Herpes simplex virus co-infection facilitates rolling circle replication of the adeno-associated virus genome

Adeno-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 replication (RHR) 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. Using Southern analysis and nanopore sequencing as a novel, high-throughput approach to study viral genome replication we demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in the presence of HSV-1, thus providing evidence for an unequivocal rolling circle replication (RCR) mechanism. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus.

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

10.1101/2020.12.23.424160 ◽

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.

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

Journal of Virology ◽

10.1128/jvi.00725-10 ◽

2010 ◽

Vol 84 (17) ◽

pp. 8871-8887 ◽

Cited By ~ 17

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.

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Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

Journal of Virology ◽

10.1128/jvi.00357-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 2

Author(s):

Francesca D. Franzoso ◽

Michael Seyffert ◽

Rebecca Vogel ◽

Artur Yakimovich ◽

Bruna de Andrade Pereira ◽

...

Keyword(s):

Cell Cycle ◽

Herpes Simplex Virus ◽

Dna Replication ◽

Herpes Simplex ◽

Helper Virus ◽

Herpes Simplex Virus 1 ◽

Adeno Associated Virus ◽

Cycle Dependent ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time-controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells.

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Impact of the MRN Complex on Adeno-Associated Virus Integration and Replication during Coinfection with Herpes Simplex Virus 1

Journal of Virology ◽

10.1128/jvi.00171-15 ◽

2015 ◽

Vol 89 (13) ◽

pp. 6824-6834 ◽

Cited By ~ 5

Author(s):

Rachel Millet ◽

Nelly Jolinon ◽

Xuan-Nhi Nguyen ◽

Gregory Berger ◽

Andrea Cimarelli ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Dna Damage Response ◽

Helper Virus ◽

Herpes Simplex Virus 1 ◽

Adeno Associated Virus ◽

Mrn Complex ◽

Damage Response ◽

Simplex Virus ◽

Hsv 1

ABSTRACTAdeno-associated virus (AAV) is a helper-dependent parvovirus that requires coinfection with adenovirus (AdV) or herpes simplex virus 1 (HSV-1) to replicate. In the absence of the helper virus, AAV can persist in an episomal or integrated form. Previous studies have analyzed the DNA damage response (DDR) induced upon AAV replication to understand how it controls AAV replication. In particular, it was shown that the Mre11-Rad50-Nbs1 (MRN) complex, a major player of the DDR induced by double-stranded DNA breaks and stalled replication forks, could negatively regulate AdV and AAV replication during coinfection. In contrast, MRN favors HSV-1 replication and is recruited to AAV replication compartments that are induced in the presence of HSV-1. In this study, we examined the role of MRN during AAV replication induced by HSV-1. Our results indicated that knockdown of MRN significantly reduced AAV DNA replication after coinfection with wild-type (wt) HSV-1 or HSV-1 with the polymerase deleted. This effect was specific to wt AAV, since it did not occur with recombinant AAV vectors. Positive regulation of AAV replication by MRN was dependent on its DNA tethering activity but did not require its nuclease activities. Importantly, knockdown of MRN also negatively regulated AAV integration within the human AAVS1 site, both in the presence and in the absence of HSV-1. Altogether, this work identifies a new function of MRN during integration of the AAV genome and demonstrates that this DNA repair complex positively regulates AAV replication in the presence of HSV-1.IMPORTANCEViral DNA genomes trigger a DNA damage response (DDR), which can be either detrimental or beneficial for virus replication. Adeno-associated virus (AAV) is a defective parvovirus that requires the help of an unrelated virus such as adenovirus (AdV) or herpes simplex virus 1 (HSV-1) for productive replication. Previous studies have demonstrated that the cellular Mre11-Rad50-Nbs1 (MRN) complex, a sensor and regulator of the DDR, negatively regulates AAV replication during coinfection with AdV, which counteracts this effect by inactivating the complex. Here, we demonstrate that MRN positively regulates AAV replication during coinfection with HSV-1. Importantly, our study also indicates that MRN also favors integration of AAV genomes within the human AAVS1 site. Altogether, this work indicates that MRN differentially regulates AAV replication depending on the helper virus which is present and identifies a new function of this DNA repair complex during AAV integration.

