Heparin binding induces conformational changes in Adeno-associated virus serotype 2

“The two resultant, essentially identical, difference density maps were inspected using Chimera (Pettersen et al., 2004) and O (Jones et al., 1991).”

“Extrusion and activity of the VP1-N terminal PLA2 domain is essential for efficient infection and is believed to occur during or shortly after virus entry into the cell (Bleker et al., 2005; Farr et al., 2006; Mani et al., 2006; Suikkanen et al., 2003; Vihinen-Ranta et al., 2002).”

“Heparan sulfate proteoglycan, the first receptor identified for an AAV virus (Summerford and Samulski, 1998), functions during infection to attach AAV2 and AAV3 to the cell surface.”

“AAV2 residues R484, R487, K527, K532, R585, and R588 have been shown to affect heparin binding in mutagenesis studies (Kern et al., 2003; Opie et al., 2003; Wu et al., 2000).”

“Heparin molecules, extracted from the atomic coordinates of the structure of a heparin hexasaccharide complexed with fibroblast growth factor (FGF- heparin) (Faham et al., 1996) (PDB Accession No.”

“Efficient AAV2 infection also requires a co-receptor, such as human fibroblast growth factor receptor 1 (FGFR1), integrins avb5 and a5b1, or hepatocyte growth factor receptor (Asokan et al., 2006; Kashiwakura et al., 2005; Summerford et al., 1999; Qing et al., 1999).”

“Bink and Pleij, 2002; Prasad et al., 1994; Speir et al., 1995; Tang et al., 2001).”

“Samples were centrifuged in a 70 Ti rotor at 350,333g for 1 h at 18 C as previously described (Zolotukhin et al., 2002).”

“These co-receptors mediate endocytosis of the virus via clathrin-coated pits ( Bartlett et al., 2000; Duan et al., 1999).”

“For MVM, the binding site for its sialic acid receptor was mapped by X-ray crystallography to the twofold depression (Cotmore and Tattersall, 1987; Lopez-Bueno et al., 2006).”

“5 mg/ml were vitrified on carbon-coated copper EM grids in liquid ethane as described (Baker et al., 1999).”

“However, alanine substitution for this stretch of four acidic residues (561-DEEE564) produces a mutant virus that does not bind heparin and is non-infectious (Wu et al., 2000).”

“The center and orientation of each boxed particle were initially estimated by means of the model-based polar Fourier transform routine (Baker and Cheng, 1996), with the AAV5 structure (Walters et al., 2004) serving as the starting search model for the procedure.”

“These mark the closest points to the heparin binding residues identified by mutagenesis (Kern et al., 2003; Opie et al., 2003) in the crystal structure of the AAV2 capsid (Xie et al.”

“In addition, the 2f pos density (Fig. 2A and B) modeled as hep2f (Fig. 2B and C) is difficult to explain on the basis of the heparin binding residues, R484, R487, K527, K532, R585, and R588 (Kern et al., 2003; Opie et al., 2003 ).”

“The N-terminal sequence unique to VP1 has a phospholipase A2 (PLA2) activity that is essential for infectivity at a step between endocytosis and nuclear entry (Girod et al., 2002) as well as nuclear localization signals.”

“Hence, since Rep-facilitated DNA packaging is proposed to occur with preformed empty capsids through the channel, these observations suggest that small portions of viral genome are pre-packaged during assembly before Rep completes the packaging (Myers and Carter, 1980; Wistuba et al., 1995; King et al., 2001).”

“A similar mechanism has been proposed for assembly of ssRNA viruses (Rudnick and Bruinsma, 2005; Zlotnick, 1994, 2004).”

“A channel at each fivefold axis (channel) is implicated in packaging of the AAV2 genome and extrusion of the PLA2 activity of the VP1 unique region (Bleker et al., 2005; Sonntag et al., 2006).”

“The binding site for the CPV transferrin receptor was mapped by mutagenesis and cryo-reconstruction to the raised portion of the capsid that lies between the icosahedral two- and fivefold axes (Hueffer et al., 2003a,b; Govindasamy et al., 2003; Hafenstein et al., 2007).”

“In addition, recent observation of NTs in baculovirus-expressed AAV8 ( Nam et al., 2007 )”

“Efficient AAV2 infection also requires a co-receptor, such as human fibroblast growth factor receptor 1 (FGFR1), integrins avb5 and a5b1, or hepatocyte growth factor receptor (Asokan et al., 2006; Kashiwakura et al., 2005; Summerford et al., 1999; Qing et al., 1999).”

