Sirika Pillay

RIP3 Induces Apoptosis Independent of Pronecrotic Kinase Activity

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

An essential receptor for adeno-associated virus infection

Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr−/− (also known as Au040320−/− and Kiaa0319l−/−) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.

Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification

Broad spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we use parallel genome-wide high-coverage shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad spectrum antiviral with unexplained cytotoxicity1–3. We show that GSK983 blocks cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduces GSK983 cytotoxicity but not antiviral activity, providing an attractive novel approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Together, our results highlight the distinct advantages and limitations of each screening method for identifying drug targets and demonstrate the utility of parallel knockdown and knockout screens for comprehensively probing drug activity.

Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor

ABSTRACT Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor. IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.

Hunting Viral Receptors Using Haploid Cells

Viruses have evolved intricate mechanisms to gain entry into the host cell. Identification of host proteins that serve as viral receptors has enabled insights into virus particle internalization, host and tissue tropism, and viral pathogenesis. In this review we discuss the most commonly employed methods for virus receptor discovery, specifically highlighting the use of forward genetic screens in human haploid cells. The ability to generate true knockout alleles at high saturation provides a sensitive means to study virus-host interactions. To illustrate the power of such haploid genetic screens, we highlight the discovery of the lysosomal proteins NPC1 and LAMP1 as intracellular receptors for Ebola virus and Lassa virus, respectively. From these studies emerges the notion that receptor usage by these viruses is highly dynamic, involving a programmed switch from cell surface receptor to intracellular receptor. Broad application of genetic knockout approaches will chart functional landscapes of receptors and endocytic pathways hijacked by viruses.

An alternate route for adeno-associated virus entry independent of AAVR

ABSTRACT Determinants and mechanisms of cell attachment and entry steer adeno-associated virus (AAV) in its utility as a gene therapy vector. Thus far, a systematic assessment of how diverse AAV serotypes engage their proteinaceous receptor AAVR (KIAA0319L) to establish transduction has been lacking, despite potential implications for cell and tissue tropism. Here, a large set of human and simian AAVs as well as in silico-reconstructed ancestral AAV capsids were interrogated for AAVR usage. We identified a distinct AAV capsid lineage comprised of AAV4 and AAVrh32.33 that can bind and transduce cells in the absence of AAVR, independent of the multiplicity of infection. Virus overlay assays and rescue experiments in nonpermissive cells demonstrate that these AAVs are unable to bind to or use the AAVR protein for entry. Further evidence for a distinct entry pathway was observed in vivo, as AAVR knockout mice were equally as permissive to transduction by AAVrh32.33 as wild-type mice upon systemic injection. We interestingly observe that some AAV capsids undergo a low level of transduction in the absence of AAVR, both in vitro and in vivo, suggesting that some capsids may have a multimodal entry pathway. In aggregate, our results demonstrate that AAVR usage is conserved among all primate AAVs except for those of the AAV4 lineage, and a non-AAVR pathway may be available to other serotypes. This work furthers our understanding of the entry of AAV, a vector system of broad utility in gene therapy. IMPORTANCE Adeno-associated virus (AAV) is a nonpathogenic virus that is used as a vehicle for gene delivery. Here, we have identified several situations in which transduction is retained in both cell lines and a mouse model in the absence of a previously defined entry receptor, AAVR. Defining the molecular determinants of the infectious pathway of this highly relevant viral vector system can help refine future applications and therapies with this vector.

Host determinants of adeno-associated viral vector entry

Viral vectors based on adeno-associated virus (AAV) are leading candidates for therapeutic gene delivery. Understanding rate-limiting steps in the entry of AAV vectors may be used in a rational approach to improve efficiency and specificity of transduction. This review describes our current understanding of AAV entry, a key step during infection. We discuss the identity and functions of AAV receptors and attachment factors, including the recently discovered multi-serotype receptor AAVR. We further provide an overview of other host factors that act during the trafficking stage of AAV vector transduction. In particular, we focus on cellular protein complexes associated with retrograde transport from endosomes to the trans-Golgi network. The novel insights in AAV-host interactions facilitated by technological advances in genetic screening approaches provide a greater depth in our understanding how AAV vectors exploit host factors to deliver its genetic cargo to the nucleus.

