In situ formation of viruses tagged with quantum dots.

Quantum dots (QDs) have great potential for applications in bio-related fields, due to their high photoluminescence, photochemical stability and size-dependent emission. QDs used for the construction of QD-virus hybrids can be harnessed as an imaging probe to reveal viral infection pathways and screen antiviral agents. In the study, human embryonic kidney (HEK) 293T cells were transfected with three plasmids, pSIN-EGFP, pMDG, and p8.91, to produce lentiviruses which can make infected cells express enhanced green fluorescent protein (EGFP). The QDs employed were CdSe-ZnS semiconductor nanocrystals emitting red fluorescence. The QD-virus hybrids, constructed as lentiviruses, were budding from the membrane surface of HEK 293T producer cells on which QDs encapsulated with alkylated chitosan (chitosan-QDs) were pre-adsorbed via electrostatic attraction force. Such in situ formation of QD-virus hybrids was confirmed by TEM micrographs indicating the lentivirus was capped with chitosan-modified QDs. To further illustrate the effectiveness (i.e., infectivity and photoluminescence) of the constructed QD-virus hybrids, NIH 3T3 cells were infected with the in situ fabricated QD-virus hybrids. Our results showed QDs were indeed entering NIH 3T3 cells along with lentiviruses as hybrids. Moreover, photoluminescence and infectivity of QD-virus hybrids remained intact, as compared to QDs and lentivirus alone. The unique approach of constructing QD-virus hybrids taking advantage of the viral budding process offers a feasible tool to create enveloped virus incorporated with nanomaterials for the study of fundamental and applied virology.

In vivo targeting of subventricular zone astrocytes.

The subventricular zone (SVZ) is a dynamic cellular niche with unique neurogenic properties that are, as of yet, not fully understood. Astrocytes residing in the SVZ have been shown to spawn migratory neuroblasts via transitory amplifying progenitor cells. These migratory neuroblasts play a role in maintaining the olfactory circuitry in healthy brains and potentially have restorative properties after brain injury. Therefore, it is imperative to understand the basic nature of these neurogenic astrocytes in order to gain a more cohesive picture of SVZ adult neurogenesis. However, one of the obstacles in this line of research is to specifically genetically modify SVZ astrocytes. Viral vector systems, based on adeno-associated viruses and lentiviruses, are flexible gene transfer systems that allow long-term transgene expression in a host cell. Electroporation allows for the transient expression of larger transgenes; whereas the cre/loxP system provides a lifetime of inherently stable genetic modulation. The benefits and drawbacks of these transduction methods and the application of various astrocyte-specific promoters are discussed with regard to their efficiency and accuracy when transducing adult SVZ astrocytes in the mouse brain. In vivo studies that manipulate gene expression in SVZ astrocytes will be essential to fully dissect and understand the complex molecular and cellular properties of the SVZ in the upcoming years.

Controlled inactivation of recombinant viruses with vitamin B2.

Inactivated viruses are important tools for vaccine development and gene transfer. 8-Methoxypsoralen (8-MOP) and long-wavelength ultraviolet irradiation (LWUVI) inactivates many viruses. Toxicity limits its use in animals and humans. Toxicological and photosensitizing properties of riboflavin make it suitable for virus inactivation in preparations for biological use. Viruses expressing beta-galactosidase were mixed with either 8-MOP (1.5mM) or riboflavin (50 microM) and exposed to LWUVI (365 nm) for 2 h. Virus activity was determined by limiting dilution. The half-life of the adenovirus preparation treated with 8-MOP was 8.28 ns(-1) and 36.5 ns(-1) after treatment with riboflavin. Despite the difference in half-life, both preparations were completely inactivated within 45 min. In contrast, the half-lives for adeno-associated virus (AAV) preparations were similar (63 ns(-1) 8-MOP vs. 67 ns(-1) riboflavin). Each AAV preparation was fully inactivated within 90 min. The half-life of lentivirus was 193.4 ns(-1) after treatment with 8-MOP and 208 ns(-1) after exposure to riboflavin. Virus treated with riboflavin was inactivated within 20 min. Virus exposed to 8-MOP was inactivated in 90 min. DNA and RNA viruses can be inactivated by riboflavin and LWUVI and used in physiological systems sensitive to other photochemicals.

