[show abstract][hide abstract] ABSTRACT: Interactions between the herpesvirus entry mediator (HVEM) and the B- and T-lymphocyte attenuator (BTLA) inhibit B and T cell activation. HVEM-BTLA interactions are blocked by herpes simplex virus (HSV) glycoprotein D (gD) through binding of its N-terminal domain to the BTLA binding site of HVEM. In this study, we inserted viral antigens into the C-terminal domain of gD and expressed these antigens with plasmid or E1-deleted (replication-defective) adenovirus vectors. Viral antigens fused to gD induced T and B cell responses to the antigen that were far more potent than those elicited by the same antigen expressed without gD. The immunopotentiating effect required binding of the gD chimeric protein to HVEM. Overall, the studies demonstrate that targeting of antigen to the BTLA binding site of HVEM augments the immunogenicity of vaccines.
Nature medicine 03/2008; 14(2):205-12. · 27.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: Primary cilia play critical roles in many aspects of biology. Specialized versions of primary cilia are involved in many aspects of sensation. The single photoreceptor sensory cilium (PSC) or outer segment elaborated by each rod and cone photoreceptor cell of the retina is a classic example. Mutations in genes that encode cilia components are common causes of disease, including retinal degenerations. The protein components of mammalian primary and sensory cilia have not been defined previously. Here we report a detailed proteomics analysis of the mouse PSC complex. The PSC complex comprises the outer segment and its cytoskeleton, including the axoneme, basal body, and ciliary rootlet, which extends into the inner segment of photoreceptor cells. The PSC complex proteome contains 1968 proteins represented by three or more unique peptides, including approximately 1500 proteins not detected in cilia from lower organisms. This includes 105 hypothetical proteins and 60 proteins encoded by genes that map within the critical intervals for 23 inherited cilia-related disorders, increasing their priority as candidate genes. The PSC complex proteome also contains many cilia proteins not identified previously in photoreceptors, including 13 proteins produced by genes that harbor mutations that cause cilia disease and seven intraflagellar transport proteins. Analyses of PSC complexes from rootletin knock-out mice, which lack ciliary rootlets, confirmed that 1185 of the identified PSC complex proteins are derived from the outer segment. The mass spectrometry data, benchmarked by 15 well characterized outer segment proteins, were used to quantify the copy number of each protein in a mouse rod outer segment. These results reveal mammalian cilia to be several times more complex than the cilia of unicellular organisms and open novel avenues for studies of how cilia are built and maintained and how these processes are disrupted in human disease.
[show abstract][hide abstract] ABSTRACT: To identify early changes in vascular gene expression mediated by CD44 that promote atherosclerotic disease in apolipoprotein E (apoE)-deficient (apoE-/-) mice.
We demonstrate that CD44 is upregulated and functionally activated in aortic arch in the atherogenic environment of apoE-/- mice relative to wild-type (C57BL/6) controls. Moreover, CD44 activation even in apoE-/- mice is selective to lesion-prone regions because neither the thoracic aorta from apoE-/- mice nor the aortic arch of C57BL/6 mice exhibited upregulation of CD44 compared with thoracic aorta of CD57BL/6 mice. Consistent with these observations, gene expression profiling using cDNA microarrays and quantitative polymerase chain reaction revealed that approximately 155 of 19,200 genes analyzed were differentially regulated in the aortic arch, but not in the thoracic aorta, in apoE-/- CD44-/- mice compared with apoE-/- CD44+/+ mice. However, these genes were not regulated by CD44 in the context of a C57BL/6 background, illustrating the selective impact of CD44 on gene expression in a proatherogenic environment. The patterns of differential gene expression implicate CD44 in focal adhesion formation, extracellular matrix deposition, and angiogenesis, processes critical to atherosclerosis.
CD44 is an early mediator of atherogenesis by virtue of its ability to regulate vascular gene expression in response to a proatherogenic environment.
[show abstract][hide abstract] ABSTRACT: Virus-specific neutralizing antibodies present an obstacle to the effective use of adenovirus vectors for gene therapy and vaccination. The specific sites recognized by neutralizing antibodies have not been identified for any adenovirus, but they have been proposed to reside within the hexon, in small regions of the molecule that are exposed on the capsid surface and possess sequences that vary among serotypes. We have mapped the epitopes recognized by a panel of seven hexon-specific monoclonal antibodies that neutralize the chimpanzee adenovirus 68 (AdC68). Surface plasmon resonance experiments revealed that the antibodies compete for a single hexon binding site, and experiments with synthetic peptides indicated that this site resides within just one small surface loop. Mutations within this loop (but not in other surface loops) permitted virus to escape neutralization by all seven monoclonal antibodies and to resist neutralization by polyclonal antisera obtained from animals immunized against AdC68. These results indicate that a single small surface loop defines a major neutralization site for AdC68 hexon.
