Identification of a heparin-binding motif on adeno-associated virus type 2 capsids

Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary.     

Novel caprine adeno-associated virus (AAV) capsid (AAV-Go.1) is closely related to the primate AAV-5 and has unique tropism and neutralization properties

Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8 administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies.     

Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies.

We examined the antigenic structure of human hepatitis A virus (HAV) by characterizing a series of 21 murine monoclonal-antibody-resistant neutralization escape mutants derived from the HM175 virus strain. The escape phenotype of each mutant was associated with reduced antibody binding in radioimmunofocus assays. Neutralization escape mutations were identified at the Asp-70 and Gln-74 residues of the capsid protein VP3, as well as at Ser-102, Val-171, Ala-176, and Lys-221 of VP1. With the exception of the Lys-221 mutants, substantial cross-resistance was evident among escape mutants tested against a panel of 22 neutralizing monoclonal antibodies, suggesting that the involved residues contribute to epitopes composing a single antigenic site. As mutations at one or more of these residues conferred resistance to 20 of 22 murine antibodies, this site appears to be immunodominant in the mouse. However, multiple mutants selected independently against any one monoclonal antibody had mutations at only one or, at the most, two amino acid residues within the capsid proteins, confirming that there are multiple epitopes within this antigenic site and suggesting that single-amino-acid residues contributing to these epitopes may play key roles in the binding of individual antibodies. A second, potentially independent antigenic site was identified by three escape mutants with different substitutions at Lys-221 of VP1. These mutants were resistant only to antibody H7C27, while H7C27 effectively neutralized all other escape mutants. These data support the existence of an immunodominant neutralization site in the antigenic structure of hepatitis A virus which involves residues of VP3 and VP1 and a second, potentially independent site involving residue 221 of VP1.     

Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection

The previously characterized monoclonal antibodies (MAbs) A1, A69, B1, and A20 are directed against assembled or nonassembled adeno-associated virus type 2 (AAV-2) capsid proteins (A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. A. Kleinschmidt, J. Virol. 71:1341-1352, 1997). Here we describe the linear epitopes of A1, A69, and B1 which reside in VP1, VP2, and VP3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. In addition, MAbs A20, C24-B, C37-B, and D3 directed against conformational epitopes on AAV-2 capsids were characterized. Epitope sequences on the capsid surface were identified by enzyme-linked immunoabsorbent assay using AAV-2 mutants and AAV serotypes, peptide scan, and peptide competition experiments. A20 neutralizes infection following receptor attachment by binding an epitope formed during AAV-2 capsid assembly. The newly isolated antibodies C24-B and C37-B inhibit AAV-2 binding to cells, probably by recognizing a loop region involved in binding of AAV-2 to the cellular receptor. In contrast, binding of D3 to a loop near the predicted threefold spike does not neutralize AAV-2 infection. The identified antigenic regions on the AAV-2 capsid surface are discussed with respect to their possible roles in different steps of the viral life cycle.     

Site-directed mutational analysis of human tumor necrosis factor-alpha receptor binding site and structure-functional relationship.

In order to define the receptor binding site and the structure-functional relationship of tumor necrosis factor (TNF), single amino acid substitutions were made by site-directed mutagenesis at selected residues of human tumor necrosis factor, using a phagemid mutagenesis/expression vector. The recombinant TNF mutants were compared to the wild type TNF in assays using crude bacterial lysates, for protein yield, solubility, subunit trimerization, receptor binding inhibition activity, and in vitro cytotoxic activity. All mutants which did not form cross-linkable trimer also showed little cytotoxic activity or receptor binding inhibition activity, indicating that trimer formation is obligatory for TNF-alpha activity. Most mutations of internal residues yielded no cross-linkable trimer, while most mutations of surface residues yielded cross-linkable trimer. Mutations at surface residues Leu29, Arg31, and Ala35 yielded cross-linkable trimers with good activities, except proline substitutions which may cause conformational changes in the polypeptide chain. This suggested that these residues are near the receptor binding site. Mutations at other strictly conserved internal residues such as Ser60, His78, and Tyr119 form cross-linkable trimer with little activity. These mutations may indirectly affect the receptor binding site by forming trimers with undetectable abnormalities. Mutants of surface residues Tyr87, Ser95, Ser133, and Ser147 affect receptor binding and cytotoxic activity but not trimer formation, suggesting that these residues are involved directly in receptor binding. The fact that residues Arg31, Ala35, Tyr87, Ser95, and Ser147, located on the opposite sides of a monomer, are clustered at the intersubunit grooves of TNF trimer supports the current notion that TNF receptor binding sites are trivalent and are located at the three intersubunit grooves. However, our finding that Ser133, which is outside the groove, can also be involved directly in receptor binding suggested that the receptor binding sites of TNF may not be confined to the intersubunit grooves, but extended to include additional surface residues.     

Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.     

Heparan sulfate proteoglycan binding properties of adeno-associated virus retargeting mutants and consequences for their in vivo tropism

Adeno-associated virus type 2 (AAV-2) targeting vectors have been generated by insertion of ligand peptides into the viral capsid at amino acid position 587. This procedure ablates binding of heparan sulfate proteoglycan (HSPG), AAV-2's primary receptor, in some but not all mutants. Using an AAV-2 display library, we investigated molecular mechanisms responsible for this phenotype, demonstrating that peptides containing a net negative charge are prone to confer an HSPG nonbinding phenotype. Interestingly, in vivo studies correlated the inability to bind to HSPG with liver and spleen detargeting in mice after systemic application, suggesting several strategies to improve efficiency of AAV-2 retargeting to alternative tissues.     

Fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the CD4 binding site of human immunodeficiency virus type 1 gp120

Alanine scanning mutagenesis was performed on monomeric gp120 of human immunodeficiency virus type 1 to systematically identify residues important for gp120 recognition by neutralizing and nonneutralizing monoclonal antibodies (MAbs) to the CD4 binding site (CD4bs). Substitutions that affected the binding of broadly neutralizing antibody b12 were compared to substitutions that affected the binding of CD4 and of two nonneutralizing anti-CD4bs antibodies (b3 and b6) with affinities for monomeric gp120 comparable to that of b12. Not surprisingly, the sensitivities to a number of amino acid changes were similar for the MAbs and for CD4. However, in contrast to what was seen for the MAbs, no enhancing mutations were observed for CD4, suggesting that the virus has evolved toward an optimal gp120-CD4 interaction. Although the epitope maps of the MAbs overlapped, a number of key differences between b12 and the other two antibodies were observed. These differences may explain why b12, in contrast to nonneutralizing antibodies, is able to interact not only with monomeric gp120 but also with functional oligomeric gp120 at the virion surface. Neutralization assays performed with pseudovirions bearing envelopes from a selection of alanine mutants mostly showed a reasonable correlation between the effects of the mutations on b12 binding to monomeric gp120 and neutralization efficacy. However, some mutations produced an effect on b12 neutralization counter to that predicted from gp120 binding data. It appears that these mutations have different effects on the b12 epitope on monomeric gp120 and functional oligomeric gp120. To determine whether monomeric gp120 can be engineered to preferentially bind MAb b12, recombinant gp120s were generated containing combinations of alanine substitutions shown to uniquely enhance b12 binding. Whereas b12 binding was maintained or increased, binding by five nonneutralizing anti-CD4bs MAbs (b3, b6, F105, 15e, and F91) was reduced or completely abolished. These reengineered gp120s are prospective immunogens that may prove capable of eliciting broadly neutralizing antibodies.     

Mutations designed to destabilize the receptor-bound conformation increase MICA-NKG2D association rate and affinity

MICA is a major histocompatibility complex-like protein that undergoes a structural transition from disorder to order upon binding its immunoreceptor, NKG2D. We redesigned the disordered region of MICA with RosettaDesign to increase NKG2D binding. Mutations that stabilize this region were expected to increase association kinetics without changing dissociation kinetics, increase affinity of interaction, and reduce entropy loss upon binding. MICA mutants were stable in solution, and they were amenable to surface plasmon resonance evaluation of NKG2D binding kinetics and thermodynamics. Several MICA mutants bound NKG2D with enhanced affinity, kinetic changes were primarily observed during association, and thermodynamic changes in entropy were as expected. However, none of the 15 combinations of mutations predicted to stabilize the receptor-bound MICA conformation enhanced NKG2D affinity, whereas all 10 mutants predicted to be destabilized bound NKG2D with increased on-rates. Five of these had affinities enhanced by 0.9-1.8 kcal/mol over wild type by one to three non-contacting substitutions. Therefore, in this case, mutations designed to mildly destabilize a protein enhanced association and affinity.     

Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding

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.     

Distribution of drug resistance mutations in type 3 poliovirus identifies three regions involved in uncoating functions.

