Dataset Information

Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way.

ABSTRACT: We determined the transcription profile of adeno-associated virus type 2 (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 to confirm not only the quality but also the robustness of the raw data. One of the genes upregulated in AAV2-infected cells was interferon-γ 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 among 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.

Importance

Adeno-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 antiviral responses, we present here the first transcriptome analysis of AAV serotype 2 (AAV2)-infected human primary fibroblasts. Our findings indicate that interferon-γ inducible factor 16 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.

SUBMITTER: Sutter SO

PROVIDER: S-EPMC11338077 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way.

Sutter Sereina O SO Tobler Kurt K Seyffert Michael M Lkharrazi Anouk A Zöllig Joël J Schraner Elisabeth M EM Vogt Bernd B Büning Hildegard H Fraefel Cornel C

Journal of virology 20240605 7

We determined the transcription profile of adeno-associated virus type 2 (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 to confirm not only the quality but also the robustness of the raw data. One of the genes ...[more]

PMID: 38837381

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Project description:Human endogenous retroviruses (HERVs) are under genomic and epigenetic control but can be expressed in normal tissues, producing RNA transcripts some of which are translated. While it has not been demonstrated experimentally in modern humans, cDNA copies from HERV RNA (namely HERV-K HML-2 or HK2) were produced after the human-chimp split and until at least 250,000 years ago. We were interested in determining if such cDNA could be a ligand for pattern recognition receptors (PRRs) of the innate immune response. The AIM-2-like receptors for DNA, interferon-γ-inducible protein 16 (IFI16) and Cyclic GMP-AMP synthase (cGAS) were candidate PRRs. IFI16 can detect cDNA produced during HIV-1 replication, causing increased T cell death. While HIV-1 has emerged relatively recently as a human pathogen, the cDNA functionality of IFI16 could have been selected for during the course of human evolution. Here we present a novel hypothesis that the products of reverse transcription of HK2, which has been proliferating in the genome of human ancestors for 30 million years, could interact with IFI16. In support of our hypothesis, we provide preliminary data showing that IFI16 (but not cGAS) interacts with synthetic single-stranded HK2 oligos corresponding to the first product of reverse transcription. Further, we show that ssDNA detection by IFI16 has variability with respect to sequence features but is not dependent on strong secondary structures mimicking dsDNA. Among the HK2 oligos, IFI16 interacts more intensely with those derived from LTRs, suggesting these oligos have undetermined structural features that allow IFI16 to bind with greater affinity. Further, cells with stem cell features that naturally allow HK2 expression were found to express many components of the innate immune system including cGAS but not IFI16. Based on the presented preliminary data we further postulate another hypothesis: that the IFI16 functionality in human cells has been acting as "second-line" defense to control abnormal HK2 replication in somatic tissues. The absence of this protein in stem cells and a stem cell line could permit these cells to express HERVs which contribute to stem cell identity. Finally, we also comment on potential studies that could support or refute our hypothesis.

Project description:Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.IMPORTANCE Host factors within the cell are the major mode of restriction of adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide short interfering RNA screen, we identified that proteins of the small ubiquitin-like modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.

Dataset Information

Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way.

Importance

Publications

Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way.

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