An ARL3-UNC119-RP2 GTPase cycle targets myristoylated NPHP3 to the primary cilium.

The membrane of the primary cilium is a highly specialized compartment that organizes proteins to achieve spatially ordered signaling. Disrupting ciliary organization leads to diseases called ciliopathies, with phenotypes ranging from retinal degeneration and cystic kidneys to neural tube defects. How proteins are selectively transported to and organized in the primary cilium remains unclear. Using a proteomic approach, we identified the ARL3 effector UNC119 as a binding partner of the myristoylated ciliopathy protein nephrocystin-3 (NPHP3). We mapped UNC119 binding to the N-terminal 200 residues of NPHP3 and found the interaction requires myristoylation. Creating directed mutants predicted from a structural model of the UNC119-myristate complex, we identified highly conserved phenylalanines within a hydrophobic ß sandwich to be essential for myristate binding. Furthermore, we found that binding of ARL3-GTP serves to release myristoylated cargo from UNC119. Finally, we showed that ARL3, UNC119b (but not UNC119a), and the ARL3 GAP Retinitis Pigmentosa 2 (RP2) are required for NPHP3 ciliary targeting and that targeting requires UNC119b myristoyl-binding activity. Our results uncover a selective, membrane targeting GTPase cycle that delivers myristoylated proteins to the ciliary membrane and suggest that other myristoylated proteins may be similarly targeted to specialized membrane domains.

Shifting players and paradigms in cell-specific transcription.

Historically, developmental-stage- and tissue-specific patterns of gene expression were assumed to be determined primarily by DNA regulatory sequences and their associated activators, while the general transcription machinery including core promoter recognition complexes, coactivators, and chromatin modifiers was held to be invariant. New evidence suggests that significant changes in these general transcription factors including TFIID, BAF, and Mediator may facilitate global changes in cell-type-specific transcription.

A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium.

Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the << molar tooth sign >>. JS is genetically heterogeneous, involving 20 genes identified to date, which are all required for cilia biogenesis and/or function. In a consanguineous family with JS associated with optic nerve coloboma, kidney hypoplasia, and polydactyly, combined exome sequencing and mapping identified a homozygous splice-site mutation in PDE6D, encoding a prenyl-binding protein. We found that pde6d depletion in zebrafish leads to renal and retinal developmental anomalies and wild-type but not mutant PDE6D is able to rescue this phenotype. Proteomic analysis identified INPP5E, whose mutations also lead to JS or mental retardation, obesity, congenital retinal dystrophy, and micropenis syndromes, as novel prenyl-dependent cargo of PDE6D. Mutant PDE6D shows reduced binding to INPP5E, which fails to localize to primary cilia in patient fibroblasts and tissues. Furthermore, mutant PDE6D is unable to bind to GTP-bound ARL3, which acts as a cargo-release factor for PDE6D-bound INPP5E. Altogether, these results indicate that PDE6D is required for INPP5E ciliary targeting and suggest a broader role for PDE6D in targeting other prenylated proteins to the cilia. This study identifies PDE6D as a novel JS disease gene and provides the first evidence of prenyl-binding-dependent trafficking in ciliopathies.

The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans.

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.

Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome.

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH(2)-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.

Wnt signaling targets ETO coactivation domain of TAF4/TFIID in vivo.

Understanding the diverse activities of the multisubunit core promoter recognition complex TFIID in vivo requires knowledge of how individual subunits contribute to overall functions of this TATA box-binding protein (TBP)/TBP-associated factor (TAF) complex. By generating altered holo-TFIID complexes in Drosophila we identify the ETO domain of TAF4 as a coactivator domain likely targeted by Pygopus, a protein that is required for Wingless-induced transcription of naked cuticle. These results establish a coactivator function of TAF4 and provide a strategy to dissect mechanisms of TFIID function in vivo.

Attenuation of recombinant vesicular stomatitis virus-human immunodeficiency virus type 1 vaccine vectors by gene translocations and g gene truncation reduces neurovirulence and enhances immunogenicity in mice.

Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.

Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert.

Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein.

Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies.

The stem of vesicular stomatitis virus G can be replaced with the HIV-1 Env membrane-proximal external region without loss of G function or membrane-proximal external region antigenic properties.

The structure of the HIV-1 envelope membrane-proximal external region (MPER) is influenced by its association with the lipid bilayer on the surface of virus particles and infected cells. To develop a replicating vaccine vector displaying MPER sequences in association with membrane, Env epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, or both were grafted into the membrane-proximal stem region of the vesicular stomatitis virus (VSV) glycoprotein (G). VSV encoding functional G-MPER chimeras based on G from the Indiana or New Jersey serotype propagated efficiently, although grafting of both epitopes (G-2F5-4E10) modestly reduced replication and resulted in the acquisition of one to two adaptive mutations in the grafted MPER sequence. Monoclonal antibodies 2F5 and 4E10 efficiently neutralized VSV G-MPER vectors and bound to virus particles in solution, indicating that the epitopes were accessible in the preattachment form of the G-MPER chimeras. Overall, our results showed that the HIV Env MPER could functionally substitute for the VSV G-stem region implying that both perform similar functions even though they are from unrelated viruses. Furthermore, we found that the MPER sequence grafts induced low but detectable MPER-specific antibody responses in rabbits vaccinated with live VSV, although additional vector and immunogen modifications or use of a heterologous prime-boost vaccination regimen will be required to increase the magnitude of the immune response.

