Herpes simplex virus 1 (HSV-1) helper co-infection affects the distribution of an amplicon encoded protein in glia

Herpes simplex virus-derived amplicon vectors simultaneously expressing the open reading frame encoding NR1 subunit of the NMDA receptor, either in sense or antisense orientation, as well as the open reading frame encoding the green fluorescent protein (GFP), as distinct transcription units, were constructed. Vector expression in cells was demonstrated by GFP-fluorescence, immunofluorescence, Western blots and RT-PCR. The vectors were inoculated into the dorsal hippocampus of adult male rats, which were then trained for habituation to an open field and for inhibitory avoidance to a foot-shock. Those animals injected with vectors expressing NR1 protein showed habituation to a new environment, and achieved the criteria for a step-down inhibitory avoidance to a foot-shock. In contrast, animals injected with vectors carrying the NR1 open reading frame in antisense position, showed neither habituation nor appropriate performance in the inhibitory avoidance task. There was no evidence for motor impairment or motivational disturbance, since all the animals exhibit similar behavior and performance in the training sessions. Hence, the impaired performance might be due to either amnesia or disability to record events. Transgene expression in brain, as revealed by GFP fluorescence, was mainly observed in pyramidal cells of CA1, but also in CA3. Therefore, our results strongly support the participation of hippocampal NR1 subunit in habituation to a new environment, but also in recording events for the inhibitory avoidance task. Hence, amplicon vectors appear to be useful tools to modify endogenous gene expression at a defined period, in restricted brain regions, and should allow investigating in vivo functions of genes.

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“…Amplicon vector stocks were prepared as already described (Lowenstein et al . 1994) using the amplicon plasmids (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Amplicon vector stocks were prepared as already described (Lowenstein et al 1994) using the amplicon plasmids ( Fig. 1a), and the highly neuroattenuated HSV-1 LaL virus as helper (Logvinoff & Epstein 2000).…”

Section: Amplicon Vectorsmentioning

confidence: 99%

Hippocampal infection with HSV‐1‐derived vectors expressing an NMDAR1 antisense modifies behavior

Adrover

Guyot-Revol

Cheli

et al. 2003

Genes Brain and Behavior

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“…The possibility to amplify the transgene expression may allow the dose of the therapeutic vector to be lowered, minimizing a possible intrinsic toxic effect, as reported in the literature for other vectors. This study shows that amplicon-mediated delivery of anti-BKVT siRNA can inhibit T-Ag expression and growth of 26 The number of living cells was determined in triplicate samples at 24, 48 and 72 h after transduction by Trypan blue staining. Compared to controls, the growth rate of pRPc cells was markedly reduced by the pA-shBKVT-GFP amplicon vector in a dose-dependent manner.…”

Section: Hsv-1 Amplicon Vector For Sirna Transfer S Sabbioni Et Almentioning

confidence: 91%

“…In in vitro experiments, pRPc cells were either non-transduced, transduced with the pA-shBKVT-GFP amplicon or with the empty control amplicon pA-EUA, derived from the pA-SF1 amplicon plasmid. 26 The cell number was determined at 24, 48 and 72 h post-transduction, using a MOT ranging from 0.5 to 5. In comparison with control amplicon or non-transduced cells, the cellular growth rate was reduced in a dose-dependent manner in cells infected by pA-shBKVT-GFP amplicon vector ( Figure 4a).…”

mentioning

confidence: 99%

Use of herpes simplex virus type 1-based amplicon vector for delivery of small interfering RNA