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Adeno-Associated Virus Type 2 Rep68 Can Bind to Consensus Rep-Binding Sites on the Herpes Simplex Virus 1 Genome

Journal of Virology ◽

10.1128/jvi.01370-15 ◽

2015 ◽

Vol 89 (21) ◽

pp. 11150-11158 ◽

Cited By ~ 3

Author(s):

Michael Seyffert ◽

Daniel L. Glauser ◽

Kurt Tobler ◽

Oleg Georgiev ◽

Rebecca Vogel ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Binding Sites ◽

Herpes Simplex Virus 1 ◽

Adeno Associated Virus ◽

Binding Domains ◽

Simplex Virus ◽

Hsv 1

Adeno-associated virus type 2 is known to inhibit replication of herpes simplex virus 1 (HSV-1). This activity has been linked to the helicase- and DNA-binding domains of the Rep68/Rep78 proteins. Here, we show that Rep68 can bind to consensus Rep-binding sites on the HSV-1 genome and that the Rep helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore, we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/Rep78 to the HSV-1 genome.

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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

Journal of Virology ◽

10.1128/jvi.76.14.7150-7162.2002 ◽

2002 ◽

Vol 76 (14) ◽

pp. 7150-7162 ◽

Cited By ~ 40

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.

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

Journal of Virology ◽

10.1128/jvi.00486-21 ◽

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.

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Adeno-associated Virus Vectors Efficiently Transduce Mouse and Rabbit Sensory Neurons Coinfected with Herpes Simplex Virus 1 following Peripheral Inoculation

Journal of Virology ◽

10.1128/jvi.01028-16 ◽

2016 ◽

Vol 90 (17) ◽

pp. 7894-7901 ◽

Cited By ~ 7

Author(s):

Zachary L. Watson ◽

Monica K. Ertel ◽

Alfred S. Lewin ◽

Sonal S. Tuli ◽

Gregory S. Schultz ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Sensory Neurons ◽

Intrathecal Injection ◽

Sensory Ganglia ◽

Herpes Simplex Virus 1 ◽

Epithelial Surface ◽

Adeno Associated Virus ◽

Simplex Virus ◽

Hsv 1

ABSTRACTFollowing infection of epithelial tissues, herpes simplex virus 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, long-term transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to that seen with HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with various amounts of rAAV as well as rAAV capsid mutants. We demonstrated that this method of inoculation can achieve a transduction rate of >90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we showed that corneal inoculation with rAAV vectors in the rabbit efficiently transduced >70% of the TG neurons in the optic tract. Finally, we demonstrated that coinfection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 resulted in colocalization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV infections or of dysfunctions of sensory ganglia.IMPORTANCEAdeno-associated virus (AAV) has been shown to transduce dorsal root ganglion sensory neurons following direct intraganglionic sciatic nerve injection and intraperitoneal and intravenous injection as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally at an epithelial surface that had been treated to expose the underlying sensory nerve termini. For this study, we chose two well-established HSV-1 infection models: mouse footpad infection and rabbit ocular infection. The results presented here provide the first description of AAV vectors transducing neurons following delivery at the skin/epithelium/eye. The ability of AAV to cotransduce HSV-1-infected neurons in both the mouse and the rabbit provides an opportunity to experimentally explore and disrupt host and viral proteins that are integral to the establishment of HSV-1 latency, to the maintenance of latency, and to reactivation from latencyin vivo.

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

Journal of Virology ◽

10.1128/jvi.01503-09 ◽

2010 ◽

Vol 84 (8) ◽

pp. 3808-3824 ◽

Cited By ~ 15

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.

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