Characteristics of DNA-AuNP networks on cell membranes and real-time movies for viral infection

AbstractThis data article provides complementary data for the article entitled “DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding” Li et al. (2016) [1]. The experimental methods for the preparation and characterization of DNA-conjugated nanoparticle networks on cell membranes were described. Confocal fluorescence images, agarose gel electrophoresis images and hydrodynamic diameter of DNA-conjugated gold nanoparticle (DNA-AuNP) networks were presented. In addition, we have prepared QDs-labeled RSV (QDs-RSV) to real-time monitor the RSV infection on HEp-2 cells in the absence and presence of DNA-AuNP networks. Finally, the cell viability of HEp-2 cells coated by six types of DNA-nanoparticle networks was determined after RSV infection

Exciton Dynamics in Perovskite CsPbBr₃ Semiconductor Nanocrystals

Semiconductor nanocrystals, or quantum dots, have attracted much attention over the past three decades due to their potential applications in optoelectronic devices, such as displays, sensors, photovoltaics, and as single-photon sources. This class of materials differs from traditional bulk semiconductors in their tunability of optical and electronic properties, which are determined by their size as well as the material composition. In the past decade, a new type of semiconductor, perovskite, has shown extraordinary photophysical properties; their low-dimensional counterpart, all-inorganic perovskite nanocrystals (CsPbBr₃ NCs), has since shown promising performances in optoelectronic devices. However, their fundamental photophysical properties, as well as energy transport properties within the NC solids, are still under active debate. Here, I measure both the temperature-dependent (35 K - 300 K) absorption and PL spectra of zwitterionic ligand-capped CsPbBr₃ NCs with four different edge lengths (L = 4.9 - 13.2 nm). The excitonic transitions observed in the absorption spectra can be explained with an effective mass model considering the quasicubic NC shape and non-parabolicity of the electronic bands. We observe a temperature-dependent Stokes shift; while the trend is similar to the Stokes shift observed in both MAPbBr₃ and CsPbBr₃ single crystals, it does not approach zero at cryogenic temperatures, pointing to an additional contribution intrinsically present in the NCs. Surprisingly, the effective dielectric constant determined from the best fit model parameters is independent of temperature. Next, I apply time-resolved photoluminescence microscopy (TPLM) to visualize exciton transport in CsPbBr₃ NC films with various surface treatments. We show that, in all samples, exciton diffusivity exhibits a striking excitation power dependence. Through fluence-, repetitionrate-, and temperature-dependent measurements, we demonstrate that this behavior does not arise from exciton-exciton annihilation or sample heating effects. Further investigation reveals that upon photoexcitation, CsPbBr₃ NCs transform into a transient configuration wherein exciton transport becomes faster. This reconfiguration memory persists for microseconds, long after electronic relaxation. Finally, I present preliminary TPLM measurements on CsPbBr₃ NC superlattices at cryogenic temperatures. We observe interesting power-dependent and temperature-dependent trends. While the exact transport mechanism in this regime is still unclear, the findings point to new transport phenomena that require further investigation.Ph.D

A time-domain view of charge carriers in semiconductor nanocrystal solids

© The Royal Society of Chemistry 2020. The movement of charge carriers within semiconductor nanocrystal solids is fundamental to the operation of nanocrystal devices, including solar cells, LEDs, lasers, photodetectors, and thermoelectric modules. In this perspective, we explain how recent advances in the measurement and simulation of charge carrier dynamics in nanocrystal solids have led to a more complete picture of mesoscale interactions. Specifically, we show how time-resolved optical spectroscopy and transient photocurrent techniques can be used to track both equilibrium and non-equilibrium dynamics in nanocrystal solids. We discuss the central role of energetic disorder, the impact of trap states, and how these critical parameters are influenced by chemical modification of the nanocrystal surface. Finally, we close with a forward-looking assessment of emerging nanocrystal systems, including anisotropic nanocrystals, such as nanoplatelets, and colloidal lead halide perovskites

Autographa californica Multiple Nucleopolyhedrovirus Ac92 (ORF92, P33) Is Required for Budded Virus Production and Multiply Enveloped Occlusion-Derived Virus Formation▿

