Mavis Agbandje-McKenna

Identification of Amino Acid Residues in the Capsid Proteins of Adeno-Associated Virus Type 2 That Contribute to Heparan Sulfate Proteoglycan Binding

ABSTRACT The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.

Functional implications of the structure of the murine parvovirus, minute virus of mice

BACKGROUNDnMinute virus of mice (MVM) is a single-stranded (ss) DNA-containing, murine parvovirus with a capsid built up of 60 icosahedrally related polypeptide chains, each of which consists of the C-terminal region common to two structural proteins, VP1 and VP2. In infectious virions, most VP2 molecules are cleaved to VP3 by the removal of about 20 amino acids from the N terminus. Of the 587 amino acids in VP2, approximately half are identical to those in the analogous capsid protein of the antigenically distinct canine parvovirus (CPV), the crystal structure of which has previously been determined. The three-dimensional structure determination of MVMi (the immunosuppressive strain of MVM) was previously reported to 3.5 A resolution.nnnRESULTSnWe report here an analysis of the MVMi virus structure and provide insights into tissue tropism, antigenicity and DNA packaging. Amino acids determining MVM tissue tropism were found to cluster on, or near, the viral surface. A conserved, glycine-rich, N-terminal peptide was seen to run through a cylindrical channel along each fivefold axis and may have implications for antigenicity. Density within the virion was interpreted as 29 ssDNA nucleotides per icosahedral asymmetric unit, and accounts for over one-third of the viral genome.nnnCONCLUSIONSnThe presence of the glycine-rich sequence in the fivefold channels of MVMi provides a possible mechanism to explain how the unique N-terminal region of VP1 becomes externalized in infectious parvovirions. Residues that determine tropism may form an attachment recognition site for a secondary host-cell factor that modulates tissue specificity. The ordering of nucleotides in a similar region of the interior surface in the CPV and MVMi capsids suggests the existence of a genomic DNA-recognition site within the parvoviral capsid.

Structure of Adeno-Associated Virus Type 4

ABSTRACT Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-Å resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology.

Combinations of two capsid regions controlling canine host range determine canine transferrin receptor binding by canine and feline parvoviruses.

ABSTRACT Feline panleukopenia virus (FPV) and its host range variant, canine parvovirus (CPV), can bind the feline transferrin receptor (TfR), while only CPV binds to the canine TfR. Introducing two CPV-specific changes into FPV (at VP2 residues 93 and 323) endowed that virus with the canine TfR binding property and allowed canine cell infection, although neither change alone altered either property. In CPV the reciprocal changes of VP2 residue 93 or 323 to the FPV sequences individually resulted in modest reductions in infectivity for canine cells. Changing both residues in CPV to the FPV amino acids blocked the canine cell infection, but that virus was still able to bind the canine TfR at low levels. This shows that both CPV-specific changes control canine TfR binding but that binding is not always sufficient to mediate infection.

Structure Determination of Minute Virus of Mice

The three-dimensional crystal structure of the single-stranded DNA-containing (full) parvovirus, minute virus of mice (MVM), has been determined to 3.5 A resolution. Both full and empty particles of MVM were crystallized in the monoclinic space group C2 with cell dimensions of a = 448.7, b = 416.7, c = 305.3 A and beta = 95.8 degrees. Diffraction data were collected at the Cornell High Energy Synchrotron Source using an oscillation camera. The crystals have a pseudo higher R32 space group in which the particles are situated at two special positions with 32 point symmetry, separated by (1/2)c in the hexagonal setting. The self-rotation function showed that the particles are rotated with respect to each other by 60 degrees around the pseudo threefold axis. Subsequently, a more detailed analysis of the structure amplitudes demonstrated that the correct space-group symmetry is C2 as given above. Only one of the three twofold axes perpendicular to the threefold axis in the pseudo R32 space group is a true crystallographic twofold axis; the other two are only local non-crystallographic symmetry axes. The known canine parvovirus (CPV) structure was used as a phasing model to initiate real-space electron-density averaging phase improvement. The electron density was easily interpretable and clearly showed the amino-acid differences between MVM and CPV, although the final overall correlation coefficient was only 0.63. The structure of MVM has a large amount of icosahedrally ordered DNA, amounting to 22% of the viral genome, which is significantly more than that found in CPV.

Structures of host range-controlling regions of the capsids of canine and feline parvoviruses and mutants.

ABSTRACT Canine parvovirus (CPV) and feline panleukopenia virus (FPV) differ in their ability to infect dogs and dog cells. Canine cell infection is a specific property of CPV and depends on the ability of the virus to bind the canine transferrin receptor (TfR), as well as other unidentified factors. Three regions in the capsid structure, located around VP2 residues 93, 300, and 323, can all influence canine TfR binding and canine cell infection. These regions were compared in the CPV and FPV capsid structures that have been determined, as well as in two new structures of CPV capsids that contain substitutions of the VP2 Asn-93 to Asp and Arg, respectively. The new structures, determined by X-ray crystallography to 3.2 and 3.3 Å resolutions, respectively, clearly showed differences in the interactions of residue 93 with an adjacent loop on the capsid surface. Each of the three regions show small differences in structure, but each appears to be structurally independent of the others, and the changes likely act together to affect the ability of the capsid to bind the canine TfR and to infect canine cells. This emphasizes the complex nature of capsid alterations that change the virus-cell interaction to allow infection of cells from different hosts.

Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 4

Adeno-associated virus (AAV) serotypes 1 to 5 are currently under development as clinical gene delivery vectors for the treatment of human diseases. However, the ubiquitous nature of their cell surface receptors, heparin sulfate (AAV2 and 3) and sialic acids (AAV4 and 5), can preclude specific tissue targeting in vivo. Structural studies of AAV4 were initiated to characterize its capsid surface for re-targeting manipulations. Crystals obtained diffracted synchrotron radiation to 3.2 A resolution. The unit cell is body-centered orthorhombic, I222, with a = 339.6, b = 319.2 and c = 285.0 A. The virus particle orientation and position have been determined.