Journal of Virology 03/2007; 81(4):1680-9. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: This chapter provides a protocol for the large-scale purification of adenovirus type 2 and 5 virions and the soluble major coat protein hexon. The purified virus particles remain intact and are suitable for vector, vaccine, or structural studies and can also be used as seed stock for further rounds of infection. The hexon may be used to produce crystals suitable for high-resolution X-ray crystallographic studies. Briefly, virus is propagated in HeLa cell suspension cultures. The infected cells are lysed, virions and hexon are separated by centrifugation, and the protein is then further purified by anion exchange chromatography. The entire purification procedure takes approx 1 wk and typically yields 10(13) virus particles and 10-20 mg of highly purified hexon.
Methods in molecular medicine 02/2007; 131:231-50.
[show abstract][hide abstract] ABSTRACT: The apolipoprotein E (ApoE) gene has been linked to maladies such as hypercholesterolemia, CNS injury and disease. In this study, we present evidence that, in addition to the known transcript (ApoE S1) that translates into ApoE, there are three additional transcripts in mice. Two of these transcripts, ApoE S2 and ApoE S3, which are predicted to be transmembrane proteins, are transcribed from the sense strand. ApoE AS1 is transcribed from the antisense strand and is complementary to exon 4 of ApoE S1. The open reading frame of ApoE AS1 is conserved between human and mouse. The antisense transcript falls within the region of the human epsilon 4 allele that has been linked to the familial onset form of Alzheimer's disease. We also demonstrate the expression of ApoE S3 and ApoE AS1 in ApoE knockout mice, and ApoE S1 and ApoE S2 do not get transcribed. We had previously identified ApoE S1 as being upregulated in mice after spinal cord injury. In this study, we show that in spinal cord-injured C57BL/6 mice, both ApoE S1 and ApoE S3 transcripts are 10-fold upregulated and the antisense ApoE AS1 is 100-fold upregulated compared with normal levels. Such data suggest that these alternate transcripts are involved in the molecular pathogenesis of CNS disease and perhaps in ApoE expression in general, as we show that ApoE S2 and AS1 are also transcribed in human.
Human Molecular Genetics 10/2005; 14(18):2661-70. · 7.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: A novel strategy, termed protein array pixelation, is described for comprehensive profiling of human plasma and serum proteomes. This strategy consists of three sequential high-resolution protein prefractionation methods (major protein depletion, solution isoelectrofocusing, and 1-DE) followed by nanocapillary RP tryptic peptide separation prior to MS/MS analysis. The analysis generates a 2-D protein array where each pixel in the array contains a group of proteins with known pI and molecular weight range. Analysis of the HUPO samples using this strategy resulted in 575 and 2890 protein identifications from plasma and serum, respectively, based on HUPO-approved criteria for high-confidence protein assignments. Most importantly, a substantial number of low-abundance proteins (low ng/mL - pg/mL range) were identified. Although larger volumes were used in initial prefractionation steps, the protein identifications were derived from fractions equivalent to approximately 0.6 microL (45 microg) of plasma and 2.4 microL (204 microg) of serum. The time required for analyzing the entire protein array for each sample is comparable to some published shotgun analyses of plasma and serum proteomes. Therefore, protein array pixelation is a highly sensitive method capable of detecting proteins differing in abundance by up to nine orders of magnitude. With further refinement, this method has the potential for even higher capacity and higher throughput.
[show abstract][hide abstract] ABSTRACT: Structural studies continue to play an essential role as the focus of adenovirus research shifts in emphasis from basic biology to adenovirus-based vector technologies. A crucial step in developing novel therapeutics for gene replacement, cancer, and vaccines is often to modify the virion. Such engineered changes are designed to retarget the virus, or to reduce the immunological responses to infection. These efforts are far more effective when they are based on detailed structural knowledge. This minireview provides a brief summary of the wealth of information that has been obtained from the combined application of X-ray crystallography and electron microscopy. This knowledge now includes a good working model for the architectural organization of the virion, and atomic resolution molecular structures for all the major capsid proteins, hexon, penton, and fiber. We highlight new developments, which include the structure of the penton base and the discovery that adenovirus has several relatives. We sketch how the structural information can be used to engineer novel virions and conclude with the prospects for future progress.