The Autographa californica multiple nucleopolyhedrovirus orf92 (p33), ac92, is one of 31 genes carried in all sequenced baculovirus genomes, thus suggesting an essential function. Ac92 has homology to the family of flavin adenine dinucleotide-linked sulfhydryl oxidases and is related to the ERV/ALR family of sulfhydryl oxidases. The role of ac92 during virus replication is unknown. Ac92 was associated with the envelope of both budded and occlusion-derived virus (ODV). To investigate the role of Ac92 during virus replication, an ac92-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration and plaque assays showed no virus spread in ac92-knockout bacmid DNA-transfected insect cells. Deletion of ac92 did not affect viral DNA replication. However, ac92-knockout bacmid DNA-transfected cells lacked multiply enveloped occlusion-derived nucleocapsids; instead, singly enveloped nucleocapsids were detected. To gain insight into the requirement for sulfhydryl oxidation during virus replication, a virus was constructed in which the Ac92 C155XXC158 amino acids, important for sulfhydryl oxidase activity, were mutated to A155XXA158. The mutant virus exhibited a phenotype similar to that of the knockout virus, suggesting that the C-X-X-C motif was essential for sulfhydryl oxidase activity and responsible for the altered ODV phenotype

Autographa californica M nucleopolyhedrovirus open reading frame 109 affects infectious budded virus production and nucleocapsid envelopment in the nucleus of cells

Autographa californica M nucleopolyhedrovirus (AcMNPV) open reading frame 109 (ac109) is conserved in all known baculovirus genomes, suggesting a crucial role in virus replication. Although viruses lacking ac109 have been previously characterized, the phenotypes differ from production of non-infectious virions to lack of virion production. To re-examine ac109 function, we constructed a recombinant AcMNPV bacmid, AcBAC109KO, with a deletion in ac109. We did not detect infectious budded virus after transfection of AcBAC109KO DNA into cells. In the nucleus, nucleocapsids had envelopment defects and polyhedra lacked virions. DNA synthesis and gene expression between AcBAC109KO and a control virus were similar. However, lower levels of non-infectious budded virus were detected from AcBAC109KO DNA-transfected cells compared to the parental virus using Q-PCR to detect viral DNA or by immunoblotting to detect a budded virus protein. Therefore, deletion of ac109 affects envelopment of nucleocapsids in the nucleus and the production of infectious budded virus.Fil: Lehiy, Christopher J.. Kansas State University. Division of Biology. Molecular, Cellular, and Developmental Biology Program; Estados UnidosFil: Wu, Wenbi. Kansas State University. Division of Biology. Molecular, Cellular, and Developmental Biology Program; Estados UnidosFil: Berretta, Marcelo Facundo. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. Kansas State University. Division of Biology. Molecular, Cellular, and Developmental Biology Program; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Passarelli, A. Lorena. Kansas State University. Division of Biology. Molecular, Cellular, and Developmental Biology Program; Estados Unido

RohrmannGeorgeMicrobiologyBaculovirusSulfhydrylOxidaseFigures1-8.pdf

The Autographa californica M nucleopolyhedrovirus (AcMNPV) sulfhydryl oxidase Ac92 is essential for production of infectious virions. Ac92 also interacts with human p53 and enhances human p53-induced apoptosis in insect cells, but it is not known whether any relationship exists between Ac92 and native p53 homologs from insect hosts of AcMNPV. We found that Ac92 interacted with SfP53 from Spodoptera frugiperda in infected cells and oxidized SfP53 in vitro. However, Ac92 did not interact with or oxidize a mutant of SfP53 predicted to lack DNA binding. Silencing Sfp53 expression did not rescue the ability of an ac92-knockout virus to produce infectious virus. Similarly, ac92 expression did not affect SfP53-stimulated caspase activity or the localization of SfP53. Thus, although Ac92 binds to SfP53 during AcMNPV replication and oxidizes SfP53 in vitro, we could not detect any effects of this interaction on AcMNPV replication in cultured cells

RohrmannGeorgeMicrobiologyBaculovirusSulfhydrylOxidase.pdf

The Autographa californica M nucleopolyhedrovirus (AcMNPV) sulfhydryl oxidase Ac92 is essential for production of infectious virions. Ac92 also interacts with human p53 and enhances human p53-induced apoptosis in insect cells, but it is not known whether any relationship exists between Ac92 and native p53 homologs from insect hosts of AcMNPV. We found that Ac92 interacted with SfP53 from Spodoptera frugiperda in infected cells and oxidized SfP53 in vitro. However, Ac92 did not interact with or oxidize a mutant of SfP53 predicted to lack DNA binding. Silencing Sfp53 expression did not rescue the ability of an ac92-knockout virus to produce infectious virus. Similarly, ac92 expression did not affect SfP53-stimulated caspase activity or the localization of SfP53. Thus, although Ac92 binds to SfP53 during AcMNPV replication and oxidizes SfP53 in vitro, we could not detect any effects of this interaction on AcMNPV replication in cultured cells

