Biology subjects

Fraefel, C.

Publications and source records attributed to Fraefel, C..

13 recordsLinked to original sources

bioRxiv2026

Unconstrained dosing agar (UDA) Reduces Stress in Mouse Oral Administration

BackgroundOral gavage is the standard method for delivering drugs and other substances orally in rodent studies, but it can cause significant stress and risk injury. To improve animal welfare and reduce confounding stress effects, this study aimed to replace oral gavage by developing and testing a new voluntary ingestion method that is easy to adopt, minimizes stress in mice, and is suitable for a wide range of compounds. ResultsWe developed a soft agar formulation with an appealing scent and taste that mice readily consumed without fasting or restraint. We called this method "unconstrained dosing agar" (UDA). Analysis of fecal corticosterone levels demonstrated that the method is associated with low stress in the animals. After training, mice quickly consumed the agar units. Body weight gain was unaffected by the treatment. ConclusionsThis study introduces a simple, low-stress method for administering substances orally in mice. By encouraging voluntary consumption and removing the need for fasting or restraint, this method provides a practical alternative to oral gavage and could improve animal welfare and experimental consistency.

animal behavior and cognition↗

bioRxiv2025

The limited effects of AAV2 vectors on host chromatin accessibility and nuclear architecture are consistent with a favorable safety profile

Recombinant adeno-associated virus (AAV) vectors are widely used for gene therapeutics, yet their early effects on host chromatin remodeling and nuclear organization remain insufficiently characterized. In contrast, several DNA viruses are known to remodel host chromatin accessibility, for example HSV-1 in mammalian cells and baculoviruses in insect systems. Here we evaluated genome accessibility and nuclear organization of cultured primary human cells at 24 and 48 hours after infection with wildtype AAV2, single-stranded (ss) and self-complementary (sc) recombinant AAV2. Genome-wide ATAC-seq showed no detectable change in host chromatin accessibility at either time point. A DNase I digestion assay at five candidate loci supported this observation. In contrast, immunofluorescence imaging revealed modest decreases in histone H3, H3K27me3, and RNA polymerase II signals, consistent with reduced polymerase engagement under stress-linked pathways. H3K4me3 deviated from this pattern in G1 cells upon scAAV2 infection, where signals increased. Nuclear geometry shifted in parallel with protein signals, with changes in area, perimeter, convexity, and eccentricity. Markers of nuclear condensates also changed, including reduced fibrillarin, SP100, and SRSF2 intensities, altered object shape metrics, and higher counts of promyelocytic leukemia (PML) bodies and nuclear splicing speckles (NSs). Collectively, at the tested doses and times, AAV2-based vectors did not remodel chromatin accessibility at scale and induced only small changes in polymerase associated readouts and nuclear architecture. These data align with a favorable safety profile while highlighting assay limits and suggesting indirect stress related mechanisms.

biochemistry↗

bioRxiv2025

Comparative analysis of NSP5/VP2-induced viroplasm-like structures in rotavirus species A to J

