scholarly journals Optimal Expression of the Envelope Glycoprotein of Orthobornaviruses Determines the Production of Mature Virus Particles

2020 ◽  
Author(s):  
Madoka Sakai ◽  
Yoko Fujita ◽  
Ryo Komorizono ◽  
Takehiro Kanda ◽  
Yumiko Komatsu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Disease Virus ◽  
Envelope Glycoprotein ◽  
Particle Production ◽  
Rna Virus ◽  
High Titer ◽  
Transduction Efficiency ◽  
Infectious Particle ◽  
Borna Disease

An RNA virus-based episomal vector (REVec) whose backbone is Borna disease virus 1 (BoDV-1) can provide long-term gene expression in transduced cells. To improve the transduction efficiency of REVec, we evaluated the role of the viral envelope glycoprotein (G) of the genus Orthobornavirus, including that of BoDV-1, in the production of infectious particles. By using G-pseudotype assay in which the lack of G in G-deficient REVec (ΔG-REVec) was compensated for expression of G, we found that excess expression of BoDV-1-G does not affect particle production itself but results in uncleaved and aberrant mature G expression in the cells, leading to the production of REVec particles with low transduction titers. We revealed that the expression of uncleaved G in the cells inhibits the incorporation of mature G and vgRNA into the particles. This feature of G was conserved among mammalian and avian orthobornaviruses; however, the cleavage efficacy of canary bornavirus 1 (CnBV-1)-G was exceptionally not impaired by its excess expression, which led to the production of the pseudotype ΔG-REVec with the highest titer. Chimeric G proteins between CnBV-1 and -2 revealed that the signal peptide of CnBV-1-G was responsible for the cleavage efficacy through the interaction with intracellular furin. We showed that CnBV-1 G leads to the development of pseudotyped REVec with high transduction efficiency and a high-titer recombinant REVec. Our study demonstrated that the restricted expression of orthobornavirus G contributes to the regulation of infectious particle production, the mechanism of which can improve the transduction efficiency of REVec. IMPORTANCE Most viruses causing persistent infection produce few infectious particles from the infected cells. Borna disease virus 1, a member of the genus Orthobornavirus, is an RNA virus that persistently infects the nucleus and has been applied to vectors for long-term gene expression. In this study, we showed that, common among orthobornaviruses, excessive G expression does not affect particle production itself but reduces the production of infectious particles with mature G and genomic RNA. This result suggested that limited G expression contributes to suppressing abnormal viral particle production. On the other hand, we found that canary bornavirus 1 has an exceptional G maturation mechanism and produces a high-titer virus. Our study will contribute to not only understanding the mechanism of infectious particle production but also improving the vector system of orthobornaviruses.

scholarly journals Plasmid-Based shRNA Lentiviral Particle Production for RNAi Applications

2014 ◽  
Vol 19 (9) ◽  
pp. 1309-1313 ◽  
Author(s):  
David Shum ◽  
Hakim Djaballah
Keyword(s):  
Particle Production ◽  
High Titer ◽  
Cell Types ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hairpin Rna ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Overall Efficiency

Lentiviral vectors have become mainstream gene transfer vehicles for their ability to deliver and integrate into host cells. In RNA interference (RNAi) applications, lentiviral constructs constitutively express double-stranded RNA molecules, usually as short hairpin RNA (shRNA), enabling long-term gene silencing, and, when pseudotyped with a broad host glycoprotein envelope, allow a multitude of cell types to be transduced. Their successful use ultimately relies on the production of lentiviral particles in high titer and uniformity. Typical methods require the transfection of three or more plasmids in which essential viral elements have been encoded separated so as to remain replication deficient. These transfection procedures are of critical importance; however, methods often vary among laboratories, making it difficult to assess the overall efficiency of lentiviral particle production. In this report, we focus exclusively on this step and compare the overall impact of the commercial transfection reagent FuGENE 6 with FuGENE HD. We found that FuGENE HD resulted in at least 5-fold improvement in viral particle titer as assessed by the p24 standard enzyme-linked immunosorbent assay. We present the complete optimized workflow and demonstrate this utility in which a single modification of this transfection step improved the lentiviral particle production.

scholarly journals Borna Disease Virus Phosphoprotein Represses p53-Mediated Transcriptional Activity by Interference with HMGB1