“A low resolution 3D map was generated from a selected subset for each set of images using the programs EMSEL and EM3DR (Baker et al., 1999; Fuller et al., 1996).”

“…and three-dimensional (3D) image reconstruction (‘‘cryo-reconstruction”) and X-ray crystallography have been used to determine the structures of several AAV serotypes, including AAV1, AAV2, AAV4, AAV5, AAV7, and AAV8 (Govindasamy et al., 2003; Kronenberg et al., 2001; Miller et al., 2006;…”

“Efficient AAV2 infection also requires a co-receptor, such as human fibroblast growth factor receptor 1 (FGFR1), integrins avb5 and a5b1, or hepatocyte growth factor receptor (Asokan et al., 2006; Kashiwakura et al., 2005; Summerford et al., 1999; Qing et al., 1999).”

“The crystal structures of CPV and the immunosuppressive strain of MVM (MVMi) showed the presence of at least 11 ordered ssDNA NTs inside the capsids (Agbandje-Mckenna et al., 1998; Kontou et al., 2005; Xie and Chapman, 1996).”

“However, the HI loop in B19 adopts a radial orientation relative to the capsid surface (Kaufmann et al., 2004), similar to the AAV2–Hep HI loop conformation described above.”

“who suggested that primary receptor binding by AAV2 might lead to structural rearrangements close to the twofold axis to facilitate co-receptor attachment (Lochrie et al., 2006).”

“A channel at each fivefold axis (channel) is implicated in packaging of the AAV2 genome and extrusion of the PLA2 activity of the VP1 unique region (Bleker et al., 2005; Sonntag et al., 2006).”

“These co-receptors mediate endocytosis of the virus via clathrin-coated pits (Bartlett et al., 2000; Duan et al., 1999).”

“Efficient AAV2 infection also requires a co-receptor, such as human fibroblast growth factor receptor 1 (FGFR1), integrins avb5 and a5b1, or hepatocyte growth factor receptor (Asokan et al., 2006; Kashiwakura et al., 2005; Summerford et al., 1999; Qing et al., 1999).”

“The diameter of the electron beam was adjusted to not exceed the limits of the Gatan 2k2 slow-scan CCD detector (0.4 lm on the specimen) to allow images to be recorded without pre-irradiating adjacent regions.”

“The crystal structures of AAV4 and AAV8 showed the ordering of a single nucleotide (NT) under each threefold axis (Govindasamy et al., 2006; Nam et al., 2007).”

“Extrusion and activity of the VP1-N terminal PLA2 domain is essential for efficient infection and is believed to occur during or shortly after virus entry into the cell (Bleker et al., 2005; Farr et al., 2006; Mani et al., 2006; Suikkanen et al., 2003; Vihinen-Ranta et al., 2002).”

“…Rep-facilitated DNA packaging is proposed to occur with preformed empty capsids through the channel, these observations suggest that small portions of viral genome are pre-packaged during assembly before Rep completes the packaging (Myers and Carter, 1980; Wistuba et al., 1995; King et al., 2001).”

“…Baker d, Mavis Agbandje-McKenna a,* a Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, College of Medicine, 1600 SW Archer Road, P.O. Box 100245, University of Florida, Gainesville, FL 32610, USA b Department of Biological Sciences,…”

“The crystal structures of CPV and the immunosuppressive strain of MVM (MVMi) showed the presence of at least 11 ordered ssDNA NTs inside the capsids (Agbandje-Mckenna et al., 1998; Kontou et al., 2005; Xie and Chapman, 1996).”

“The diameter of the electron beam was adjusted to not exceed the limits of the Gatan 2k2 slow-scan CCD detector (0.4 lm on the specimen) to allow images to be recorded without pre-irradiating adjacent regions.”

“AAV terminal repeats (TRs) rather than heterologous DNA sequences are known to form the signal used to package AAV (McLaughlin et al., 1988).”

“The binding site for the CPV transferrin receptor was mapped by mutagenesis and cryo-reconstruction to the raised portion of the capsid that lies between the icosahedral two- and fivefold axes (Hueffer et al., 2003a,b; Govindasamy et al., 2003; Hafenstein et al., 2007).”

