Adeno-associated virus type 5 exploits two different entry pathways in human embryo fibroblasts

Abstract

The helper-dependent adeno-associated viruses (AAVs) have attracted great interest as vectors for gene therapy. Uptake and intracellular trafficking pathways of AAV are of importance, since they are often rate-limiting steps in infection. Here, we have investigated the entry of AAV type 5 (AAV5) in primary human embryo fibroblasts. At low binding temperatures, numerous virions are concentrated between cells, at contact points between cells and cellular protrusions, and at filopodia. When the temperature is raised to 37 6C, uptake of AAV5 takes place but up to 80 % of the bound virions dissociate from the cells. Uptake is achieved by cellular structures that are part of at least two different entry pathways. In addition to the common clathrin-dependent route, caveolar endocytosis and caveosome-like organelles are involved in a second pathway not yet described for parvoviruses. Both pathways can be used in parallel to enter an individual cell.Adeno-associated virus (AAV) is a small, non-enveloped parvovirus with single-stranded DNA that requires a viral helper for productive replication (Berns & Parrish, 2007). AAVs have attracted great interest as vectors for gene therapy and major advances have been made in recent years. AAV serotypes have been isolated from human and various non-human primate species and many have been developed to generate a variety of recombinant AAV (rAAV) vectors. The basic biology of AAV and its associated vectors is well-studied and clinical trials are now under investigation (for reviews, see Gao et al., 2005;Grieger et al., 2006;Grimm & Kay, 2003;Hendrie & Russell, 2005;Muzyczka & Warrington, 2005;Srivastava, 2005;Warrington & Herzog, 2006;Wu et al., 2006;Zhong et al., 2006). In gene therapy, targeting of viral vectors to the correct tissues and cells is essential. Uptake and intracellular trafficking pathways of AAV are important, especially since they may be rate-limiting in transduction (Ding et al., 2006(Ding et al., , 2005Greber, 2002;Harbison et al., 2008;Vihinen-Ranta et al., 2004). We were interested in the early steps of the entry pathway exploited by AAV type 5 (AAV5), a virus isolated from human material (BantelSchaal & zur Hausen, 1984;Bantel-Schaal et al., 1999;Chiorini et al., 1999;Georg-Fries et al., 1984), using primary human embryo fibroblasts (D7) as targets. D7 cells (established by J.R. Schlehofer, Deutsches Krebsforschungszentrum, Heidelberg;Bantel-Schaal, 1999; Bantel-Schaal & Stoehr, 1992) were grown as monolayers in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum, penicillin, streptomycin and glutamine at 37 u C either on coverslips or in plastic tissue culture dishes. Absence of mycoplasma was demonstrated by PCR. Permissiveness for infection by AAV5 was determined by detection of AAV5 capsid antigen (GeorgFries et al., 1984) by using immunofluorescence assays of cultures co-infected with the helper adenovirus type 2 (AAV2). The relative infection efficiency of D7 cells with other permissive cell types, such as HeLa, proved difficul...

Multiple Pathways Involved in Porcine Parvovirus Cellular Entry and Trafficking toward the Nucleus

“…Most parvoviruses are known to use clathrin-mediated endocytosis extensively, if not exclusively. Nevertheless, it was recently shown that AAV5 uses both clathrin and caveolar endocytosis to enter cells (1). Inhibition of caveolae had no effect on PPV infection.…”

“…Caveolar endocytosis is not constitutive and needs to be triggered. This entry mode has been shown to be responsible for simian virus 40 (SV40) infection (37), and among the parvoviruses, only adeno-associated virus 5 (AAV5) is known to use it (1). A third mechanism for virus entry into a cell is macropinocytosis (53).…”

Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis

“…19 As a positive control, we included rAAV5, a serotype capable of entering cells by both clathrin-and caveolin-mediated endocytosis. 20 Although rAAV5 transduction was inhibited by 40% in the presence of genistein, rAAV2-mediated transduction was unaffected and transduction of the capsid insertion mutants was even increased by up to 43% (D5) (Supplementary Figure 1).…”

Gene Therapy in Heart Failure

“…1). The receptors for some of the AAV serotypes have been described in the literature (see 6 for a description of the AAV receptors) Receptor-binding is then followed by endocytosis, which for AAV2 is via the so-called CLIC/GEEC (Clathrin Independent Carriers/GPI-anchored protein Enriched Endocytic Compartment) pathway 7 whereas for AAV5 it has been reported that endocytosis can occur both by clathrin coated vesicles or caveolae 8 .…”