2003 ◽  
Vol 77 (22) ◽  
pp. 12243-12251 ◽  
Author(s):  
Guoqi Zhang ◽  
Takeshi Kobayashi ◽  
Wataru Kamitani ◽  
Satoshi Komoto ◽  
Makiko Yamashita ◽  
...  
Keyword(s):  
Disease Virus ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Rna Virus ◽  
Broad Host Range ◽  
Neural Cells ◽  
Cellular Functions ◽  
Borna Disease Virus ◽  
Multifunctional Protein ◽  
Borna Disease

ABSTRACT Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that has a broad host range in warm-blooded animals, probably including humans. Recently, it was demonstrated that a 24-kDa phosphoprotein (P) of BDV directly binds to a multifunctional protein, amphoterin-HMGB1, and inhibits its function in cultured neural cells (W. Kamitani, Y. Shoya, T. Kobayashi, M. Watanabe, B. J. Lee, G. Zhang, K. Tomonaga, and K. Ikuta, J. Virol. 75:8742-8751, 2001). This observation suggested that expression of BDV P may cause deleterious effects in cellular functions by interference with HMGB1. In this study, we further investigated the significance of the binding between P and HMGB1. We demonstrated that P directly binds to the A-box domain on HMGB1, which is also responsible for interaction with a tumor suppression factor, p53. Recent works have demonstrated that binding between HMGB1 and p53 enhances p53-mediated transcriptional activity. Thus, we examined whether BDV P affects the transcriptional activity of p53 by interference with HMGB1. Mammalian two-hybrid analysis revealed that p53 and P competitively interfere with the binding of each protein to HMGB1 in a p53-deficient cell line, NCI-H1299. In addition, P was able to significantly decrease p53-mediated transcriptional activation of the cyclin G promoter. Furthermore, we showed that activation of p21waf1 expression was repressed in cyclosporine-treated BDV-infected cells, as well as p53-transduced NCI-H1299 cells. These results suggested that BDV P may be a unique inhibitor of p53 activity via binding to HMGB1.

scholarly journals 1-β-d-Arabinofuranosylcytosine Inhibits Borna Disease Virus Replication and Spread

2002 ◽  
Vol 76 (12) ◽  
pp. 6268-6276 ◽  
Author(s):  
Jeffrey J. Bajramovic ◽  
Sylvie Syan ◽  
Michel Brahic ◽  
Juan Carlos de la Torre ◽  
Daniel Gonzalez-Dunia
Keyword(s):  
Disease Virus ◽  
Measles Virus ◽  
Rna Virus ◽  
Neurological Diseases ◽  
Borna Disease Virus ◽  
Negative Strand ◽  
Neuropsychiatric Diseases ◽  
Borna Disease

ABSTRACT Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that causes neurological diseases in a variety of warm-blooded animal species. There is general consensus that BDV can also infect humans, being a possible zoonosis. Although the clinical consequences of human BDV infection are still controversial, experimental BDV infection is a well-described model for human neuropsychiatric diseases. To date, there is no effective treatment against BDV. In this paper, we demonstrate that the nucleoside analog 1-β-d-arabinofuranosylcytosine (Ara-C), a known inhibitor of DNA polymerases, inhibits BDV replication. Ara-C treatment inhibited BDV RNA and protein synthesis and prevented BDV cell-to-cell spread in vitro. Replication of other negative-strand RNA viruses such as influenza virus or measles virus was not inhibited by Ara-C, underscoring the particularity of the replication machinery of BDV. Strikingly, Ara-C treatment induced nuclear retention of viral ribonucleoparticles. These findings could not be attributed to known effects of Ara-C on the host cell, suggesting that Ara-C directly inhibits the BDV polymerase. Finally, we show that Ara-C inhibits BDV replication in vivo in the brain of infected rats, preventing persistent infection of the central nervous system as well as the development of clinical disease. These findings open the way to the development of effective antiviral therapy against BDV.

scholarly journals Fine Structure and Morphogenesis of Borna Disease Virus

1999 ◽  
Vol 73 (1) ◽  
pp. 760-766 ◽  
Author(s):  
Takehiro Kohno ◽  
Toshiyuki Goto ◽  
Tomohiko Takasaki ◽  
Chizuko Morita ◽  
Takaaki Nakaya ◽  
...  
Keyword(s):  
Fine Structure ◽  
Cell Surface ◽  
Disease Virus ◽  
Mdck Cells ◽  
Rna Virus ◽  
Borna Disease Virus ◽  
Persistently Infected ◽  
Virus Like Particles ◽  
Hemagglutinating Virus ◽  
Borna Disease