Tubulovesicular structures within vesicular stomatitis virus G protein-pseudotyped lentiviral vector preparations carry DNA and stimulate antiviral responses via Toll-like receptor 9.

Recombinant lentiviral vectors (LVs) are commonly used as research tools and are being tested in the clinic as delivery agents for gene therapy. Here, we show that Vesicular stomatitis virus G protein (VSV-G)-pseudotyped LV preparations produced by transient transfection are heavily contaminated with tubulovesicular structures (TVS) of cellular origin, which carry nucleic acids, including the DNA plasmids originally used for LV generation. The DNA carried by TVS can act as a stimulus for innate antiviral responses, triggering Toll-like receptor 9 and inducing alpha/beta interferon production by plasmacytoid dendritic cells (pDC). Removal of TVS markedly reduces the ability of VSV-G-pseudotyped LV preparations to activate pDC. Conversely, virus-free TVS are sufficient to stimulate pDC and act as potent adjuvants in vivo, eliciting T- and B-cell responses to coadministered proteins. These results highlight the role of by-products of virus production in determining the immunostimulatory properties of recombinant virus preparations and suggest possible strategies for diminishing responses to LVs in gene therapy and in research use.

The zinc-finger protein ZFR is critical for Staufen 2 isoform specific nucleocytoplasmic shuttling in neurons.

In mammalian neurons, transport and translation of mRNA to individual potentiated synapses is believed to occur via a heterogeneous population of RNA granules. To identify components of Staufen2-containing granules, we used the yeast two-hybrid system. A mouse fetal cDNA library was screened with the N-terminal fragment of Staufen2 as bait. ZFR, a three zinc finger protein, was identified as an interacting protein. Confocal microscopy showed that ZFR, although mainly nuclear, was also found in the somatodendritic compartment of primary hippocampal neurons where it localized as granule-like structures. Co-localization with Staufen2 was observed in several granules. Biochemical analyses (immunoprecipitation, cell fractionation) further confirmed the ZFR/Staufen2 association. ZFR was shown to interact with at least the Staufen2(62) isoform, but not with Staufen1. ZFR also co-fractionated with ribosomes and Staufen2(59) and Staufen2(52) in a sucrose gradient. Interestingly, knockdown expression of ZFR through RNA interference in neurons relocated specifically the Staufen2(62), but not the Staufen2(59), isoform to the nucleus. Our results demonstrate that ZFR is a native component of Staufen2-containing granules and likely plays its role during early steps of RNA transport and localization. They also suggest that one of these roles may be linked to Staufen2(62)-containing RNA granule formation in the nucleus and/or to their nucleo-cytoplasmic shuttling.

Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer.

Recombinant lentiviral vectors stably transduce both dividing and nondividing cells. Virus pseudotyping with vesicular stomatitis virus envelope G (VSV-G) protein broadens the host range of lentiviral vector and enables vector concentration by ultra-centrifugation. However, as a result of virus vector concentration, contaminating protein debris derived from vector-producing cell culture media is toxic to target cells and reduces the transduction efficiency. Here we report a new and rapid technique for purifying lentivirus vector using the strong anion exchange column that significantly improves gene transfer rates. We purified VSV-G pseudotyped self-inactivating lentivirus vector and obtained two protein elution peaks (Peak 1 and Peak 2) corresponding to transducing activity. Peak 1 viral particles were 4-8 times more effective in transducing target cells than Peak 2 or non-purified (pre-HPLC) viral particles. We used purified lentivirus vector expressing the human Fanconi anemia group A (FANCA) gene to transduce murine hematopoietic stem/progenitor cells. We observed a consistent 2- to 3-fold increase in gene transfer rates using Peak 1 purified virus compared with non-purified virus. We conclude that the purification method using the HPLC system provides the highly purified virus vector that reduces cell toxicity and significantly improves gene transfer in primary cells.