Effects of Manipulating Fibroblast Growth Factor Expression on Sindbis Virus Replication In Vitro and in Aedes aegypti Mosquitoes

Fibroblast growth factors (FGFs) are conserved among vertebrate and invertebrate animals and function in cell proliferation, cell differentiation, tissue repair, and embryonic development. A viral fibroblast growth factor (vFGF) homolog encoded by baculoviruses, a group of insect viruses, is involved in escape of baculoviruses from the insect midgut by stimulating basal lamina remodeling. This led us to investigate whether cellular FGF is involved in the escape of an arbovirus from mosquito midgut. In this study, the effects of manipulating FGF expression on Sindbis virus (SINV) replication and escape from the midgut of the mosquito vector Aedes aegypti were examined. RNAi-mediated silencing of either Ae. aegypti FGF (AeFGF) or FGF receptor (AeFGFR) expression reduced SINV replication following oral infection of Ae. aegypti mosquitoes. However, overexpression of baculovirus vFGF using recombinant SINV constructs had no effect on replication of these viruses in cultured mosquito or vertebrate cells, or in orally infected Ae. aegypti mosquitoes. We conclude that reducing FGF signaling decreases the ability of SINV to replicate in mosquitoes, but that overexpression of vFGF has no effect, possibly because endogenous FGF levels are already sufficient for optimal virus replication. These results support the hypothesis that FGF signaling, possibly by inducing remodeling of midgut basal lamina, is involved in arbovirus midgut escape following virus acquisition from a blood meal

Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Assembly Is Interrupted upon Deletion of the 38K Gene

38K (ac98) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose function is unknown. To determine the role of 38K in the baculovirus life cycle, a 38K knockout bacmid containing the AcMNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a 38K repair bacmid was constructed by transposing the 38K open reading frame with its native promoter region into the polyhedrin locus of the 38K knockout bacmid. After transfection of these viruses into Spodoptera frugiperda cells, the 38K knockout bacmid led to a defect in production of infectious budded virus, while the 38K repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Slot blot analysis indicated that 38K deletion did not affect the levels of viral DNA replication. Subsequent immunoelectron-microscopic analysis revealed that masses of electron-lucent tubular structures containing the capsid protein VP39 were present in cells transfected with 38K knockout bacmids, suggesting that nucleocapsid assembly was interrupted. In contrast, the production of normal nucleocapsids was restored when the 38K knockout bacmid was rescued with a copy of 38K. Recombinant virus that expresses 38K fused to green fluorescent protein as a visual marker was constructed to monitor protein transport and localization within the nucleus during infection. Fluorescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized to the center of the nucleus. These results demonstrate that 38K plays a role in nucleocapsid assembly and is essential for viral replication in the AcMNPV life cycle

Autographa californica Multiple Nucleopolyhedrovirus ac76 Is Involved in Intranuclear Microvesicle Formation▿

In this study, we characterized Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf76 (ac76), which is a highly conserved gene of unknown function in lepidopteran baculoviruses. Transcriptional analysis of ac76 revealed that transcription of multiple overlapping multicistronic transcripts initiates from a canonical TAAG late-transcription start motif but terminates at different 3′ ends at 24 h postinfection in AcMNPV-infected Sf9 cells. To investigate the role of ac76 in the baculovirus life cycle, an ac76-knockout virus was constructed using an AcMNPV bacmid system. Microscopy, titration assays, and Western blot analysis demonstrated that the resulting ac76-knockout virus was unable to produce budded viruses. Quantitative real-time PCR analysis demonstrated that ac76 deletion did not affect viral DNA synthesis. Electron microscopy showed that virus-induced intranuclear microvesicles as well as occlusion-derived virions were never observed in cells transfected with the ac76-knockout virus. Confocal microscopy analysis revealed that Ac76 was predominantly localized to the ring zone of nuclei during the late phase of infection. This suggests that ac76 plays a role in intranuclear microvesicle formation. To the best of our knowledge, this is the first baculovirus gene identified to be involved in intranuclear microvesicle formation