials have been widely used in biomedical sciences; however, the mechanism of interaction between nanoparticles and biomolecules is still not fully understood. In the present study, we report the interaction mechanism between differently sized Ag nanoparticles and the improved light-oxygen-voltage (iLOV) protein. The steady-state and time-resolved fluorescence results demonstrated that the fluorescence intensity and lifetime of the iLOV protein decreased upon its adsorption onto Ag nanoparticles, and this decrease was dependent upon nanoparticle size. Further, we showed that the decrease of fluorescence intensity and lifetime arose from electron transfer between iLOV and Ag nanoparticles. Moreover, through point mutation and controlled experimentation, we demonstrated for the first time that electron transfer between iLOV and Ag nanoparticles is mediated by the tryptophan residue in the iLOV protein. These results are of great importance in revealing the function of iLOV protein as it applies to biomolecular sensors, the field of nano-photonics, and the interaction mechanism between the protein and nanoparticles.

Asunto(s)

RESUMEN

Intracellular pH and redox states are critical for multiple processes and partly determine cell behavior. Here, we developed a genetically encoded dual-function probe, named pHand redox-sensitive fluorescent protein (pHaROS), for simultaneous real-time detection of changes in redox potential and pH in living cells. pHaROS consists of the Arabidopsis flavin mononucleotide-binding fluorescent protein iLOV and an mKATE variant, mBeRFP. Using pHaROS in Saccharomyces cerevisiae cells, we confirmed that H2O2 raises the overall redox potential of the cell and found that this increase is accompanied by a decrease in cytosolic pH. Furthermore, we observed spatiotemporal pH and redox homeostasis within the nucleus at various stages of the cell cycle in budding yeast (Saccharomyces cerevisiae) during cellular development and responses to oxidative stress. Importantly, we could tailor pHaROS to specific applications, including measurements in different organelles and cell types and the GSH/GSSG ratio, highlighting pHaROS's high flexibility and versatility. In summary, we have developed pHaROS as a dual-function probe that can be used for simultaneously measuring cellular pH and redox potential, representing a very promising tool for determining the cross-talk between intracellular redox- and pH-signaling processes in yeast and mammalian U87 cell.

Asunto(s)

RESUMEN

Recombinant SHFV infectious cDNA clones expressing a foreign gene from an additional sg mRNA were constructed. Two 3' genomic region sites, between ORF4' and ORF2b and between ORF4 and ORF5, were utilized for insertion of the myxoma M013 gene with a C-terminal V5 tag followed by one of the three inserted transcription regulatory sequences (TRS), TRS2', TRS4' or TRS7. M013 insertion at the ORF4'/ORF2b site but not the ORF4/ORF5 site generated progeny virus but only the recombinant virus with an inserted TRS2' retained the entire M013 gene through passage four. Insertion of an auto-fluorescent protein gene, iLOV, with an inserted TRS2' at the ORF4'/ORF2b site, generated viable progeny virus. iLOV expression was maintained through passage eight. Although regulation of SHFV subgenomic RNA synthesis is complex, the ORF4'/ORF2b site, which is located between the two sets of minor structural proteins, is able to tolerate foreign gene insertion.

Asunto(s)

RESUMEN

BACKGROUND: Rhinovirus (RV) causes the common cold and asthma exacerbations. The RV genome is a 7.3 kb single-strand positive-sense RNA. OBJECTIVE: Using minor group RV1A as a backbone, we sought to design and generate a recombinant RV1A accommodating fluorescent marker expression, thereby allowing tracking of viral infection. METHOD: Recombinant RV1A infectious cDNA clones harboring the coding sequence of green fluorescent protein (GFP), Renilla luciferase, or iLOV (for light, oxygen, or voltage sensing) were engineered and constructed. RV-infected cells were determined by flow cytometry, immunohistochemistry, and immunofluorescence microscopy. RESULTS: RV1A-GFP showed a cytopathic effect in HeLa cells but failed to express GFP or Renilla luciferase due to deletion. The smaller fluorescent protein construct, RV1A-iLOV, was stably expressed in infected cells. RV1A-iLOV expression was used to examine the antiviral effect of bafilomycin in HeLa cells. Compared to parental virus, RV1A-iLOV infection of BALB/c mice yielded a similar viral load and level of cytokine mRNA expression. However, imaging of fixed lung tissue failed to reveal a fluorescent signal, likely due to the oxidation and bleaching of iLOV-bound flavin mononucleotide. We therefore employed an anti-iLOV antibody for immunohistochemical and immunofluorescence imaging. The iLOV signal was identified in airway epithelial cells and CD45+ CD11b+ lung macrophages. CONCLUSIONS: These results suggest that RV1A-iLOV is a useful molecular tool for studying RV pathogenesis. The construction strategy for RV1A-iLOV could be applied to other RV serotypes. However, the detection of iLOV-expressing RV in fixed tissue required the use of an anti-iLOV antibody, limiting the value of this construct.

