scholarly journals Herpes Simplex Virus Type 1/Adeno-Associated Virus rep+ Hybrid Amplicon Vector Improves the Stability of Transgene Expression in Human Cells by Site-Specific Integration

2002 ◽  
Vol 76 (14) ◽  
pp. 7150-7162 ◽  
Author(s):  
Y. Wang ◽  
S. M. Camp ◽  
M. Niwano ◽  
X. Shen ◽  
J. C. Bakowska ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transgene Expression ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Primary Myoblasts ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep − HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep + HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep − vector 3′ AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep +, but not the rep −, hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep + hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by “deconcatenating” the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells.

scholarly journals Efficient Site-Specific Integration of Large Transgenes by an Enhanced Herpes Simplex Virus/Adeno-Associated Virus Hybrid Amplicon Vector

2006 ◽  
Vol 80 (4) ◽  
pp. 1672-1679 ◽  
Author(s):  
Qiang Liu ◽  
Claudio F. Perez ◽  
Yaming Wang
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transgene Expression ◽  
Vector System ◽  
Aav Vector ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT We previously demonstrated that a herpes simplex virus type 1 (HSV-1)/adeno-associated virus (AAV) hybrid amplicon vector constructed by inserting the sequences of regulatory protein (rep) and inverted terminal repeats of AAV into an HSV amplicon vector resulted in the enhanced stability of transgene expression compared to the original HSV-1 amplicon vector. However, problems related to the expression of Rep compromised its therapeutic applications. We report here a new HSV/AAV hybrid amplicon vector system that not only solved problems associated with Rep expression but also markedly improved the stable transduction efficiency of this vector. This new HSV/AAV vector is designed in a way that little or no Rep would be expressed in packaging cells, but it can be expressed in transduced cells if Cre recombinase is provided. Furthermore, Rep expression will be automatically suppressed as a consequence of Rep-mediated integration. Our results showed that the new hybrid amplicon vector yielded titers comparable to those of standard amplicon vectors. When Cre-expressing 293 cells were transduced, a low level of Rep expression was detected, and stable transduction was achieved in ∼22% of transduced cells; of those cells, ∼70% transduction was achieved by Rep-mediated site-specific integration. In the majority of the stably transduced cells, Rep expression was no longer observed. Our results also proved that this vector system is capable of efficiently accommodating and site-specifically integrating large transgenes, such as the full-length dystrophin expression cassette. Thus, the new HSV/AAV vector demonstrated unique advantages in safe and effective delivery of long-lasting transgene expression into human cells.

scholarly journals Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

2010 ◽  
Vol 84 (17) ◽  
pp. 8871-8887 ◽  
Author(s):  
Armel Nicolas ◽  
Nathalie Alazard-Dany ◽  
Coline Biollay ◽  
Loredana Arata ◽  
Nelly Jolinon ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Particle Production ◽  
Helper Virus ◽  
Adeno Associated Virus ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase δ was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

scholarly journals Herpes Simplex Virus Type 1 Co-Infection Modifies Adeno-Associated Virus Genome End Recombination

2021 ◽  
Author(s):  
Anita Felicitas Meier ◽  
Kurt Tobler ◽  
Kevin Michaelsen ◽  
Bernd Vogt ◽  
Els Henckaerts ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Molecular Structures ◽  
Adeno Associated Virus ◽  
Genome Recombination ◽  
Simplex Virus ◽  
Hsv 1

Wildtype adeno-associated virus (AAV) can only replicate in the presence of helper factors, which can be provided by co-infecting helper viruses such as adenoviruses and herpes viruses. The AAV genome consists of a linear, single-stranded DNA (ssDNA), which is converted into different molecular structures within the host cell. Using high throughput sequencing we found that herpes simplex virus type 1 (HSV-1) co-infection leads to a shift in the type of AAV genome end recombination. In particular, open-end ITR recombination was enhanced whereas open-closed ITR recombination was reduced in the presence of HSV-1. We demonstrate that the HSV-1 protein ICP8 plays an essential role in HSV-1 mediated interference with AAV genome end recombination, indicating that the previously described ICP8-driven mechanism of HSV-1 genome recombination may be underlying the observed changes. We also provide evidence that additional factors, such as products of true late genes, are involved. Although HSV-1 co-infection significantly changed the type of AAV genome end recombination, no significant change in the amount of circular AAV genomes was identified. IMPORTANCE AAV-mediated gene therapy represents one of the most promising approaches for the treatment of genetic diseases. Currently, various GMP-compatible production methods can be applied to manufacture clinical grade vector, including methods that employ helper factors derived form HSV-1. Yet to date we do not fully understand how HSV-1 interacts with AAV. We observed that HSV-1 modulates AAV genome ends similarly to the genome recombination events observed during HSV-1 replication and postulate that further improvements of the HSV-1 production platform may enhance packaging of the recombinant AAV particles.

scholarly journals Cleavage in and around the DR1 Element of the aSequence of Herpes Simplex Virus Type 1 Relevant to the Excision of DNA Fragments with Length Corresponding to One and Two Units of the a Sequence