Human Gene Therapy 01/2005; 15(12):1167-76. · 4.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Small interfering RNA (siRNA) is used in functional genomics applications to decrease the expression of a target gene, which may yield a biological effect that suggests a function for the target gene. The siRNA design tool scans a target gene for candidate siRNA sequences that satisfy user-adjustable rules. Selected candidates are then screened to identify those siRNA sequences that are specific to the gene of interest.
[show abstract][hide abstract] ABSTRACT: A major impediment to the use of adenovirus as a gene therapy vector and for vaccine applications is the host immune response to adenovirus hexon-the major protein component of the icosahedral capsid. A solution may lie in novel vectors with modified or chimeric hexons designed to evade the immune response. To facilitate this approach, we have distinguished the portion of hexon that all serotypes have in common from the hypervariable regions that are responsible for capsid diversity and type-specific immunogenicity. The common hexon core-conserved because it forms the viral capsid-sets boundaries to the regions where modifications can be made to produce nonnative hexons. The core has been defined from the large and diverse set of known hexon sequences by an accurate alignment based on the newly refined crystal structures of human adenovirus types 2 (Ad2) and Ad5 hexon. Comparison of the two hexon models, which are the most accurate so far, reveals that over 90% of the residues in each have three-dimensional positions that closely match. Structures for more distant hexons were predicted by building molecular models of human Ad4, chimpanzee adenovirus (AdC68), and fowl adenovirus 1 (FAV1 or CELO). The five structures were then used to guide the alignment of the 40 full-length (>900 residues) hexon sequences in public databases. Distance- and parsimony-based phylogenetic trees are consistent and reveal evolutionary relationships between adenovirus types that parallel those of their animal hosts. The combination of crystallography, molecular modeling, and phylogenetic analysis defines a conserved molecular core that can serve as the armature for the directed design of novel hexons.
Journal of Virology 09/2003; 77(17):9553-66. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adeno-associated viruses (AAVs) are single-stranded DNA viruses that are endemic in human populations without known clinical sequelae and are being evaluated as vectors for human gene therapy. To better understand the biology of this virus, we examined a number of nonhuman primate species for the presence of previously uncharacterized AAVs and characterized their structure and distribution. AAV genomes were widely disseminated throughout multiple tissues of a variety of nonhuman primate species. Surprising diversity of sequence, primarily localized to hypervariable regions of the capsid protein, was detected. This diversity of sequence is caused, in part, by homologous recombination of co-infecting parental viruses that modify the serologic reactivity and tropism of the virus. This is an example of rapid molecular evolution of a DNA virus in a way that was formerly thought to be restricted to RNA viruses.
Proceedings of the National Academy of Sciences 06/2003; 100(10):6081-6. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rab3a is the most abundant Rab (ras-associated binding) protein in the brain and has a regulatory role in synaptic vesicle trafficking. Mice with a targeted loss-of-function mutation in Rab3a have defects in Ca(2+)-dependent synaptic transmission: the number of vesicles released in response to an action potential is greater than in wildtype mice, resulting in greater synaptic depression and the abolishment of CA3 mossy-fiber long term potentiation. The effect of these changes on behavior is unknown. In a screen for mouse mutants with abnormal rest-activity and sleep patterns, we identified a semidominant mutation, called earlybird, that shortens the circadian period of locomotor activity. Sequence analysis of Rab3a identified a point mutation in the conserved amino acid (Asp77Gly) within the GTP-binding domain of this protein in earlybird mutants, resulting in significantly reduced levels of Rab3a protein. Phenotypic assessment of earlybird mice and a null allele of Rab3a revealed anomalies in circadian period and sleep homeostasis, providing evidence that Rab3a-mediated synaptic transmission is involved in these behaviors.