Asunto(s)

RESUMEN

Plant viruses are suitable as building blocks for nanomaterials and nanoparticles because they are easy to modify and can be expressed and purified using plants or heterologous expression systems. Plant virus nanoparticles have been utilized for epitope presentation in vaccines, for drug delivery, as nanospheres and nanowires, and for biomedical imaging applications. Fluorescent protein fusions have been instrumental for the tagging of plant virus particles. The monomeric non-oxygen-dependent fluorescent protein iLOV can be used as an alternative to green fluorescent protein. In this study, the iLOV sequence was genetically fused either directly or via a glycine-serine linker to the C-terminus of the Tobacco mosaic virus (TMV) coat protein (CP) and also carried an N-terminal Foot-and-mouth disease virus (FMDV) 2A sequence. Nicotiana benthamiana plants were inoculated with recombinant viral vectors and a systemic infection was achieved. The presence of iLOV fusion proteins and hybrid particles was confirmed by western blot analysis and transmission electron microscopy. Our data suggest that TMV-based vectors are suitable for the production of proteins at least as large as iLOV when combined with the FMDV 2A sequence. This approach allowed the simultaneous production of foreign proteins fused to the CP as well as free CP subunits.

RESUMEN

Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. There is thus a need for robust and unbiased molecular biological approaches for the construction of the requisite comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and comprehensive, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple protocol.

Asunto(s)

RESUMEN

Trichomonas vaginalis is a flagellated protozoan causing a notorious urogenital infection in humans. Due to its anaerobic metabolism, an alternative fluorescent protein that can be readily expressed in oxygen-deprived conditions is ideal. This study assessed the performance of iLOV, which does not require oxygen to function, as compared to the conventional enhanced green fluorescent protein (eGFP) in T. vaginalis. The results indicated that iLOV outperforms eGFP in both transient and stable expression, being detectable earlier and producing higher fluorescent intensity than eGFP in T. vaginalis. This finding facilitates forthcoming genetic studies that will advance the knowledge on this human parasitic infection.

Asunto(s)

RESUMEN

Over the past few decades, genetically encoded fluorescent proteins have been widely used as efficient probes to explore and investigate the roles of metal ions in biological processes. The discovery of small FMN-based fluorescent proteins, such as iLOV and FbFP, has enabled researchers to exploit these fluorescent reporter proteins for metal-sensing applications. In this study, we report the inherent binding properties of iLOV towards arsenic ions. The fluorescence quenching of iLOV was linearly related to the concentration of arsenic ions, and engineered proteins showed better sensitivity than the wild-type protein. Engineering key residues around the chromophore converted the iLOV protein into a highly sensitive sensor for As3+ ions. iLOVN468S exhibited an improved binding affinity with a dissociation constant of 1.5 µM. Furthermore, the circular dichroism spectra indicated that the fluorescence quenching mechanism might be related to arsenic-protein complex formation. Thus, the reagentless sensing of arsenic can potentially be exploited to determine intracellular or environmental arsenic using a genetically encoded biosensing approach.

Asunto(s)

RESUMEN

JC virus (JCV) is highly prevalent in humans, and may cause progressive multifocal leukoencephalopathy (PML), JCV granule cell neuronopathy (JCV GCN), JCV encephalopathy (JCVE) and JCV meningitis (JCVM) in immunocompromised individuals. There is no treatment for JCV, and a growing number of multiple sclerosis patients treated with immunomodulatory medications have developed PML. Antiviral agents against JCV are therefore highly desirable but remain elusive, due to the difficulty of determining their effect in vitro. A JCV strain carrying a fluorescent protein gene would greatly simplify and accelerate the drug screening process. To achieve this goal, we selected the 366bp improved Light, Oxygen or Voltage-sensing domain (iLOV) of plant phototropin gene and created two full-length JCV-iLOV constructs on the prototype JCV Mad1 backbone. The iLOV gene was inserted either before the early regulatory T gene (iLOV-T), or after the late Agno gene (iLOV-Agno). Both JCV iLOV strains were replication-competent in vitro and emitted a fluorescent signal detectable by confocal microscope, but JCV iLOV-T exhibited higher cellular and supernatant viral loads compared to JCV iLOV-Agno. JCV iLOV-T could also produce infectious pseudovirions. These data suggest that JCV iLOV constructs may become valuable tools for anti-JCV drug screening.

Asunto(s)

RESUMEN

In the filamentous heterocyst-forming cyanobacterium Anabaena PCC 7120, vegetative cells and heterocysts are interdependent on each other and engaged in exchanges of metabolites for survival when grown under diazotrophic conditions. In this organism, the periplasm appears to be continuous along each filament, with a shared outer membrane; however, barriers exist preventing free diffusion of the fluorescent protein GFP (27 kDa) targeted into the periplasmic space. Here we expressed a smaller fluorescent protein iLOV (≈ 13 kDa) fused to the All3333 (a putative homologue of NrtA) signal sequence corresponding to those recognized by the TAT protein translocation system, which exports iLOV to the periplasm of either heterocysts or vegetative cells. Fluorescence microscopy and immunoblot analysis indicated that the iLOV protein is translocated into the periplasm of the producing cell and properly processed, but does not diffuse to neighboring cells via the periplasm. Thus, periplasmic barriers appear to block diffusion of molecules with a size of 13 kDa, the minimum size tested thus far. Assuming that the physical barrier is the peptidoglycan sacculus, its pores might allow diffusion of molecules within the size range between the PatS pentapeptide and iLOV, thus between 0.53 kDa and 13 kDa.

Asunto(s)