Rotavirus (RV) is classified into nine species, A-D and F-J, with RV species A (RVA) being the most extensively studied. While RVA infects infants and young animals, non-RVA species infect adult humans, various mammals, and birds. However, the lack of appropriate research tools has limited our understanding of non-RVA life cycles. RVA replication and assembly occur in cytosolic inclusions termed viroplasms. We recently identified viroplasm-like structures (VLS) composed of NSP5 and NSP2, designated as VLS(NSP2)i, in non-RVA. In this context, globular VLS(NSP2)i formed in RVA, RVB, RVD, RVF, RVG, and RVI, but not in RVC, RVH, and RVJ. Additionally, in RVA, VLS can also be formed through co-expression of NSP5 with VP2, referred to as VLS(VP2)i. Here, we report VLS(VP2)i formation in all non-RVA species except RVB, with notable VLS formation in RVH and RVJ. Moreover, NSP2 RVH or RVJ are recruited into VLS(VP2)i. The NSP5 C-terminal region in non-RVA is required for association with VP2 and forming VLS(VP2)i. Mutation of conserved VP2-L141 in RVA to alanine disrupts viroplasm formation, impairing RV replication. Equivalent residues within the same predicted VP2 region disrupt VLS formation across non-RVA. We also observed interspecies VLS formation, particularly between closely related RVA and RVC, RVH and RVJ, and RVD and RVF. Interestingly, substituting the N-terminal region of VP2 RVB with that of the closely related VP2 RVG restores its ability to form VLS with NSP5 RVB. Elucidating the formation of viroplasms is essential for developing strategies to halt infection across RV species A to J. ImportanceRotaviruses (RV) are a group of viruses classified into species A through J, with species A being the best understood. Other RV species infecting animals and humans are less studied due to limited research tools. In RVA, the virus replicates in specialized compartments called viroplasms formed in the cytoplasm by viral proteins, including NSP5, NSP2, and VP2. In this study, we explored how similar structures, termed viroplasm-like structures (VLS), are formed by proteins of other RV species. We found that in most species, NSP5 and VP2 form VLSs. One exception was RVB, where VLS formation was observed only when the VP2 protein was mutated to resemble that of a related species. We also identified key regions in the VP2 protein that are essential for forming these structures. Understanding how viroplasms form across different RV species may help develop new strategies to block infection in humans and animals.

microbiology↗

bioRxiv2025

Assembly-Activating Protein Phase Separation Properties Are Required for Adeno-Associated Virus Type 2 Assembly

Adeno-associated virus serotype 2 (AAV2), a non-pathogenic parvovirus reliant on helper viruses, is studied extensively as a potential gene delivery vector. A +1 open reading frame within the cap gene encodes a nonstructural protein of 204-amino-acids termed assembly-activating protein (AAP), which has been attributed a critical role in transporting the viral capsid protein VP3 into the nucleolus for assembly. However, AAP remains poorly characterized because of its relatively late discovery and lack of commercial antibodies. In the absence of other virus proteins, AAP localizes in the nucleolus due to five redundant nuclear and nucleolar localization signals. Additionally, AAP, a predicted intrinsically disordered protein, forms spontaneous dose-dependent nuclear globular condensates, a trait of liquid-liquid phase separated inclusions. Consistent with LLPS biophysical properties, the AAP condensates recovered rapidly from photobleaching and are sensitive to aliphatic diol treatment--moreover, AAP self-oligomerizes. We produced an AAP-specific antibody to analyze the role of this protein during productive AAV2 replication. In this context, we observed that AAP also forms nuclear globular condensates with LLPS biophysical properties in cells co-infected with AAV2 and either herpes simplex virus type 1 (HSV-1) or adenovirus type 5 (AdV-5) as the helper viruses. The screening of AAP deletion mutants revealed that the N-terminal region (amino acids 1-61) is necessary for condensate formation and self-oligomerization. Interestingly, this AAP region contains a predicted alpha-helix spanning amino acids 16 to 45. The substitution in this region of the hydrophobic residues by alanines drastically impaired AAP-LLPS biophysical properties and its ability to trigger AAV2 capsid assembly. Identifying the amino acids involved in assembly and LLPS may improve AAV vector production. Author SummaryAdeno-associated virus serotype 2 (AAV2) is a non-pathogenic virus extensively studied for its potential in gene therapy. It relies on a protein called assembly-activating protein (AAP) to transport its capsid protein, VP3, to the nucleolus for assembly. The 204-amino-acid AAP is not well characterized because it was discovered only relatively recently and commercial antibodies are not availabe, making it challenging to study. Here, we demonstrate that AAP localizes in the nucleolus and forms globular condensates through liquid-liquid phase separation (LLPS), a property characterized by rapid recovery from photobleaching and sensitivity to aliphatic diol treatment. Additionally, we prepared a specific antibody to study AAP during AAV2 co-infection with helper viruses like herpes simplex virus type 1 (HSV-1) or adenovirus type 5 (AdV-5). We found that AAP also forms nuclear condensates with LLPS properties in co-infected cells. We demonstrate that the N-terminal region of AAP (amino acids 1-61) is crucial for condensate formation and self-oligomerization. Within this region, a predicted alpha-helix (amino acids 16-45) is essential, as substituting its hydrophobic residues with alanines significantly impaired the LLPS properties of AAP and its ability to facilitate AAV2 capsid assembly. Identifying these key amino acids may enhance AAV vector production for gene therapy applications.