ABSTRACT Borna disease virus (BDV), a negative nonsegmented single-stranded RNA virus, has not been fully characterized morphologically. Here we present what is to our knowledge the first data on the fine ultrastructure and morphogenesis of BDV. The supernatant of MDCK cells persistently infected with BDV treated with n-butyrate contained many virus-like particles and more BDV-specific RNA than that of untreated samples. The particles were spherical, enveloped, and approximately 130 nm in diameter; had spikes 7 nm in length; and reacted with BDV p40 antibody. A thin nucleocapsid, 4 nm in width, was present peripherally in contrast to the thick nucleocapsid of hemagglutinating virus of Japan. The BDV particles reproduced by budding on the cell surface.

scholarly journals Authentic Borna disease virus transcripts are spliced less efficiently than cDNA-derived viral RNAs

2000 ◽  
Vol 81 (8) ◽  
pp. 1947-1954 ◽  
Author(s):  
Christian Jehle ◽  
W. Ian Lipkin ◽  
Peter Staeheli ◽  
Rosa M. Marion ◽  
Martin Schwemmle
Keyword(s):  
Disease Virus ◽  
Rna Virus ◽  
Borna Disease Virus ◽  
Viral Rnas ◽  
Negative Strand ◽  
Intron 1 ◽  
Splicing Pattern ◽  
Alternatively Spliced ◽  
Infected Cells ◽  
Borna Disease

Borna disease virus (BDV) is a non-segmented, negative-strand RNA virus that replicates and transcribes its genome in the nucleus of infected cells. It uses the cellular splicing machinery to generate a set of alternatively spliced mRNAs from the 2·8 and 7·1 kb primary transcripts, each harbouring two introns. To determine whether splicing of these transcripts is regulated by viral factors, the extent of splicing was studied in infected cells and COS-7 cells transiently transfected with plasmids encoding the 2·8 kb RNA of BDV. Unspliced RNA was found to be the most abundant RNA species in infected cells, whereas viral transcripts lacking both introns were only found in minute amounts. In sharp contrast, plasmid-derived 2·8 kb RNA was predominantly intron 1-spliced and double-spliced. Co-expression of the BDV proteins P, N and X did not influence splicing of plasmid-expressed 2·8 kb RNA. Furthermore, the splicing pattern did not change when the 2·8 kb RNA was expressed in BDV-infected cells. Based on these results we speculate that splicing of authentic BDV transcripts is tightly linked to transcription by the viral polymerase.

scholarly journals Evidence of Borna disease virus genome detection in French domestic animals and in foxes (Vulpes vulpes)

2001 ◽  
Vol 82 (9) ◽  
pp. 2199-2204 ◽  
Author(s):  
G. Dauphin ◽  
V. Legay ◽  
C. Sailleau ◽  
S. Smondack ◽  
S. Hammoumi ◽  
...  
Keyword(s):  
Disease Virus ◽  
Roe Deer ◽  
Rna Virus ◽  
Internal Standard ◽  
Virus Genome ◽  
Red Foxes ◽  
Borna Disease Virus ◽  
Blood Samples ◽  
Horse Blood ◽  
Borna Disease

Borna disease virus (BDV) is an enveloped, non-segmented negative-stranded RNA virus which belongs to the Bornaviridae family. BDV is an aetiological agent of encephalitis in horses, sheep and several other vertebrate species. In order to extend our knowledge about the presence of BDV in France, a study based on BDV RNA detection by RT–nested-PCR was done with 196 animal tissues: 171 brain samples collected from different animal species (75 horses, 59 foxes, 31 cattle, 4 dogs, 1 sheep, 1 roe deer) and 25 horse blood samples. An RNA internal standard molecule was constructed and was co-amplified with the test template. This study reports the first detection of BDV RNA in France in 10 brain samples collected from horses, foxes and cattle, and from 14 horse blood samples. Detection of the BDV genome in the brains of six red foxes is the first evidence of BDV infection in this species.

scholarly journals Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3394-3404 ◽  
Author(s):  
DG Hafenrichter ◽  
X Wu ◽  
SD Rettinger ◽  
SC Kennedy ◽  
MW Flye ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Retroviral Vectors ◽  
Transduction Efficiency ◽  
Fatty Acid Binding ◽  
Retroviral Transduction ◽  
Rat Livers

Abstract Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347- bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596- bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35- , 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins.

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