Super-resolution imaging of nuclear import of adeno-associated virus in live cells
The SRV9 belongs to living attenuated rabies vaccine strain with good safety and immunogenicity, resulting from the alteration of Arg→Ser in the 333 position of glycoprotein of rabies viruses31. L. HSV-1 internalization required neither endosomal acidification nor clathrin- or caveolin-mediated endocytosis. H., Achor, D. They allow direct observation of the pathway a nanoparticle or virus particle takes on their way into and through the cell. Virus-mediated transduction of murine retina with adeno-associated virus: effects of viral capsid and genome size. J Virol 76: 7651–7660.
The use of motor-specific inhibitors indicated that traffic was predominantly kinesin mediated, consistent with the reconstitution of anterograde traffic. Until recently, three-dimensional electron microscopic analysis of the native states of viruses without intrinsic symmetry such as retroviruses and other enveloped viruses has remained largely unexplored. Minson, and D. Links to PubMed are also available for Selected References. Links to PubMed are also available for Selected References. The role of actin cytoskeleton in clathrin-mediated endocytosis in mammalian cells remains unclear. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.
Nat Genet 8: 148–154. Although AcMNPV has long been used as an efficient gene expression vector in insect cells (41, 45), recombinant baculoviruses were shown to be capable of entering into various mammalian cells without any replication and of expressing foreign genes under the control of mammalian promoters (11, 17, 65, 70, 71). Upon replication of the viral genome and subsequent packaging of genomes into viral nucleocapsids, immature particles egress the nucleus and again must be efficiently transported to the trans-Golgi network (TGN) or an endosomal compartment, the site of secondary envelopment (62). In vitro, HSVs are pantropic, causing lytic infections in various tissues and cell types of a broad range of host species . These axonal mRNAs are transported from the cell body as messenger ribonucleoprotein (mRNP) granules and their local translation in axons enables spatial control of homeostasis and production of rapid responses (reviewed in (Lin and Holt, 2008; Wang et al., 2010)). Depending on the purpose of study, viral membrane, capsid proteins or viral genome contents are often labeled separately or simultaneously (). Other studies, however, reported that HSV-1 infection can also induce autophagy [15, 16].
This could be due in part to a lack in consensus over the rAAV entry process or to the assumption that entry and early trafficking are universal, fixed processes. The occasional sharp declines in the intensity were likely due to large numbers of HSV moving out of the focal plane of the region of interest (ROI). In this process, many viruses have been reported to exploit the microtubules as transport tracks, such as adenovirus, herpes simplex virus, HIV, and influenza virus10,15,22,23. Our objectives were (i) to investigate the susceptibility of cultured human CF to productive HSV-1 entry and replication, (ii) to determine the expression of HSV-1 gD receptors in cultured CF, and (iii) to identify a gD receptor(s) crucial for HSV-1 entry into cultured CF. To discriminate between these possibilities, ubiquitination was inhibited during PRV infection in a neuron culture model (). The hydrodynamic size distribution of PRV was analyzed by dynamic light scattering (DLS). Filopodia are active in the neuronal growth cones of neurons, which guide axons and dendrites to their proper targets (Dent et al., 2011).
(2011). Prove that this would work so we are able to show the viral outer structure to resolution that wasn’t possible for this pleomorphic virus as before. Mol Ther 19: 1440–1448. Ali, RR, Reichel, MB, Thrasher, AJ, Levinsky, RJ, Kinnon, C, Kanuga, N et al. (1996). To explore the mechanisms of viral entry, a new infectivity assay was developed utilizing recombinant HVS expressing secreted, engineered alkaline phosphatase (SEAP) under the control of a HCMV promoter. Previously, Hutchinson et al.
Wang, Z, Ma, HI, Li, J, Sun, L, Zhang, J and Xiao, X (2003). Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo. Overall, this project will provide an innovative approach to both the study of inheritance and the role of epigenetic mechanisms in sustaining environmental effects within and across generations. Aschauer, DF, Kreuz, S and Rumpel, S (2013). Cell fusion was assayed by measuring β-gal by both in situ staining (photomicrographs) and calorimetric assay of NP-40 lysates of parallel co-cultures (values below, duplicate samples ± SD). These results suggest that down-regulation does not rely on the direct interaction of nectin-1 with cytoplasmic proteins, and highlights the importance of the nectin-1 ectodomain in this process. Schmidt, M, Voutetakis, A, Afione, S, Zheng, C, Mandikian, D and Chiorini, JA (2008).
Adeno-associated virus type 12 (AAV12): a novel AAV serotype with sialic acid- and heparan sulfate proteoglycan-independent transduction activity. Retina development in zebrafish requires the heparan sulfate proteoglycan Agrin Dev Neurobiol 2008; 68()(7 ): 877-98. Naim, B, Brumfeld, V, Kapon, R, Kiss, V, Nevo, R and Reich, Z (2007). Observations that HSV gE/gI and US9 can promote the anterograde axonal transport of unenveloped capsids, which are not obviously membrane associated, provided some clues as to how gE/gI and US9 (both membrane proteins) might function (6, 27, 28). J Biol Chem 282: 3881–3888. After 1 h at 37°C virus inocula were removed, cells were washed with PBS and the number of internalized virions was determined by an infectious centre assay. We used a monoclonal antibody directed against the KSHV small capsid protein Orf65 to stain the viral capsid in an IFA.
Nonenveloped viruses gain entry into host cells by endocytic mechanisms that may include clathrin- or caveolar-mediated endocytosis, and macropinocytosis . 1). The UL36 insert was confirmed by DNA sequencing. Subsequently, the supernatant was removed. Further, we found that gB, gD, gL, and 824L gH are insufficient for lipid mixing. These pseudotyped systems have limitations because the morphology of the virions differs significantly from that of authentic Ebola virions (spherical for retrovirus or VSV-pseudotyped virions versus filamentous for authentic Ebola virions). Y.
Phosphotyrosine 416 (phospho-Y416)-specific Src antibody, phosphotyrosine 527 (phospho-Y527)-specific Src antibody, total Src clone 36D10 antibody (Cell Signaling Technology), JAM-A-specific monoclonal antibody (provided by Charles Parkos, Emory University), human β1 integrin-specific monoclonal antibody MAB2253Z (Millipore), glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific antibody (Sigma), lysosome-associated membrane protein 1 (LAMP1)-specific antibody (Abcam), and actin-specific antibody (Santa Cruz Biotechnology) were used in indirect immunofluorescence and immunoblotting assays. Plasmid pGFP-hEB3 was a kind gift of John Victor Small (Institute of Molecular Biotechnology, Vienna, Austria). 2A). Virus infection and entry assays.For both infection and entry experiments, cells were plated on eight-well chamber slides coated with collagen (BD Biosciences) at 4 × 104 cells/well. For vector infusions into mouse brain, animals were anesthetized with isoflurane and placed into a stereotaxic frame. J Virol 84: 8888–8902. epub Yuan et al.
C. Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. Heparin and cycloheximide were purchased from Sigma. Rabbit phospho-C3G antibodies were from Santa Cruz Biotechnology Inc., Santa Cruz, CA. One-step RT-qPCR was carried out in CFX96™ Real-Time PCR Detection System using the iScriptTM One-Step RT-PCR Kit with SYBR® Green (Bio-Rad, USA). J Biomed Opt 15: 066005.