[show abstract][hide abstract] ABSTRACT: More than 220 Kruppel-associated box-zinc finger protein (KRAB-ZFP) genes are encoded in the human genome. KRAB-ZFPs function as transcriptionalrepressors by binding DNA through their tandem zinc finger motifs.Gene silencing is mediated by the highly conserved KRAB domain, which recruits histone deacetylase complexes, histone methylases, and heterochromatin proteins. However, little is known of the biological programs regulated by KRAB-ZFPs, in large part because of the difficulty in identifying DNA-binding sites recognized by long arrays of zinc fingers. In an attempt to identify the natural target genes for a KRAB-ZFP, we chose SZF1, a hematopoietic progenitor-restricted, KRAB-ZFP that contains only four C(2)H(2) zinc finger motifs. Using recombinant SZF1 protein and a PCR-based binding site selection strategy, we identified a 15-bp consensus DNA sequence recognized by SZF1. Remarkably, this sequence is similar to the core DNA-binding site described recently for ZBRK1, a KRAB-ZFP that binds to BRCA1 and is involved in coordinating the cellular DNA damage response. The SZF1 and ZBRK1 proteins bind to both the experimentally derived SZF1 site and the canonical ZBRK1 site. The KRAB domain from SZF1 bound directly to the KAP-1 corepressor and displayed intrinsic silencing activity. Moreover, full-length SZF1 repressed a promoter containing ZBRK1 recognition sequences. Thus, SZF1 and ZBRK1 may regulate a common set of target genes in vivo.
Cancer Research 08/2002; 62(13):3773-81. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.
Journal of Virology 01/2002; 75(23):11603-13. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: A major obstacle to the use of adenovirus as a vector for gene therapy is the host immune response to hexon, the major protein component of the icosahedral capsid. A solution lies in creating novel vectors with modified or chimeric hexons to evade the immune response to native hexon. The crystal structure of hexon from human adenovirus type 5 (ad5), the type primarily used for gene therapy, has been determined to facilitate the design of such molecules. As the 951-amino-acid (aa) ad5 hexon sequence is closely related to that of ad2 (967 aa; 86% aa identity), the ad5 structure was solved by molecular replacement with a model constructed from the known ad2 hexon. During refinement, greater than 25% of the sequence was reassigned, resulting in a relocation of two epitope regions, from buried positions in the ad2 model to external sites at the top of the ad5 molecule. The resultant model is in better agreement with crystallographic data, while maintaining the overall topology of ad2 hexon. This work suggests that all hexons have the same basic fold and that the ad5 hexon structure provides an accurate and representative model for designing new adenovirus vectors.
[show abstract][hide abstract] ABSTRACT: In the past two years, structural studies on spherical viruses have experienced a significant advance with the dramatic increase in the resolution attainable by cryo-electron microscopy and image reconstruction. X-ray crystallography, both alone and, increasingly, in combination with electron microscopy, continues to play a crucial role in elucidating how viruses function.
Current Opinion in Structural Biology 05/1998; 8(2):142-9. · 8.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: An Fab fragment of a virus-neutralizing monoclonal antibody (DL11) that binds to herpes simplex virus glycoprotein D (HSV gD) has been purified, sequenced and crystallized. The biological activity of the purified Fab was verified by enzyme-linked immunosorbant assay, flow cytometry and by neutralization of HSV infectivity. The crystals have the space group P1 with cell dimensions a = 40.2, b = 49.2, c = 63.9 A, alpha = 76.1, beta = 77.4, gamma = 71.6 degrees. The unit-cell volume is consistent with it containing a single Fab molecule. The crystals grow to a maximum size of 0.7 x 0.3 x 0.3 mm and diffract X-rays to greater than 2.2 A resolution. The amino-acid sequences of the variable regions of the heavy and light chains of DL11 have been determined. These have been compared to those for other known Fab structures in the Protein Data Bank for selection of a starting model for crystallographic refinement by the molecular-replacement method.
