scholarly journals High-Titer, Wild-Type Free Recombinant Adeno-Associated Virus Vector Production Using Intron-Containing Helper Plasmids

2000 ◽  
Vol 74 (24) ◽  
pp. 11456-11463 ◽  
Author(s):  
Lei Cao ◽  
Yuhong Liu ◽  
Matthew J. During ◽  
Weidong Xiao
Keyword(s):  
Gene Expression ◽  
High Titer ◽  
Infectious Clone ◽  
Wild Type ◽  
Helper Plasmid ◽  
Intron Insertion ◽  
Adeno Associated Virus ◽  
Vector Systems ◽  
Simplex Virus ◽  
High Level

ABSTRACT Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate therep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.

scholarly journals Herpes Simplex Virus Type 1 ICP0 Protein Mediates Activation of Adeno-Associated Virus Type 2 rep Gene Expression from a Latent Integrated Form

2004 ◽  
Vol 78 (20) ◽  
pp. 10977-10986 ◽  
Author(s):  
Marie-Claude Geoffroy ◽  
Alberto L. Epstein ◽  
Estelle Toublanc ◽  
Philippe Moullier ◽  
Anna Salvetti
Keyword(s):  
Gene Expression ◽  
Herpes Simplex ◽  
Virus Type ◽  
Helper Virus ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Adeno-associated virus type 2 (AAV-2) is a human parvovirus that requires the presence of a helper virus, such as the herpes simplex virus type 1 (HSV-1) to accomplish a complete productive cycle. In the absence of helper virus, AAV-2 can establish a latent infection that is characterized by the absence of expression of viral genes. So far, four HSV-1 early genes, UL5/8/52 (helicase primase complex) and UL29 (single-stranded DNA-binding protein), were defined as sufficient for AAV replication when cells were transfected with a plasmid carrying the wild-type AAV-2 genome. However, none of these viral products was shown to behave as a transcriptional factor able to activate AAV gene expression. Our study provides the first evidence that the immediate-early HSV-1 protein ICP0 can promote rep gene expression in cells latently infected with wild-type AAV-2. This ICP0-mediated effect occurs at the transcriptional level and involves the ubiquitin-proteasome pathway. Furthermore, using deletion mutants, we demonstrate that the localization of ICP0 to ND10 and their disruption is not required for the activation of the rep promoter, whereas binding of ICP0 to the ubiquitin-specific protease HAUSP makes a significant contribution to this effect.

Activation of immediate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4

1985 ◽  
Vol 5 (8) ◽  
pp. 1997-2008 ◽  
Author(s):  
N A DeLuca ◽  
P A Schaffer
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Immediate Early Genes ◽  
Early Gene ◽  
Immediate Early ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Early Genes ◽  
Simplex Virus

To better define the activities on herpes simplex virus type 1 gene expression of temperature-sensitive and wild-type forms of the transcriptional regulatory protein ICP4, regulatory sequences from immediate-early, early, and late herpes simplex virus genes were fused to the gene for chloramphenicol acetyltransferase (CAT). These constructs were used in trans induction and cotransfection experiments with wild-type and temperature-sensitive mutant alleles of ICP4. The ICP4 genes used in this study were cloned from the KOS strain (wild type) and two phenotypically distinct temperature-sensitive ICP4 mutants, tsB32 and tsL14 (DeLuca et al., J. Virol. 52:767-776, 1984), both alone and in conjunction with three other immediate-early genes. The latter series of plasmids was used to assess the influence of additional immediate-early gene products on gene expression in the presence of a given ICP4 allele. The results of this study demonstrate that the phenotypes of these ICP4 mutants observed in cell culture at the nonpermissive temperature were determined in part by activities associated with the mutant ICP4 polypeptides and that these activities differed from those of wild-type ICP4. Low levels of wild-type ICP4 had a marginal but reproducible stimulatory effect on immediate-early CAT gene expression, especially the pIE4/5CAT chimera. This effect was diminished with increasing quantities of ICP4, suggesting an inhibitory role for the wild-type form of the protein. The ICP4 mutants had a strong stimulatory effect on immediate-early CAT expression, consistent with their phenotypes at 39 degrees C. The mutant forms of the ICP4 polypeptide differed in their ability to induce CAT activity from an early chimeric gene. Thus, the tsL14 form of ICP4 was effective in early gene induction (i.e., ptkCAT was induced), whereas the ICP4 derived from tsB32 was slightly inhibitory. Cotransfection of tsB32 ICP4 simultaneously with other immediate-early genes resulted in a marginal increase in ptkCAT induction. This induction was enhanced when the gene for ICP4 was inactivated by restriction enzyme cleavage, substantiating the inhibitory effect of the tsB32 form of ICP4. The two mutant ICP4 genes (tsB32 and tsL14) were unable to trans-activate either of the late CAT constructs (p5CAT and pL42CAT) tested. Cotransfecting tsL14 ICP4 with the other immediate-early genes resulted in activation of p5CAT but not pL42CAT. Taken together, these studies demonstrate that (i) low levels of wild-type ICP4 have stimulatory effect on immediate-early promoters and that higher concentrations of wild-type ICP4 have an inhibitory effect on these promoters, (ii) isolated mutant form of ICP4 exhibit activities that reflect the phenotypes of the mutants from which they were isolated, and (iii) immediate-early gene products other than ICP4 are involved in determining the distinct phenotypes of the two mutants at 39 degrees Celsius.