microbiology↗

bioRxiv2024

Characterization of Viroplasm-Like Structures by Co-Expression of NSP5 and NSP2 Across Rotavirus Species A to J

Rotaviruses (RV) are classified into nine species, A-C and D-J, with species A being the most studied. In rotavirus of species A (RVA), replication occurs in viroplasms, which are cytosolic globular inclusions primarily composed of the proteins NSP5, NSP2, and VP2. The co-expression of NSP5 with either NSP2 or VP2 leads to the formation of viroplasm-like structures (VLS). Although morphologically identical to viroplasms, VLSs cannot replicate, but they serve as excellent simplified tools for studying complex viroplasms. There is a knowledge gap regarding viroplasms of non-RVA species due to a lack of research tools, such as specific antibodies and tissue culture systems. In this study, we explored the ability of NSP5 and NSP2 from non-RVA species to form VLSs. The co-expression of these two proteins led to globular VLSs in RV species A, B, D, F, G, and I, while RVC formed filamentous VLSs. The co-expression of NSP5 and NSP2 of RV species H and J did not result in VLS formation. Interestingly, NSP5 of all RV species self-oligomerizes, with the ordered C-terminal region, termed the tail, being necessary for self-oligomerization of RV species A-C and G-J. Except for NSP5 from species J, all NSP5 bound with their respective NSP2. We also found that interspecies VLS are formed between closely related RV species B with G and D with F. Additionally, VLS from RVH and RVJ formed when the tail of NSP5 RVH and RVJ was replaced by the tail of NSP5 from RVA and co-expressed with their respective NSP2. ImportanceRotaviruses (RV) are classified into nine species, A-D and F-J, infecting mammals and birds. Due to the lack of research tools, all cumulative knowledge on RV replication is based on RV species A (RVA). The RV replication compartments are globular cytosolic structures named viroplasms, which have only been identified in RV species A. In this study, we examined the formation of viroplasm-like structures (VLS) by the expression of NSP5 with NSP2 across RV species A to J. Globular VLSs formed for RV species A, B, D, F, G, and I, while RV species C formed filamentous structures. The RV species H and J did not form VLS with NSP5 and NSP2. Similar to RVA, NSP5 self-oligomerizes in all RV species, which is a requirement for VLS formation. This study provides basic knowledge of the non-RVA replication mechanisms, which could help develop strategies to halt virus infection across RV species.

microbiology↗

bioRxiv2024

The interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus serotype 2 (AAV2) transduction in an immune-modulatory independent way

We determined the transcription profile of AAV2-infected primary human fibroblasts. Subsequent analysis revealed that cells respond to AAV infection through changes in several significantly affected pathways including cell cycle regulation, chromatin modulation, and innate immune responses. Various assays were performed to validate selected differentially expressed genes and confirmed not only the quality but also the robustness of the raw data. One of the genes upregulated in AAV2 infected cells was the interferon-{gamma} inducible factor 16 (IFI16). IFI16 is known as a multifunctional cytosolic and nuclear innate immune sensor for double-stranded, as well as single-stranded DNA, exerting its effects through various mechanisms, such as interferon response, epigenetic modifications, or transcriptional regulation. IFI16 thereby constitutes a restriction factor for many different viruses amongst them, as shown here, AAV2 and thereof derived vectors. Indeed, the post-transcriptional silencing of IFI16 significantly increased AAV2 transduction efficiency, independent of the structure of the virus/vector genome. We also show that IFI16 exerts its inhibitory effect on AAV2 transduction in an immune-modulatory independent way, by interfering with Sp1-dependent transactivation of wild-type AAV2 and AAV2 vector promoters. IMPORTANCEAdeno-associated virus (AAV) vectors are among the most frequently used viral vectors for gene therapy. The lack of pathogenicity of the parental virus, the long-term persistence as episomes in non-proliferating cells, and the availability of a variety of AAV serotypes differing in their cellular tropism are advantageous features of this biological nanoparticle. To deepen our understanding of virus-host interactions, especially in terms of innate immune responses, we present here the first transcriptome analysis of AAV serotype 2 (AAV2) infected human primary fibroblasts. Our findings indicate that the interferon-{gamma} inducible factor 16 (IFI16) acts as an antiviral factor in AAV2 infection and AAV2 vector-mediated cell transduction in an immune-modulatory independent way by interrupting the Sp1-dependent gene expression from viral or vector genomes.

