Hepatitis C virus (HCV) cannot be grown in vitro, making biochemical identification of new drug targets especially important. HCV p7 is a small hydrophobic protein of unknown function, yet necessary ...for particle infectivity in related viruses Harada, T. et al., (2000) J. Virol. 74, 9498–9506. We show that p7 can be cross-linked in vivo as hexamers. Escherichia coli expressed p7 fusion proteins also form hexamers in vitro. These and HIS-tagged p7 function as calcium ion channels in black lipid membranes. This activity is abrogated by Amantadine, a compound that inhibits ion channels of influenza Hay, A.J. et al. (1985) EMBO J. 4, 3021–3024; Duff, K.C. and Ashley, R.H. (1992) Virology 190, 485–489 and has recently been shown to be active in combination with current HCV therapies.
Dimerization of retroviral genomic RNA is essential for efficient viral replication and is mediated by structural interactions between identical RNA motifs in the viral leader region. We have ...visualized, by electron microscopy, RNA dimers formed from the leader region of the prototype lentivirus, maedi visna virus. Characterization by in vitro assays of the domains responsible for this interaction has identified a 20 nucleotide sequence that functions as the core dimerization initiation site. This region is predicted to form a GACG tetraloop and therefore differs significantly from the kissing loop palindromes utilized to initiate dimerization in primate lentiviruses. The motif is strongly conserved across the ovine and caprine lentiviruses, implying a critical functional role. Furthermore, the proposed GACG tetraloop exhibits marked structural homology with similar structural motifs present in the leader regions of the α- and γ-retroviruses, and the maedi visna virus dimer linkage region is capable of forming heterodimeric species with the Moloney murine leukemia virus Ψ domain. This may be indicative of commonality of origin of the two viruses or convergent evolution.
Translation of hepatitis C virus (HCV) RNA is initiated
via the internal ribosome entry site (IRES), located
within the 5′ untranslated region. Although the secondary
structure of this element has ...been predicted, little information
on the tertiary structure is available. Here we report
the first structural characterization of the HCV IRES using
electron microscopy. In vitro transcribed RNA appeared
as particles with characteristic morphology and gold labeling
using a specific oligonucleotide confirmed them to be HCV
IRES. Dimerization of the IRES by hybridization with tandem
repeat oligonucleotides allowed the identification of domain
III and an assignment of domains II and IV to distinct
regions within the molecule. Using immunogold labeling,
the pyrimidine tract binding protein (PTB) was shown to
bind to domain III. Structure–function relationships
based on the flexible hinge between domains II and III
are suggested. Finally, the architecture of the HCV IRES
was seen to be markedly different from that of a picornavirus,
foot-and-mouth disease virus (FMDV).
The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major ...insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.
By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. ...Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.
Congenital heart defects (CHDs) are the most common birth defect worldwide and are a leading cause of neonatal mortality. Nonsyndromic atrioventricular septal defects (AVSDs) are an important subtype ...of CHDs for which the genetic architecture is poorly understood. We performed exome sequencing in 13 parent-offspring trios and 112 unrelated individuals with nonsyndromic AVSDs and identified five rare missense variants (two of which arose de novo) in the highly conserved gene NR2F2, a very significant enrichment (p = 7.7 × 10−7) compared to 5,194 control subjects. We identified three additional CHD-affected families with other variants in NR2F2 including a de novo balanced chromosomal translocation, a de novo substitution disrupting a splice donor site, and a 3 bp duplication that cosegregated in a multiplex family. NR2F2 encodes a pleiotropic developmental transcription factor, and decreased dosage of NR2F2 in mice has been shown to result in abnormal development of atrioventricular septa. Via luciferase assays, we showed that all six coding sequence variants observed in individuals significantly alter the activity of NR2F2 on target promoters.
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