We have previously described the use of an uncoating inhibitor, WIN 51711, to select drug-resistant mutants of the Sabin strain of poliovirus type 3. Two-thirds of the mutants proved to be dependent on the drug for plaque formation because of extreme thermolability (A. G. Mosser and R. R. Rueckert, J. Virol. 67:1246-1254, 1993). Here we report the responsible mutations; all were traced to single amino acid substitutions. Mutations conferring dependence and thermolability occurred in all four capsid proteins (VP1 to VP4), but all were clustered near residue 53 of VP4 at the inner capsid surface. Amino acid substitutions of the remaining non-drug-dependent mutants were mapped to three distinct loci: (i) on or near the inner capsid surface, at VP4 residue 46 or VP1 residue 129, in the vicinity of the drug dependence substitutions; (ii) at residues 192, 194, and 260 in the lining of the VP1 beta barrel, which is the drug-binding site; and (iii) at VP1 residue 105 on the edge of the canyon surrounding the fivefold axis of symmetry, the putative receptor-binding site. All of the mutations increased the eclipse rate of cell-attached virus. Such mutants help identify parts of the capsid that play a role in viral uncoating functions.     

Targeted Mutagenesis at Charged Residues in <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> BinA Toxin Affects Mosquito-Larvicidal Activity

The mosquito-larvicidal binary toxin from Bacillus sphaericus is composed of two polypeptides called BinA and BinB with molecular masses of approximately 42 and 51 kDa. Both components are required for full activity, with BinB acting as a specificity determinant and BinA being responsible for toxic action. To investigate the role of the selected charged residues in BinA, four mutants were generated by replacing charged amino acids with alanine (R97A, E98A, R101A, and E114A). All mutant proteins were produced at high levels and formed inclusion bodies similar to that of the wild type. Mosquito-larvicidal assays against Culex quinquefasciatus larvae revealed that the mutant R97A completely lost its activity and mutants E98A, R101A, and E114A showed significantly reduced toxicity. Intrinsic fluorescence spectroscopy analysis indicated that alanine substitutions at these positions did not alter the overall structure of the toxin. Binding of the mutants to BinB was not different from that of the wild type, suggesting that these mutations did not affect BinA-BinB interaction. Results demonstrated that R97, E98, R101, and E114 neither play a direct role in maintenance of BinA structure nor are involved in BinA-BinB interaction. Since these residues are required for full activity, they may play an important role during toxin internalization and/or toxic action of BinA inside the target cells.     

Infection with adeno-associated virus type 5 inhibits mutagenicity of herpes simplex virus type 1 or 4-nitroquinoline-1-oxide

Infection with adeno-associated virus type 5 (AAV-5) reduced the number of mutants arising in the hypoxanthine phosphoribosyltransferase locus of human RD 176 cells after infection with herpes simplex virus type 1 (HSV-1; partially inactivated) or 4-nitroquinoline-1-oxide (4-NQO). The mutation frequency was reduced by AAV-5 infection from 11.4 to 1.8 after mutation with HSV-1 and from 3.2 to 2.5 when mutation was induced by 4-NQO. This was analyzed by determination of the number of cells resistant to 8-azaguanine when infected with AAV-5 prior to induction of mutations with HSV-1 or 4-NQO.     

Alanine Scan of Core Positions in Ubiquitin Reveals Links between Dynamics,Stability, and Function

Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. Both the two null mutants (I30A and L43A) were less stable to temperature-induced unfolding in vitro than wild type (WT) but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to WT. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high-molecular-weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high-molecular-weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings, we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation.     

Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases

The effects of various protoporphyrinogen oxidase (PPOX) mutations responsible for variegate porphyria (VP), the roles of the arginine-59 residue and the glycines in the conserved flavin binding site, in catalysis and/or cofactor binding, were examined. Wild-type recombinant human PPOX and a selection of mutants were generated, expressed, purified and partially characterised. All mutants had reduced PPOX activity to varying degrees. However, the activity data did not correlate with the ability/inability to bind flavin. The positive charge at arginine-59 appears to be directly involved in catalysis and not in flavin-cofactor binding alone. The K(m)s for the arginine-59 mutants suggested a substrate-binding problem. T(1/2) indicated that arginine-59 is required for the integrity of the active site. The dominant alpha-helical content was decreased in the mutants. The degree of alpha-helix did not correlate linearly with T(1/2) nor T(m) values, supporting the suggestion that arginine-59 is important for catalysis at the active site. Examination of the conserved dinucleotide-binding sequence showed that substitution of glycine in codon 14 was less disruptive than substitutions in codons 9 and 11. Ultraviolet melting curves generally showed a two-state transition suggesting formation of a multi-domain structure. All mutants studied were more resistant to thermal denaturation compared to wild type, except for R168C.     