microbiology↗

bioRxiv2024

Non-canonical induction of autophagy increases adeno-associated virus type 2 (AAV2) transduction efficiency

Adeno-associated virus (AAV) serotypes infect a wide range of cell types, making this member of the parvovirus family a versatile tool in gene therapy. Infection as well as transduction is set in motion by means of specific receptors in conjunction with trafficking pathways, particularly endocytosis, a main cell entry pathway of non-enveloped viruses. Here, we report that efficacy of transduction is enhanced upon treating cells with hyperosmotic sucrose, a known blocker of clathrin-mediated endocytosis, through the non-canonical induction of autophagy. This mechanism of autophagy induction, however, is different from the previously reported AAV2-mediated induction of autophagy, which relies on a canonical, phosphoinositide 3-kinase class III (PI3K-III) complex-dependent pathway and appears to be dependent on the virus intrinsic secreted phospholipase A2 (sPLA2) domain, particularly its catalytic center activity. IMPORTANCEAdeno-associated virus (AAV) vectors are among the most frequently applied virus-based delivery vehicles for gene therapy. Lack of pathogenicity for humans, availability of a huge number of AAV serotypes differing in their cellular tropism, and the mainly episomal persistence of AAV vector genomes are clear advantages of these biological nanoparticles. By exploring non-pharmacological inducers of autophagy, we provide evidence for a potent and easy to apply strategy to significantly improve the efficacy of recombinant AAV-based gene delivery in hepatic and structural cells. Besides, our data also demonstrate the importance of autophagy for AAV2 infection and vector-mediated transduction in non-hepatic cells.

microbiology↗

bioRxiv2023

Adeno-associated virus (AAV2) can replicate its DNA by a rolling hairpin or rolling circle mechanism, depending on the helper virus.

Adeno-associated virus type 2 (AAV2) is a small, non-pathogenic, helper virus-dependent parvovirus with a single-stranded (ss) DNA genome of approximately 4.7 kb. AAV2 DNA replication requires the presence of a helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1) and is generally assumed to occur as a strand-displacement rolling hairpin (RHR) mechanism initiated at the AAV2 3 inverted terminal repeat (ITR). We have recently shown that AAV2 replication supported by HSV-1 leads to the formation of double-stranded head-to-tail concatemers, which provides evidence for a rolling circle replication (RCR) mechanism. We have revisited AAV2 DNA replication and specifically compared the formation of AAV2 replication intermediates in presence of either HSV-1 or AdV5 as the helper virus. The results confirmed that the AAV2 DNA replication mechanism is helper virus-dependent and follows a strand-displacement RHR mechanism when AdV5 is the helper virus and primarily an RCR mechanism when HSV-1 is the helper virus. We also demonstrate that recombination plays a negligible role in AAV2 genome replication. Interestingly, the formation of high molecular weight AAV2 DNA concatemers in presence of HSV-1 as the helper virus was dependent on an intact HSV-1 DNA polymerase. ImportanceAAV is a small helper virus-dependent, non-pathogenic parvovirus. The AAV genome replication mechanism was extensively studied in presence of AdV as the helper virus and described to proceed using RHR. Surprisingly, HSV-1 co-infection facilitates RCR of the AAV2 DNA. We directly compared AdV5 and HSV-1 supported AAV2 DNA replication and show that AAV2 can adapt its replication mechanism to the helper virus. Detailed understanding of the AAV replication mechanism expands our knowledge of virus biology and can contribute to increase gene therapy vector production.

molecular biology↗

bioRxiv2022

Recruitment of TRiC chaperonin in rotavirus viroplasms directly associates with virus replication.