[show abstract][hide abstract] ABSTRACT: The crystal structure of hexon, the major coat protein from adenovirus type 2, has been refined at 2.9 A resolution. Hexon is a homo-trimer (molecular mass 3 x 109,077 Da) and crystallizes in the cubic space group P2(1)3, with a cell edge of 150.5 A. There are four molecules in the unit cell so that the crystallographic asymmetric unit contains one subunit of the trimer. The electron density in most regions is well-defined and 880 amino acid residues, of the 967 in this unusually long polypeptide chain, have been located and fitted. The N terminus (1 to 43) and three internal stretches (192 to 203, 270 to 291 and 444 to 453) are not defined, and a stretch (168 to 207) with unclear side-chain density is modelled as poly(Ala/Gly). The current refined model, consisting of 6943 non-hydrogen protein atoms and 85 water molecules, yields an R-factor of 19.9% for 18,176 reflections in the resolution range 5.0 to 2.9 A. The model has reasonable geometry with root-mean-square deviations from ideal bond lengths of 0.022 A and angle-related 1-3 distances of 0.056 A. The overall shape of the trimeric hexon molecule is unusual and may be divided into a pseudo-hexagonal base rich in beta-structure, and a triangular top formed from three long loops containing some secondary structure. The base contains two similar pedestal domains, P1 and P2, each of which is a flattened eight-stranded beta-barrel with the "jelly-roll greek key" topology characteristic of other viral coat proteins. P1 and P2 are related by an approximate 6-fold operation about the molecular 3-fold axis so that six barrels form the walls of the tubular hexon base. The hexon bases form close-packed p3 arrays on each facet of the icosahedral adenovirus virion. Unlike other viral capsids, the barrel axes are almost perpendicular to rather than parallel with the capsid surface. The hexon top, which consists of intimately interacting loops emerging from P1 and P2 in the base, has a triangular outline and so does not exhibit the pseudo-symmetry of the base. The structure of the hexon trimer shows how economically it meets the demands of its function as a stable protective viral coat, reveals the significance of the special features in its unusual amino acid sequence, and explains its biochemical and immunological properties. The molecule is hollow, with a large central cavity, and so has a high effective volume for its mass.(ABSTRACT TRUNCATED AT 400 WORDS)
Journal of Molecular Biology 10/1994; 242(4):430-55. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The crystal structure of hexon, the major coat protein from adenovirus type 2, has been refined at 2·9 Å resolution. Hexon is a homo-trimer (molecular mass 3×109,077 Da) and crystallizes in the cubic space group P 21 3, with a cell edge of 150·5 Å. There are four molecules in the unit cell so that the crystallographic asymmetric unit contains one subunit of the trimer. The electron density in most regions is well-defined and 880 amino acid residues, of the 967 in this unusually long polypeptide chain, have been located and fitted. The N terminus (1 to 43) and three internal stretches (192 to 203, 270 to 291 and 444 to 453) are not defined, and a stretch (168 to 207) with unclear side-chain density is modelled as poly(Ala/Gly). The current refined model, consisting of 6943 non-hydrogen protein atoms and 85 water molecules, yields an R-factor of 19·9% for 18,176 reflections in the resolution range 5·0 to 2·9 Å. The model has reasonable geometry with root-mean-square deviations from ideal bond lengths of 0·022 Å and angle-related 1-3 distances of 0·056 Å.The overall shape of the trimeric hexon molecule is unusual and may be divided into a pseudo-hexagonal base rich in β-structure, and a triangular top formed from three long loops containing some secondary structure. The base contains two similar pedestal domains, P1 and P2, each of which is a flattened eight-stranded β-barrel with the "jelly-roll greek key" topology characteristic of other viral coat proteins. P1 and P2 are related by an approximate 6-fold operation about the molecular 3-fold axis so that six barrels form the walls of the tubular hexon base. The hexon bases form close-packed p3 arrays on each facet of the icosahedral adenovirus virion. Unlike other vital capsids, the barrel axes are almost perpendicular to rather than parallel with the capsid surface. The hexon top, which consists of intimately interacting loops emerging from P1 and P2 in the base, has a triangular outline and so does not exhibit the pseudo-symmetry of the base.The structure of the hexon trimer shows how economically it meets the demands of its function as a stable protective viral coat, reveals the significance of the special features in its unusual amino acid sequence, and explains its biochemical and immunological properties. The molecule is hollow, with a large central cavity, and so has a high effective volume for its mass. Hydrophobic residues are clustered together in the interior of P1 and P2, and at the central depression in the top of the molecule. The latter is a "hydrophobic plug" that seals the central cavity and so shields the viral interior from the outside environment. Intra and intersubunit salt bridges, hydrogen bonds and hydrophobic contacts all contribute to the extreme stability of hexon. In the trimer, each hexon subunit is locked in position to confer additional stability. The interaction of the loops at the top mad the N-terminal arm at the bottom prevents any relative upward or downward displacement of a subunit with respect to its neighbors. The intricate topology of the subunits suggests why their correct folding into the trimeric molecule requires the initial formation of a complex of hexon polypeptides with the adenovirus 100 K protein.
Journal of Molecular Biology - J MOL BIOL. 01/1994; 242(4):430-455.