scholarly journals Impact of the Interaction between Herpes Simplex Virus Type 1 Regulatory Protein ICP0 and Ubiquitin-Specific Protease USP7 on Activation of Adeno-Associated Virus Type 2 rep Gene Expression

2006 ◽  
Vol 80 (7) ◽  
pp. 3650-3654 ◽  
Author(s):  
Marie-Claude Geoffroy ◽  
Gilliane Chadeuf ◽  
Anne Orr ◽  
Anna Salvetti ◽  
Roger D. Everett
Keyword(s):  
Gene Expression ◽  
Virus Type ◽  
Regulatory Protein ◽  
Ring Finger ◽  
Adeno Associated Virus ◽  
Protein Icp0 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Expression of the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 in transfected cells reactivates rep gene expression from integrated adeno-associated virus (AAV) type 2 genomes via a mechanism that requires both its RING finger and USP7 interaction domains. In this study, we found that the rep reactivation defect of USP7-binding-negative ICP0 mutants can be overcome by further deletion of sequences in the C-terminal domain of ICP0, indicating that binding of USP7 to ICP0 is not directly required. Unlike the case in transfected cells, only the RING finger domain of ICP0 was essential for rep gene reactivation during HSV-1 infection. However, mutants unable to bind to USP7 activate HSV-1 gene expression and reactivate rep gene expression with reduced efficiencies. These results further elucidate the role of ICP0 as a helper factor for AAV replication and illustrate that care is required when extrapolating from the properties of ICP0 in transfection assays to events occurring during HSV-1 infection.

scholarly journals Addition of Six-His-Tagged Peptide to the C Terminus of Adeno-Associated Virus VP3 Does Not Affect Viral Tropism or Production

2002 ◽  
Vol 76 (23) ◽  
pp. 12023-12031 ◽  
Author(s):  
Huang-Ge Zhang ◽  
Jinfu Xie ◽  
Igor Dmitriev ◽  
Elena Kashentseva ◽  
David T. Curiel ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Affinity Purification ◽  
High Titer ◽  
Plasmid Vector ◽  
Nitrilotriacetic Acid ◽  
Start Codon ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Viral Tropism ◽  
His Tag

ABSTRACT Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6×His tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6×His)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6×His-tagged VP3mutant recombinant AAV. The 6×His-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6×His-modified AAV were equivalent to those of wild-type particles. The 6×His-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6×His tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6×His-tagged AAV VP3 capsid protein and to utilize the 6×His-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

scholarly journals Accumulation of Viral Transcripts and DNA during Establishment of Latency by Herpes Simplex Virus

1998 ◽  
Vol 72 (2) ◽  
pp. 1177-1185 ◽  
Author(s):  
Martha F. Kramer ◽  
Shun-Hua Chen ◽  
David M. Knipe ◽  
Donald M. Coen
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Latent Infection ◽  
Acute Infection ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Simplex Virus

ABSTRACT Latent infection of mice with wild-type herpes simplex virus is established during an acute phase of ganglionic infection in which there is abundant viral replication and productive-cycle gene expression. Thymidine kinase-negative mutants establish latent infections but are severely impaired for acute ganglionic replication and productive-cycle gene expression. Indeed, by in situ hybridization assays, acute infection by these mutants resembles latency. To assess events during establishment of latency by wild-type and thymidine kinase-negative viruses, we quantified specific viral nucleic acid sequences in mouse trigeminal ganglia during acute ganglionic infection by using sensitive PCR-based assays. Through 32 h postinfection, viral DNA and transcripts representative of the three kinetic classes of productive-cycle genes accumulated to comparable levels in wild-type- and mutant-infected ganglia. At 48 and 72 h, although latency-associated transcripts accumulated to comparable levels in ganglia infected with wild-type or mutant virus, levels of DNA accumulating in wild-type-infected ganglia exceeded those in mutant-infected ganglia by 2 to 3 orders of magnitude. Coincident with this increase in DNA, wild-type-infected ganglia exhibited abundant expression of productive-cycle genes and high titers of infectious progeny. Nevertheless, the levels of productive-cycle RNAs expressed by mutant virus during acute infection greatly exceeded those expressed by wild-type virus during latency. The results thus distinguish acute infection of ganglia by a replication-compromised mutant from latent infection and may have implications for mechanisms of latency.

scholarly journals Oct-1 Is Posttranslationally Modified and Exhibits Reduced Capacity To Bind Cognate Sites at Late Times after Infection with Herpes Simplex Virus 1

2003 ◽  
Vol 77 (22) ◽  
pp. 11927-11932 ◽  
Author(s):  
Sunil J. Advani ◽  
Lizette O. Durand ◽  
Ralph R. Weichselbaum ◽  
Bernard Roizman
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transcriptional Factors ◽  
Dependent Manner ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Cycle Dependent ◽  
Simplex Virus ◽  
High Level