Rotavirus replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which is the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. To investigate the relationship between rotavirus proteins and host components of the viroplasms, we performed a pull-down assay of lysates from rotavirus-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the TRiC complex, a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our validated results show that TRiC is recruited in viroplasms and specifically surrounds newly formed double-layered particles (DLPs). Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Interestingly, TRiC-inhibited RV-infected cells lacked triple-layered particles (TLPs) but harbored empty DLPs. Through sequence-specific direct RNA nanopore sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly (-)ssRNA. Moreover, TRiC associates and regulates the folding of VP2, a cofactor allowing dsRNA synthesis. This study provides in-cell culture evidence of the regulatory mechanism by which dsRNA genome segment replication is controlled and coordinated in the rotavirus viroplasms. ImportanceThe replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the eleven double-stranded RNA genome segments are synthesized and packaged individually into the newly generated virus particles. In this study, we show for the first time that the TRiC complex, a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral (-)ssRNA. Specifically, TRiC interacts and assists in folding VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited in viroplasm to assist replication.

microbiology↗

bioRxiv2022

Rotavirus spike protein VP4 mediates viroplasm assembly by association to actin filaments

The formation of viroplasms is a well-conserved step in the rotavirus (RV) life cycle. In these structures, both virus genome replication and progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are required for the viroplasm formation, which involves several virus proteins. The viral spike protein VP4 has not previously been shown to have a direct role in viroplasm formation. However, it is involved with virus-cell attachment, endocytic internalization, and virion morphogenesis. Moreover, VP4 interacts with actin cytoskeleton components, mainly in processes involving virus entrance and egress, and thereby may have an indirect role in viroplasm formation. In this study, we used reverse genetics to construct a recombinant RV, rRV/VP4-BAP, which contains a biotin acceptor peptide (BAP) in the K145-G150 loop of the VP4 lectin domain, permitting live monitoring. The recombinant virus was replication competent but showed a reduced fitness. We demonstrate that rRV/VP4-BAP infection, as opposed to rRV/wt infection, did not lead to a reorganized actin cytoskeleton as viroplasms formed were insensitive to drugs that depolymerize actin and inhibit myosin. Moreover, wt VP4, but not VP4-BAP, appeared to associate with actin filaments. Similarly, VP4 in co-expression with NSP5 and NSP2 induced a significant increase in the number of viroplasm-like structures. Interestingly, a small peptide mimicking loop K145-G150 rescued the phenotype of rRV/VP4-BAP by increasing its ability to form viroplasms and hence, improve virus progeny formation. Collectively, these results provide a direct link between VP4 and the actin cytoskeleton to catalyze viroplasm assembly. IMPORTANCEThe spike protein VP4 participates in diverse steps of the rotavirus (RV) life cycle, including virus-cell attachment, internalization, modulation of endocytosis, virion morphogenesis, and virus egress. Using reverse genetics, we constructed for the first time a recombinant RV, rRV/VP4-BAP, harboring a heterologous peptide in the lectin domain (loop K145-G150) of VP4. The rRV/VP4-BAP was replication-competent but with reduced fitness due to a defect in the ability to reorganize the actin cytoskeleton, which affected the efficiency of viroplasm assembly. This defect was rescued by adding a permeable small-peptide mimicking the wild-type VP4 loop K145-G150. In addition to revealing a new role of VP4, our findings suggest that rRV harboring an engineered VP4 could be used as a new dual vaccination platform providing immunity against RV and additional heterologous antigens.