“Cryo-electron microscopy and three-dimensional (3D) image reconstruction (‘‘cryo-reconstruction”) and X-ray crystallography have been used to determine the structures of several AAV serotypes, including AAV1, AAV2, AAV4, AAV5, AAV7, and AAV8 (Govindasamy et al., 2003; Kronenberg et al., 2001; Miller et al., 2006; Nam…”

“Hence, since Rep-facilitated DNA packaging is proposed to occur with preformed empty capsids through the channel, these observations suggest that small portions of viral genome are pre-packaged during assembly before Rep completes the packaging (Myers and Carter, 1980; Wistuba et al., 1995; King et al., 2001).”

“Regions of the capsid surface that bind to host-cell receptors have been identified for three autonomous parvoviruses: human parvovirus B19 (B19), canine parvovirus (CPV), and minute virus of mice (MVM) (Chipman et al., 1996; Hafenstein et al., 2007; Lopez-Bueno et al., 2006).”

“The crystal structures of CPV and the immunosuppressive strain of MVM (MVMi) showed the presence of at least 11 ordered ssDNA NTs inside the capsids (Agbandje-Mckenna et al., 1998; Kontou et al., 2005; Xie and Chapman, 1996).”

“…and X-ray crystallography have been used to determine the structures of several AAV serotypes, including AAV1, AAV2, AAV4, AAV5, AAV7, and AAV8 (Govindasamy et al., 2003; Kronenberg et al., 2001; Miller et al., 2006; Nam et al., 2007; Padron et al., 2005; Quesada et al., 2007; Walters et…”

“The crystal structures of CPV and the immunosuppressive strain of MVM (MVMi) showed the presence of at least 11 ordered ssDNA NTs inside the capsids (Agbandje-Mckenna et al., 1998; Kontou et al., 2005; Xie and Chapman, 1996).”

“Soluble heparin has been shown to compete with permissive cells for AAV2 binding and de-sulfating heparin reduces binding to AAV2 ( Qiu et al., 2000 ).”

“Significantly, the fivefold channels of parvovirus capsids have been implicated as conduits for extrusion of the N-terminal portions of VP1 and VP2 (both of which are reported to normally lie inside the capsid and for genome packaging (Bleker et al., 2005; Farr and Tattersall, 2004; Farr et al., 2006).”

“Significantly, the fivefold channels of parvovirus capsids have been implicated as conduits for extrusion of the N-terminal portions of VP1 and VP2 (both of which are reported to normally lie inside the capsid and for genome packaging (Bleker et al., 2005; Farr and Tattersall, 2004; Farr et al., 2006).”

“A total of 2335 particle images was boxed from ten VLP micrographs and 1523 particle images were boxed from eight AAV2–Hep micrographs as previously described (Bowman et al., 2002; Walters et al., 2004).”

“Recently, mutagenesis, confocal microscopy, and VP structural alignment studies reported a role for the HI loop in genome packaging (DiPrimio et al., 2008).”

“A similar mechanism has been proposed for assembly of ssRNA viruses (Rudnick and Bruinsma, 2005; Zlotnick, 1994, 2004).”

“Several investigators have shown that AAV DNA and heterologous, chromosomal DNA that contains a Rep binding element and a terminal resolution site, but no TRs, can also be packaged into AAV virions (Huser et al., 2003; Nony et al., 2003).”

“A similar mechanism has been proposed for assembly of ssRNA viruses (Rudnick and Bruinsma, 2005; Zlotnick, 1994, 2004).”

“Several investigators have shown that AAV DNA and heterologous, chromosomal DNA that contains a Rep binding element and a terminal resolution site, but no TRs, can also be packaged into AAV virions ( Huser et al., 2003; Nony et al., 2003).”

“The estimated resolution achieved with each reconstruction was defined by the lowest spatial frequency at which the FSC first dropped below 0.5 and the average phase difference exceeded 45 .”

“The diameter of the electron beam was adjusted to not exceed the limits of the Gatan 2k2 slow-scan CCD detector (0.4 lm on the specimen) to allow images to be recorded without pre-irradiating adjacent regions.”

“The second study (Levy et al., 2009), using a partially depolymerized heparin, suffered the same problems, perhaps to a lesser extent; however, both studies agreed on the location of the major heparin binding site in the valleys between protrusions at the 3-fold axis of the icosahedral capsid.”

“Receptor binding not only enables cell entry, but also primes the capsid for the later release of its genetic payload (uncoating)(46) and prepares the cell for viral particle transport and processing.(46) Since AAV serotypes differ in receptors used for cell attachment and internalization, serotypes other than AAV2, t…”