2001 ◽  
Vol 75 (13) ◽  
pp. 5870-5878 ◽  
Author(s):  
Kenichi Umene
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Cleavage Site ◽  
Dna Fragments ◽  
Site Specific ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The a sequence of herpes simplex virus type 1 (HSV-1) is a region bracketed by two direct repeats named DR1. Concatemeric HSV-1 DNA, the product of DNA replication, is cleaved at a specific site on the second DR1 distal from the S component (authentic cleavage) to yield unit-length linear HSV-1 DNA prior to or during packaging of HSV-1 DNA. The presence of two DNA bands, of 0.25 kb (shorter band) and 0.5 kb (longer band), the lengths of which correspond to one and two units of the a sequence, was identified using acrylamide gel electrophoresis of HSV-1 DNA preparations extracted by the method of Hirt. Twelve DNA fragments from each band were molecularly cloned, and nucleotide sequences were determined. Both termini of eight (67%) DNA clones from the shorter band corresponded to the specific cleavage site on DR1. Five (41%) DNA clones from the longer band had a terminus corresponding to the specific cleavage site on DR1 on one side, but not on the opposite side. Thirteen (54%) of 24 termini of 12 analyzed DNA clones from the longer band were in and around DR1. Thus, cleavage events of DR1 can be classified into three categories: (i) authentic cleavage; (ii) site-specific cleavage on the third DR1 distal from the S component (secondary site-specific cleavage), which is related to the generation of the shorter DNA band in combination with authentic cleavage; and (iii) less-specific cleavage events in and around other DR1 elements which relate to the generation of the longer DNA band.

scholarly journals Inhibition of Herpes Simplex Virus Type 1 Replication by Adeno-Associated Virus Rep Proteins Depends on Their Combined DNA-Binding and ATPase/Helicase Activities

2010 ◽  
Vol 84 (8) ◽  
pp. 3808-3824 ◽  
Author(s):  
Daniel L. Glauser ◽  
Michael Seyffert ◽  
Regina Strasser ◽  
Marco Franchini ◽  
Andrea S. Laimbacher ◽  
...  
Keyword(s):  
Dna Damage ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Binding ◽  
Herpes Simplex Virus Type ◽  
Adeno Associated Virus ◽  
Rep Proteins ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Adeno-associated virus (AAV) has previously been shown to inhibit the replication of its helper virus herpes simplex virus type 1 (HSV-1), and the inhibitory activity has been attributed to the expression of the AAV Rep proteins. In the present study, we assessed the Rep activities required for inhibition of HSV-1 replication using a panel of wild-type and mutant Rep proteins lacking defined domains and activities. We found that the inhibition of HSV-1 replication required Rep DNA-binding and ATPase/helicase activities but not endonuclease activity. The Rep activities required for inhibition of HSV-1 replication precisely coincided with the activities that were responsible for induction of cellular DNA damage and apoptosis, suggesting that these three processes are closely linked. Notably, the presence of Rep induced the hyperphosphorylation of a DNA damage marker, replication protein A (RPA), which has been reported not to be normally hyperphosphorylated during HSV-1 infection and to be sequestered away from HSV-1 replication compartments during infection. Finally, we demonstrate that the execution of apoptosis is not required for inhibition of HSV-1 replication and that the hyperphosphorylation of RPA per se is not inhibitory for HSV-1 replication, suggesting that these two processes are not directly responsible for the inhibition of HSV-1 replication by Rep.

scholarly journals Herpes Simplex Virus Type 1 Co-Infection Leads to the Formation of Rolling Circle Amplification-Like Adeno-Associated Virus DNA Replication Products

2020 ◽  
Author(s):  
Anita F. Meier ◽  
Kurt Tobler ◽  
Remo Leisi ◽  
Anouk Lkharrazi ◽  
Carlos Ros ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rolling Circle Amplification ◽  
Helper Virus ◽  
Genome Replication ◽  
Adeno Associated Virus ◽  
Rolling Circle ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTAdeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin mechanism initiated at the AAV 3’ inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. We demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in presence of HSV-1, and thus provide evidence for an unequivocal rolling circle amplification (RCA) mechanism. This study reveals the ability of AAV to modify the canonical rolling hairpin replication mechanism and to mimic the replication strategy of a co-infecting herpesvirus. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus. Furthermore, we introduce nanopore sequencing as a novel, high-throughput approach to study viral genome replication in unprecedented detail.

scholarly journals Live Covisualization of Competing Adeno-Associated Virus and Herpes Simplex Virus Type 1 DNA Replication: Molecular Mechanisms of Interaction

2007 ◽  
Vol 81 (9) ◽  
pp. 4732-4743 ◽  
Author(s):  
Daniel L. Glauser ◽  
Regina Strasser ◽  
Andrea S. Laimbacher ◽  
Okay Saydam ◽  
Nathalie Clément ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Early Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT We performed live cell visualization assays to directly assess the interaction between competing adeno-associated virus (AAV) and herpes simplex virus type 1 (HSV-1) DNA replication. Our studies reveal the formation of separate AAV and HSV-1 replication compartments and the inhibition of HSV-1 replication compartment formation in the presence of AAV. AAV Rep is recruited into AAV replication compartments but not into those of HSV-1, while the single-stranded DNA-binding protein HSV-1 ICP8 is recruited into both AAV and HSV-1 replication compartments, although with differential staining patterns. Slot blot analysis of coinfected cells revealed a dose-dependent inhibition of HSV-1 DNA replication by wild-type AAV but not by rep-negative recombinant AAV. Consistent with this, Western blot analysis indicated that wild-type AAV affects the levels of the HSV-1 immediate-early protein ICP4 and the early protein ICP8 only modestly but strongly inhibits the accumulation of the late proteins VP16 and gC. Furthermore, we demonstrate that the presence of Rep in the absence of AAV DNA replication is sufficient for the inhibition of HSV-1. In particular, Rep68/78 proteins severely inhibit the formation of mature HSV-1 replication compartments and lead to the accumulation of ICP8 at sites of cellular DNA synthesis, a phenomenon previously observed in the presence of viral polymerase inhibitors. Taken together, our results suggest that AAV and HSV-1 replicate in separate compartments and that AAV Rep inhibits HSV-1 at the level of DNA replication.

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