A novel, live-attenuated vesicular stomatitis virus vector displaying conformationally intact, functional HIV-1 envelope trimers that elicits potent cellular and humoral responses in mice.

Though vaccination with live-attenuated SIV provides the greatest protection from progressive disease caused by SIV challenge in rhesus macaques, attenuated HIV presents safety concerns as a vaccine; therefore, live viral vectors carrying HIV immunogens must be considered. We have designed a replication-competent vesicular stomatitis virus (VSV) displaying immunogenic HIV-1 Env trimers and attenuating quantities of the native surface glycoprotein (G). The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5'terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG. Western blot analysis indicated that ∼75% of incorporated EnvG was a mature proteolytically processed form. Flow cytometry showed that surface EnvG bound various conformationally- and trimer-specific antibodies (Abs), and in-vitro growth assays on CD4+CCR5+ cells demonstrated EnvG functionality. Neither intranasal (IN) or intramuscular (IM) administration in mice induced any observable pathology and all regimens tested generated potent Env-specific ELISA titers of 10(4)-10(5), with an IM VSV prime/IN VSV boost regimen eliciting the highest binding and neutralizing Ab titers. Significant quantities of Env-specific CD4+ T cells were also detected, which were augmented as much as 70-fold by priming with IM electroporated plasmids encoding EnvG and IL-12. These data suggest that our novel vector can achieve balanced safety and immunogenicity and should be considered as an HIV vaccine platform.

Membrane-bound SIV envelope trimers are immunogenic in ferrets after intranasal vaccination with a replication-competent canine distemper virus vector.

We are investigating canine distemper virus (CDV) as a vaccine vector for the delivery of HIV envelope (Env) that closely resembles the native trimeric spike. We selected CDV because it will promote vaccine delivery to lymphoid tissues, and because human exposure is infrequent, reducing potential effects of pre-existing immunity. Using SIV Env as a model, we tested a number of vector and gene insert designs. Vectors containing a gene inserted between the CDV H and L genes, which encoded Env lacking most of its cytoplasmic tail, propagated efficiently in Vero cells, expressed the immunogen on the cell surface, and incorporated the SIV glycoprotein into progeny virus particles. When ferrets were vaccinated intranasally, there were no signs of distress, vector replication was observed in the gut-associated lymphoid tissues, and the animals produced anti-SIV Env antibodies. These data show that live CDV-SIV Env vectors can safely induce anti-Env immune responses following intranasal vaccination.

In vivo biodistribution of a highly attenuated recombinant vesicular stomatitis virus expressing HIV-1 Gag following intramuscular, intranasal, or intravenous inoculation.

Recombinant vesicular stomatitis viruses (rVSVs) are being developed as potential HIV-1 vaccine candidates. To characterize the in vivo replication and dissemination of rVSV vectors in mice, high doses of a highly attenuated vector expressing HIV-1 Gag, rVSV(IN)-N4CT9-Gag1, and a prototypic reference virus, rVSV(IN)-HIVGag5, were delivered intramuscularly (IM), intranasally (IN), or intravenously (IV). We used quantitative, real-time RT-PCR (Q-PCR) and standard plaque assays to measure the temporal dissemination of these viruses to various tissues. Following IM inoculation, both viruses were detected primarily at the injection site as well as in draining lymph nodes; neither virus induced significant weight loss, pathologic signs, or evidence of neuroinvasion. In contrast, following IN inoculation, the prototypic virus was detected in all tissues tested and caused significant weight loss leading to death. IN administration of rVSV(IN)-N4CT9-Gag1 resulted in detection in numerous tissues (brain, lung, nasal turbinates, and lymph nodes) albeit in significantly reduced levels, which caused little or no weight loss nor any mortality. Following IV inoculation, both prototypic and attenuated viruses were detected by Q-PCR in all tissues tested. In contrast to the prototype, rVSV(IN)-N4CT9-Gag1 viral loads were significantly lower in all organs tested, and no infectious virus was detected in the brain following IV inoculation, despite the presence of viral RNA. These studies demonstrated significant differences in the biodistribution patterns of and the associated pathogenicity engendered by the prototypic and attenuated vectors in a highly susceptible host.

TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription.