microbiology↗

bioRxiv2021

Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus

Nucleoli are membrane-less structures located within the nucleus and are known to be involved in many cellular functions, including stress response and cell cycle regulation. Besides, many viruses can employ the nucleolus or nucleolar proteins to promote different steps of their life cycle such as replication, transcription and assembly. While adeno-associated virus type 2 (AAV2) capsids have previously been reported to enter the host cell nucleus and accumulate in the nucleolus, both the role of the nucleolus in AAV2 infection, and the viral uncoating mechanism remain elusive. In all prior studies on AAV uncoating, viral capsids and viral genomes were not directly correlated on the single cell level, at least not in absence of a helper virus. To elucidate the properties of the nucleolus during AAV2 infection and to assess viral uncoating on a single cell level, we combined immunofluorescence analysis for detection of intact AAV2 capsids and capsid proteins with fluorescence in situ hybridization for detection of AAV2 genomes. The results of our experiments provide evidence that uncoating of AAV2 particles occurs in a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar structure. Author SummaryAdeno-associated virus (AAV) capsids have been reported to enter the host cell nucleus and accumulate in the nucleolus. However, both the role of the nucleolus in AAV2 infection as well as the viral uncoating mechanism remain unknown. Here, we provide evidence that uncoating of the AAV2 particle is a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar morphology.

molecular biology↗

bioRxiv2021

A recombinant rotavirus harboring a spike protein with a heterologous peptide reveals a novel role of VP4 in viroplasm stability

The rotavirus (RV) VP4 spike protrudes as a trimeric structure from the five-fold axes of the virion triple-layer. Infectious RV particles need to be proteolytically cleaved in VP4 into two subunits, VP8* and VP5*, constituting both the distal part and central body of the virus spike. Modification of VP4 has been challenging as it is involved in biological process including the interaction with sialic acid and integrins, cell tropism and hemagglutinin activity. Here, we engineered a loop at position K145-G150 in the lectin domain of the VP8* subunit to harbor a small biotin acceptor peptide (BAP) tag and rescued viable viral particles using RV reverse genetics system. This rRV/VP4-BAP internalizes, replicates, and generates virus progeny, demonstrating that the VP4 spike of RV particles can be genetically manipulated by the incorporation of at least 15 exogenous amino acids. Although, VP4-BAP had a similar distribution as VP4 in infected cells by localizing in the cytoskeleton and surrounding viroplasms. However, compared to wild-type RV, rRV/VP4-BAP featured a reduced replication fitness and impaired viroplasm stability. Upon treatment of viroplasms with 1,6-hexanediol, a drug disrupting liquid-liquid phase-separated condensates, the kinetic of rRV/VP4-BAP viroplasm recovery was delayed, and their size and numbers reduced when compared to viroplasms of wild type RV. Moreover, siRNA silencing of VP4 expression in RV strain SA11 showed similar recovery patterns as rRV/VP4-BAP, revealing a novel function of VP4 in viroplasm stability. IMPORTANCEThe rotavirus (RV) spike protein, VP4, has a relevant role in several steps involving virion internalization. The strategic position of VP4 in the virion resulted in a challenge for the addition of an exogenous peptide producing infectious particles. The identification of a specific loop in position K145-G150 in the VP8* subunit of VP4 allowed the rescue by RV reverse genetics of a recombinant RV harboring VP4 containing a 15 amino acids tag. This study demonstrates this recombinant virus has similar replication properties as a wild-type virus. Moreover, we also discovered that VP4 is necessary for the assembly and stabilization of the cytosolic replication compartments, the viroplasms, demonstrating a novel role of this protein in the RV life cycle.

microbiology↗

bioRxiv2020

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

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 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. SIGNIFICANCEEfficient adeno-associated virus (AAV) replication requires the presence of helper factors, which can be provided by co-infecting helper viruses such as adenoviruses or herpesviruses. AAV replication has been described to occur as a rolling hairpin replication mechanism. However, we show that during a herpes simplex virus type 1 (HSV-1) supported replication, AAV rolling circle-like replication intermediates are formed. Thus, this study stands in contrast to the textbook model of AAV genome replication. Additionally, we introduce nanopore sequencing as a novel, high-throughput approach to study viral genome replication in unprecedented detail.

microbiology↗