Control of adeno-associated virus type 2 cap gene expression: relative influence of helper virus, terminal repeats, and Rep proteins.

Adeno-associated virus type 2 (AAV-2) gene expression is tightly controlled by functions of the helper virus as well as by the products of its own viral rep gene. Double-immunofluorescence studies of Rep and VP protein expression in cells coinfected with AAV-2 and adenovirus type 2 showed that a large proportion of these cells expressed Rep78 and Rep52 but no capsid proteins. The percentage of Rep78/Rep52- and capsid protein-positive cells was strongly influenced by the relative ratio of AAV-2 to adenovirus type 2. In contrast, nearly all cells positive for Rep68/Rep40 were also positive for capsid protein expression. Examination of p40 promoter transactivation by individual Rep proteins in the presence of adenovirus, however, showed that both Rep78 and Rep68 efficiently stimulated p40 mRNA accumulation and capsid protein expression. This strong transactivation was reliant upon the presence of terminal repeats and correlated with template amplification. In replication-deficient expression constructs, transactivation was observed only with Rep68 and was dependent on the linear Rep binding site within the left terminal repeat which was detected in the presence of high adenovirus concentrations. In the absence of any terminal repeat sequences, Rep68 expression again led to a minor transactivation of capsid protein expression which was detectable only at low adenovirus concentrations. This low level of transactivation of capsid protein expression by Rep proteins in the absence of terminal repeats resulted in a lower efficiency of capsid assembly. The data show a dominant influence of adenovirus type 2 functions on AAV-2 gene expression, a requirement for terminal repeats for strong transactivation of the p40 promoter by Rep proteins, and differential influences of Rep78 and Rep68 on AAV-2 promoters. Implications for the production of recombinant AAV-2 vectors are discussed.     

Substitutions in the hydrophobic core of the alpha-factor receptor of Saccharomyces cerevisiae permit response to Saccharomyces kluyveri alpha-factor and to antagonist.

Mutations in the Saccharomyces cerevisiae alpha-factor receptor that lead to improved response to Saccharomyces kluyveri alpha-factor were identified and sequenced. Mutants were isolated from cells bearing randomly mutagenized receptor gene (STE2) plasmids by an in vivo screen. Five mutations lead to substitutions in hydrophobic segments in the core of the receptor (M54I, S145L, S145L-S219L, A229V, L255S-S288P). Remarkably, strains expressing these mutant receptors exhibited positive pheromone responses to desTrp1,Ala3-alpha-factor, an analog that normally blocks these responses. The M54I mutation appeared to affect only ligand specificity. The other mutations conferred additional effects on signaling or recovery. Two mutants were more sensitive to alpha-factor than wild type (S145L, A229V). One mutant was more sensitive to alpha-factor-induced cell cycle arrest initially, but then recovered more efficiently (S145L-S219L). One mutant (L255S-S288P) conferred positive pheromone responses to alpha-factor as assayed by FUS1-lacZ reporter induction, but did not display growth arrest. The hydrophobic receptor core thus appears to control activation by some ligands and to play roles in aspects of signal transduction and recovery.     

Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human alpha-tropomyosin.

Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+. However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.     

Cloning and characterization of a bovine adeno-associated virus

To better understand the relationship between primate adeno-associated viruses (AAVs) and those of other mammals, we have cloned and sequenced the genome of an AAV found as a contaminant in two isolates of bovine adenovirus that was reported to be serologically distinct from primate AAVs. The bovine AAV (BAAV) genome has 4,693 bp, and its organization is similar to that of other AAV isolates. The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively). However, the right-hand ORF was only 55% identical to the AAV-5 capsid ORF; it had the highest homology with the capsid ORF of AAV-4 (76%). By comparing the BAAV cap sequence with a model of an AAV-4 capsid, we mapped the regions of BAAV VP1 that are divergent from AAV-4. These regions are located on the outside of the capsid and are partially located in exposed loops. BAAV was not neutralized by antisera raised against recombinant AAV-2, AAV-4, or AAV-5, and it demonstrated a unique cell tropism profile in four human cancer cell lines, suggesting that BAAV might have transduction activity distinct from that of other isolates. A murine model of salivary gland gene transfer was used to evaluate the in vivo performance of recombinant BAAV. Recombinant BAAV-mediated gene transfer was 11 times more efficient than that with AAV-2. Overall, these data suggest that vectors based on BAAV could be useful for gene transfer applications.