et al. 2006

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Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. The use of DNA-based plasmid vectors to achieve transient and stable expression of siRNA has been developed to avoid the problems of double-stranded oligonucleotides transfection. These vectors direct the transcription of small hairpin RNAs (shRNAs) from a polymerase-III (H1 or U6)-RNA gene promoter. However, numerous disadvantages remain, including low transfection efficiency and difficulty in transfecting primary cells. To overcome some of these problems, the use of viral vectors for siRNA delivery has been described. Retroviral, adenoviral, adeno-associated and herpes viral shRNAs delivery systems have been successfully used to silence genes, in vitro and in vivo. The use of a herpes simplex virus type 1 (HSV-1)-based amplicon vector for siRNA delivery into mammalian cells, using human polyomavirus BK (BKV)-transformed cells as a model system is described. The results demonstrate the ability of amplicon vectors to inhibit the expression of BKV T-Ag and tumorigenicity of BKV-transformed cells. We show that the use of the amplicon vector is highly efficient for the delivery of siRNA molecules. The unique ability of these vectors to deliver multiple copies of siRNA may provide a useful tool in the development of novel anticancer therapy.

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“…Compared to the parental MoMLV genome, these GPE transcription units lacked primer binding sites and polypurine tracts, both long terminal repeats (LTRs), and the packaging sequence E. This design of this defective-retrovirus genome was adopted to abolish or minimize its packaging, reverse transcription (RT), and integration. Each transcription unit was then cloned into a plasmid containing one HSV-1 origin of replication (ori-S) and one HSV-1 packaging signal ("a") (23). The resulting amplicon plasmids were called pA-TK-GPE, pA-IE3-GPE, and pA-CMV-GPE.…”

mentioning

confidence: 99%

Defective herpes simplex virus type 1 vectors harboring gag, pol, and env genes can be used to rescue defective retrovirus vectors

Savard

Cosset

Epstein

1997

J Virol

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A retroviral packaging transcription unit was constructed in which the Moloney murine leukemia virus (MoMLV) gag-pol and env genes are expressed under the control of herpesvirus regulatory sequences. This transcription unit, lacking long terminal repeats, primer binding sites, and most of the retrovirus packaging signal but retaining both retroviral donor and acceptor splice sites, was cloned into a herpes simplex virus type 1 (HSV-1) amplicon plasmid, and amplicon vectors (the gag-pol-env [GPE] vectors) were generated by using a defective HSV-1 vector as helper virus. The GPE vector population was used to infect human TE671 cells (ATCC CRL 8805), harboring a lacZ provirus (TE-lac2 cells), and supernatants of infected cells were collected and filtered at different times after infection. These supernatants were found to contain infectious ecotropic lacZ retroviral particles, as shown both by reverse transcription-PCR and by their ability to transduce a ␤-galactosidase activity to murine NIH 3T3 cells but not to human TE671 cells. The titer of retroviral vectors released by GPE vector-infected TE-lac2 cells increased with the dose of infectious amplicon particles. Retrovirus vector production was inhibited by superinfection with helper virus, indicating that helper virus coinfection negatively interfered with retrovirus production. Induction of retrovirus vectors by GPE vectors was neutralized by anti-HSV-1 but not by anti-MoMLV antiserum, while transduction of ␤-galactosidase activity to NIH 3T3 cells by supernatants of GPE vector-infected TE-lac2 cells was neutralized by anti-MoMLV antiserum. These results demonstrate that HSV-1 GPE amplicon vectors can rescue defective lacZ retrovirus vectors and suggest that they could be used as a sort of launching ramp to fire defective retrovirus vectors from within virtually any in vitro or in vivo cell type containing defective retroviral vectors.

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Herpes simplex virus 1 (HSV-1) helper co-infection affects the distribution of an amplicon encoded protein in glia

Cited by 18 publications

References 0 publications

Hippocampal infection with HSV‐1‐derived vectors expressing an NMDAR1 antisense modifies behavior

Hippocampal infection with HSV‐1‐derived vectors expressing an NMDAR1 antisense modifies behavior

Use of herpes simplex virus type 1-based amplicon vector for delivery of small interfering RNA

Defective herpes simplex virus type 1 vectors harboring gag, pol, and env genes can be used to rescue defective retrovirus vectors

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