John J. Rux

Adeno-associated viruses undergo substantial evolution in primates during natural infections

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.

The Proteome of the Mouse Photoreceptor Sensory Cilium Complex

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 ∼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.

Replication-Defective Vector Based on a Chimpanzee Adenovirus

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.

Structural and Phylogenetic Analysis of Adenovirus Hexons by Use of High-Resolution X-Ray Crystallographic, Molecular Modeling, and Sequence-Based Methods

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.

Gene specific siRNA selector

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.

Mutations in Rab3a alter circadian period and homeostatic response to sleep loss in the mouse.

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 Ca2+-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.

Structure-Based Identification of a Major Neutralizing Site in an Adenovirus Hexon

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.

Genome wide ChIP-chip analyses reveal important roles for CTCF in Drosophila genome organization.

Insulators or chromatin boundary elements are defined by their ability to block transcriptional activation by an enhancer and to prevent the spread of active or silenced chromatin. Recent studies have increasingly suggested that insulator proteins play a role in large-scale genome organization. To better understand insulator function on the global scale, we conducted a genome-wide analysis of the binding sites for the insulator protein CTCF in Drosophila by Chromatin Immunoprecipitation (ChIP) followed by a tiling-array analysis. The analysis revealed CTCF binding to many known domain boundaries within the Abd-B gene of the BX-C including previously characterized Fab-8 and MCP insulators, and the Fab-6 region. Based on this finding, we characterized the Fab-6 insulator element. In genome-wide analysis, we found that dCTCF-binding sites are often situated between closely positioned gene promoters, consistent with the role of CTCF as an insulator protein. Importantly, CTCF tends to bind gene promoters just upstream of transcription start sites, in contrast to the predicted binding sites of the insulator protein Su(Hw). These findings suggest that CTCF plays more active roles in regulating gene activity and it functions differently from other insulator proteins in organizing the Drosophila genome.

Targeting of antigen to the herpesvirus entry mediator augments primary adaptive immune responses

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.

BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery

Quantitative analyses of melanosome cargo localization and trafficking and of endosomal membrane dynamics in immortalized melanocytes from mouse Hermansky–Pudlak syndrome models show that BLOC-2 functions to specify the delivery of recycling endosomal cargo transport intermediates to maturing melanosomes.