Corrigendum: An essential receptor for adeno-associated virus infection

This corrects the article DOI: 10.1038/nature16465

478. An Essential and Ubiquitous Protein Receptor for AAV; Glycans as Attachment Receptors

Motivated by unsuccessful attempts to observe physical binding between AAV-2 and heterologously expressed domains of previously reported co-receptors, we set out to identify novel protein receptor(s) for AAV2 through an unbiased genome-wide knockout screen in human cells. Using an mCherry AAV vector, resistant cells were iteratively selected by FACS for gene trap screening in a library of mutagenized haploid cells. Upon deep sequencing, refractory cells had significantly high frequencies of mutation in genes encoding glycan synthesis and retrograde transport, but most significantly in a hitherto poorly characterized transmembrane protein, now termed AAVR. Genetic confirmation of AAVRs role in the entry of multiple AAV serotypes has come through CRISPR-Cas9 knockouts in multiple cell lines then restoration of susceptibility through complementation; infection of poorly permissive cells following AAVR transduction; and creation of a mouse knockout with greatly diminished susceptibility.Various AAVR ectodomain constructs have been heterologously expressed and purified as fusion proteins, and these have been shown to inhibit in vitro viral transduction at concentrations consistent with effective nM binding constants (between AAV & AAVR) measured by surface plasmon resonance (SPR). Pre-incubation with antibodies to AAVR also inhibits infection or transduction. AAVR is transiently expressed on the plasma membrane. Expression of chimeric constructs suggests that AAV takes advantage of its trafficking to the perinuclear trans Golgi network as the dominant, but non-exclusive, entry pathway. Identification of AAVR and its apparently ubiquitous use has interesting implications for AAVs cell specificity. Progress towards structure of complexes will be reported.AAVR exhibits the classic characteristics of a viral receptor, casting the roles ascribed to glycan “primary” receptors in new light. Electron microscopy has been used to visualize AAV-DJ in complex with various heparin analogs at increasingly high resolution. A structure at 2.8 A resolution, as a pentasaccharide complex, shows some disorder in the glycan, but the side chains of viral amino acids are clearly resolved and in different conformations from those seen in a sucrose octasulfate complex. With little change to the backbone, the binding site accommodates diverse glycan sequences through adjustments to side chains, consistent with SPR binding assays of AAV-2 to a library of heparanoids. This, together with comparisons of heparan and AAVR cell knock-outs, indicates a more accessory role for glycans than is implied by the term “primary”. As for several other viruses, in AAV-2 at least, the glycan is an attachment receptor that likely elevates the AAV concentration proximal to the membrane, improving the efficiency with which the virus then binds to AAVR.

293. Discovery of an Essential Receptor for Adeno-Associated Virus Infection

Cellular entry of adeno-associated viruses (AAV) is poorly understood, despite the prominent use of AAV vectors in gene therapy for several monogenic diseases. Using an unbiased, haploid genetic screen, we identified critical players in AAV serotype 2 (AAV2) entry including members of distinct protein complexes involved in retrograde trafficking as well as genes involved in the biosynthesis of the AAV2 attachment factor, heparan sulphate. We focused on the single most significantly enriched gene of the screen, an uncharacterized type-I transmembrane protein. Based on the evidence below we renamed this gene AAV receptor (AAVR). We discovered AAVR as capable of rapidly endocytosing from the plasma membrane and trafficking to the trans-Golgi network, taking a similar path as AAV particles utilize. Genetic ablation of AAVR using CRISPR/Cas9 technology demonstrated a robust resistance to AAV2 infection in a wide range of mammalian cell types, which could be reversed upon AAVR complementation. This confirmed the essentiality of AAVR in AAV2 infection. Further investigation revealed that AAVR was also required for the infections of all tested human and simian-derived AAV serotypes including AAV1, 3B, 5, 6, 8 and 9. Deeper characterization of AAVR showed it to contain Ig-like domains, which are commonly found in many virus receptors including those for poliovirus and measles. We observed that these domains are capable of binding to AAV2 particles, and anti-AAVR antibodies efficiently block AAV2 infection. Moreover, the importance of AAVR for AAV infection in vivo was demonstrated by the strong resistance of AAVR−/− mice to AAV9 infection, recapitulating what we showed in vitro. Collectively, the data indicates that AAVR is a universal receptor involved in AAV infection. This has significant implications for the improvement of future AAV vector design.