The RNA polymerase II core promoter is a structurally and functionally diverse transcriptional module. RNAi depletion and overexpression experiments revealed a genetic circuit that controls the balance of transcription from two core promoter motifs, the TATA box and the downstream core promoter element (DPE). In this circuit, TBP activates TATA-dependent transcription and represses DPE-dependent transcription, whereas Mot1 and NC2 block TBP function and thus repress TATA-dependent transcription and activate DPE-dependent transcription. This regulatory circuit is likely to be one means by which biological networks can transmit transcriptional signals, such as those from DPE-specific and TATA-specific enhancers, via distinct pathways.

TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter.

Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. Here, we use RNAi to knock down specific TFIID subunits in Drosophila tissue culture cells to determine which subunits are most critical for maintaining stability of TFIID in vivo. Contrary to expectations, we find that TAF4 rather than TBP or TAF1 plays the most critical role in maintaining stability of the complex. Our analysis also indicates that TAF5, TAF6, TAF9, and TAF12 play key roles in stability of the complex, whereas TBP, TAF1, TAF2, and TAF11 contribute very little to complex stability. Based on our results, we propose that holo-TFIID comprises a stable core subcomplex containing TAF4, TAF5, TAF6, TAF9, and TAF12 decorated with peripheral subunits TAF1, TAF2, TAF11, and TBP. Our initial functional studies indicate a specific and significant role for TAF1 and TAF4 in mediating transcription from a TATA-less, downstream core promoter element (DPE)-containing promoter, whereas a TATA-containing, DPE-less promoter was far less dependent on these subunits. In contrast to both TAF1 and TAF4, RNAi knockdown of TAF5 had little effect on transcription from either class of promoter. These studies significantly alter previous models for the assembly, structure, and function of TFIID.

Coactivator cross-talk specifies transcriptional output.

Cells often fine-tune gene expression at the level of transcription to generate the appropriate response to a given environmental or developmental stimulus. Both positive and negative influences on gene expression must be balanced to produce the correct level of mRNA synthesis. To this end, the cell uses several classes of regulatory coactivator complexes including two central players, TFIID and Mediator (MED), in potentiating activated transcription. Both of these complexes integrate activator signals and convey them to the basal apparatus. Interestingly, many promoters require both regulatory complexes, although at first glance they may seem to be redundant. Here we have used RNA interference (RNAi) in Drosophila cells to selectively deplete subunits of the MED and TFIID complexes to dissect the contribution of each of these complexes in modulating activated transcription. We exploited the robust response of the metallothionein genes to heavy metal as a model for transcriptional activation by analyzing direct factor recruitment in both heterogeneous cell populations and at the single-cell level. Intriguingly, we find that MED and TFIID interact functionally to modulate transcriptional response to metal. The metal response element-binding transcription factor-1 (MTF-1) recruits TFIID, which then binds promoter DNA, setting up a "checkpoint complex" for the initiation of transcription that is subsequently activated upon recruitment of the MED complex. The appropriate expression level of the endogenous metallothionein genes is achieved only when the activities of these two coactivators are balanced. Surprisingly, we find that the same activator (MTF-1) requires different coactivator subunits depending on the context of the core promoter. Finally, we find that the stability of multi-subunit coactivator complexes can be compromised by loss of a single subunit, underscoring the potential for combinatorial control of transcription activation.

Recombinant vesicular stomatitis virus vectors expressing herpes simplex virus type 2 gD elicit robust CD4+ Th1 immune responses and are protective in mouse and guinea pig models of vaginal challenge.

Recombinant vesicular stomatitis virus (rVSV) vectors offer an attractive approach for the induction of robust cellular and humoral immune responses directed against human pathogen target antigens. We evaluated rVSV vectors expressing full-length glycoprotein D (gD) from herpes simplex virus type 2 (HSV-2) in mice and guinea pigs for immunogenicity and protective efficacy against genital challenge with wild-type HSV-2. Robust Th1-polarized anti-gD immune responses were demonstrated in the murine model as measured by induction of gD-specific cytotoxic T lymphocytes and increased gamma interferon expression. The isotype makeup of the serum anti-gD immunoglobulin G (IgG) response was consistent with the presence of a Th1-CD4+ anti-gD response, characterized by a high IgG2a/IgG1 IgG subclass ratio. Functional anti-HSV-2 neutralizing serum antibody responses were readily demonstrated in both guinea pigs and mice that had been immunized with rVSV-gD vaccines. Furthermore, guinea pigs and mice were prophylactically protected from genital challenge with high doses of wild-type HSV-2. In addition, guinea pigs were highly protected against the establishment of latent infection as evidenced by low or absent HSV-2 genome copies in dorsal root ganglia after virus challenge. In summary, rVSV-gD vectors were successfully used to elicit potent anti-gD Th1-like cellular and humoral immune responses that were protective against HSV-2 disease in guinea pigs and mice.