ABSTRACT In herpes simplex virus 1-infected cells, a high level of α gene expression requires the transactivation of the genes by a complex containing the viral α transinducing factor (αTIF) and two cellular proteins. The latter two, HCF-1 and octamer binding protein Oct-1, are transcriptional factors regulated in a cell cycle-dependent manner. αTIF is a protein made late in infection but packaged with the virion to transactivate viral genes in newly infected cells. In light of the accumulation of large amounts of αTIF, the absence of α gene expression late in infection suggested the possibility that one or more transcriptional factors required for α gene expression is modified late in infection. Here we report that Oct-1 is posttranscriptionally modified late in infection, that the modification is mediated by the virus but does not involve viral protein kinases or cdc2 kinase activated by the virus late in infection, and that the modified Oct-1 has a reduced affinity for its cognate DNA site. These results are consistent with the hypothesis that modification of Oct-1 transcriptional factor could account at least in part for the shutoff of α gene expression late in infection.

scholarly journals Role of the 3′ untranslated region of baculovirus p10 mRNA in high-level expression of foreign genes

1999 ◽  
Vol 80 (8) ◽  
pp. 2253-2262 ◽  
Author(s):  
Monique M. van Oers ◽  
Just M. Vlak ◽  
Harry O. Voorma ◽  
Adri A. M. Thomas
Keyword(s):  
Gene Expression ◽  
Gene Cassette ◽  
Expression Vectors ◽  
Foreign Gene ◽  
Sequence Motif ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Foreign Genes ◽  
High Level ◽  
Terminator Sequence

The p10 gene of Autographa californica nucleopolyhedrovirus has two putative AATAAA polyadenylation signals. The downstream signal is used predominantly, as was determined by analysing 3′ cDNA ends. This downstream motif is followed by a GT-rich sequence, known to be important for efficient polyadenylation in mammalian systems. To analyse the importance of polyadenylation for p10 gene expression, recombinant viruses with altered 3′ untranslated regions (UTRs) were tested using chloramphenicol acetyltransferase (CAT) as a reporter. Surprisingly, after inactivation of the downstream AATAAA motif, CAT expression remained at the same high level as observed with a wild-type 3′ UTR. Polyadenylation occurred 24–28 nucleotides further downstream, probably due to an ATTAAA sequence motif. Replacing the p10 3′ UTR with the SV40 early terminator sequence as part of an hsp70–lacZ–SV40 gene cassette, which is commonly used in baculovirus expression vectors, resulted in a reduction in reporter gene expression. Polyadenylation occurred far more efficiently in the original p10 3′ UTR than in the SV40 terminator sequence, as was shown by testing the SV40 terminator separately. These results indicate that in order to obtain high levels of foreign gene expression, vectors that provide a wild-type p10 3′ UTR are to be preferred over those containing the hsp70–lacZ–SV40 gene cassette. Comparison of the p10 genes of various baculoviruses showed the presence of at least one AATAAA or ATTAAA motif in combination with a GT-rich sequence in the 3′ UTR, suggesting an evolutionary conservation of these two elements, thereby maintaining the high level of p10 gene expression.

scholarly journals HSV-1 as a Model for Emerging Gene Delivery Vehicles

ISRN Virology ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Filip Lim
Keyword(s):  
Viral Vectors ◽  
High Titer ◽  
Ease Of Use ◽  
Type I ◽  
Human Virus ◽  
Vector Systems ◽  
Rapid Transition ◽  
Low Efficiency ◽  
Simplex Virus ◽  
Hsv 1

The majority of viral vectors currently used possess modest cargo capability (up to 40 kb) being based on retroviruses, lentiviruses, adenoviruses, and adenoassociated viruses. These vectors have made the most rapid transition from laboratory to clinic because their small genomes have simplified their characterization and modification. However, there is now an increasing need both in research and therapy to complement this repertoire with larger capacity vectors able to deliver multiple transgenes or to encode complex regulatory regions, constructs which can easily span more than 100 kb. Herpes Simplex Virus Type I (HSV-1) is a well-characterized human virus which is able to package about 150 kb of DNA, and several vector systems are currently in development for gene transfer applications, particularly in neurons where other systems have low efficiency. However, to reach the same level of versatility and ease of use as that of smaller genome viral vectors, simple systems for high-titer production must be developed. This paper reviews the major HSV-1 vector systems and analyses the common elements which may be most important to manipulate to achieve this goal.

scholarly journals The PK Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Is Required for Immediate-Early Gene Expression and Virus Growth

1998 ◽  
Vol 72 (11) ◽  
pp. 9131-9141 ◽  
Author(s):  
C. C. Smith ◽  
T. Peng ◽  
M. Kulka ◽  
L. Aurelian
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Replication ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Wild Type ◽  
Virus Growth ◽  
Simplex Virus

ABSTRACT The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10ΔPK). ICP10ΔPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10ΔPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10ΔPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.

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