We have used defined mutant CHO cells defective in GAG synthesis and standard biochemical assays to establish that membrane-associated HS proteoglycan serves as a receptor for AAV-2 and mediates both viral attachment to and subsequent infection of target cells. We have shown that binding and infection of cells by AAV is sensitive to (i) competitive inhibition with heparin, a soluble receptor analog, (ii) enzymatic removal of HS but not chondroitin sulfate moieties from the cell surface, and (iii) genetic defects in the cellular pathway for the production of HS. In addition, the use of mutant cell lines provided genetic evidence that HS, and not chondroitin sulfate, proteoglycans are responsible for a productive AAV infection. This is the first report of a role for proteoglycan in cellular attachment of a parvovirus and provides an explanation for the broad host range of AAV.
We have demonstrated that HS proteoglycan serves as a principal attachment receptor for AAV-2. However, additional factors could also participate in AAV host cell attachment. Some viruses can use more than one distinct attachment receptor. For example, HIV uses CD4 as its main attachment receptor but can also attach to glycolipid galactosyl ceramide to mediate infection (10
). Further, Ad attachment to target cells can be mediated by αMβ2 integrin as well as CAR (6
). Since removal of HS moieties from the cell surface did not completely abolish AAV infectivity and AAV still exhibits some specific binding to cell lines that do not produce HS proteoglycans, it is possible that AAV attachment and infection can also be mediated by an as yet unidentified receptor, albeit inefficiently.
While the inefficient binding and poor infection by AAV in the absence of HS GAGs suggests that HS proteoglycan could mediate both AAV attachment and entry, it remains to be determined whether AAV attachment to HS proteoglycan is sufficient for viral entry. For example, it is well established that the initial interaction of HSV with its host cell is mediated through HS proteoglycans (35
) and that another secondary event is responsible for promoting entry (20
). Recently, a novel member of the tumor necrosis factor/nerve growth factor receptor family was demonstrated to serve as a mediator of HSV entry (42
). In addition, Ad infection is initiated by attachment to CAR (6
) followed by subsequent interaction with secondary receptors, identified as αV integrins, that are known to facilitate virus internalization (60
). On the other hand, a large percentage of HS proteoglycans are known to undergo endocytosis (26
) and could be involved in direct AAV internalization. Such a mechanism of entry has been described for other HS proteoglycan ligands, including basic fibroblast growth factor and lipoprotein lipase (47
). AAV may use either or both of these proposed mechanisms of entry. Further, the possibility that a large functional multireceptor complex is required for efficient AAV entry should not be ruled out, since cell surface proteoglycans have been implicated as members of multimeric complexes (7
). That is, our results show the HS proteoglycans are required for AAV infection but do not address whether they are in fact sufficient.
The GAG structures can be complex, exhibiting a diversity of disaccharide sequences with heterogeneous sulfation. Some GAG ligands require the presence of a specific sugar sequence for high affinity binding, as is the case for antithrombin, which binds a distinct sequence present in heparin/HS (3
). In addition, a recent report identified HS GAGs as a receptor for the pathogenic RNA virus dengue virus. However, the virus appears to require a highly sulfated form of HS GAG in order to be infectious (12
). Although we have not identified a specific sugar sequence requirement for AAV binding, our data indicate that AAV requires HS and not chondroitin sulfate moieties. This conclusion is based on the inability of chondroitinase ABC enzymatic digestion to inhibit AAV binding and infection as well as the inability of AAV to appreciably bind and infect mutant CHO cells that lack HS yet have an excess of chondroitin sulfate proteoglycans. HS GAGs consist of repeating disaccharide units composed of alternating glucosamine and hexuronic acid (either glucuronic acid or iduronic acid) monsacharides. The chondroitin sulfate disaccharide units contain a galactosamine monosacharide in place of glucosamine. Chondroitin sulfate B (dermatan sulfate) is the only chondroitin that contains iduronic acid monosacharides that are also found in HS GAGs. The specificity exhibited by AAV for HS moieties demonstrates that AAV prefers an interaction with a glucosamine-hexuronic acid backbone. Further, since excess soluble dermatan sulfate could inhibit AAV binding and infection, AAV may prefer a HS backbone that contains iduronic acid.
Our data may indicate that multiple receptor molecules mediate AAV infection. In cases where the amount of cell-associated HS GAG was reduced, either by enzymatic digestion or in mutant cell lines, the reduction in AAV infection was more sensitive than the reduction of AAV attachment. One possible explanation is that the density of receptors, and thus the increased probability of virus or HS proteoglycan interactions with some other receptor molecule(s), may be an important factor influencing AAV entry. It will be interesting to determine if the amount of cell surface HS proteoglycan influences the ratio of internalized virus to bound virus. Alternatively, another explanation for our results may be that AAV can attach to a subset of surface molecules that are not capable of mediating AAV infection.
Since the degree of HS sulfation affects the amount of AAV that can bind the cell surface, there appears to be an important charge component to the specific AAV-HS interaction. A majority of ligand-proteoglycan interactions are mediated by a cluster of basic amino acids displayed by the ligand and the high density of charge found on sulfated GAGs (27
). Until the crystal structure of AAV-2 is determined, we cannot be certain which basic residues are exposed to the virion surface, nor can we address the noncolinear basic amino acids that may be close in space in the intact virion. However, there is a high density of positively charged amino acids within the first 170 residues of the VP1 capsid protein, including three strings of basic amino acids (either K/RX4/5
KKR or KX6
RKR) that, if exposed on the virion surface, could be involved in an ionic interaction with the cell surface. It is also interesting that viral mutants that map in this region are referred to as low-infectious-particle mutants (44
Recently, we have demonstrated long-term (1.5-year) gene expression after direct rAAV injection into immunocompetent mouse muscle and brain (37
). These data have provided preclinical results suggesting that this viral delivery system may provide an attractive alternative to other vectors. In fact, rAAV has recently been tested in a clinical trial for gene therapy of cystic fibrosis without any signs of toxicity of immune complications (19a
). Identification of the AAV receptor should now help facilitate maximum use of this vector with appropriate target cells (bone marrow stem cells, airway epithelia cells, etc.). Fluorescence-activated cell sorting analysis of various human cells has shown a correlation between HS GAGs and virus binding consistent with this report (58a
). Identification of the AAV receptor should provide further information concerning primary events involved in AAV infection and future development of this virus as a viral vector.