Epigenetic Maternal Effects on Endogenous Rhythms in Precocial Birds

Development involves interactions between genetic and environmental influences. Vertebrate mothers are generally the first individuals to encounter and interact with young animals. Thus, their role is primordial during ontogeny. The present study evaluated non‐genomic effects of mothers on the development of rhythms of precocial Japanese quail (Coturnix c. japonica). First, we investigated the influence of mothering on the ontogeny of endogenous rhythms of young. We compared circadian and ultradian rhythms of feeding activity of quail reared with or without adoptive mothers. More brooded than non‐brooded quail presented a circadian and/or an ultradian rhythm. Thus, the presence of the mother during the normal brooding period favors, in the long term, expression of rhythms in the young. Second, we investigated the influence of rhythmic phenotype of the mother on the development of endogenous rhythms of young by comparing quail brooded by circadian‐rhythmic adoptive mothers (R) to quail brooded by circadian‐arrhythmic adoptive mothers (A). More R‐brooded than A‐brooded quail expressed circadian rhythmicity, and circadian rhythm clarities were greater in R‐brooded than A‐brooded quail. Ultradian rhythmicity did not differ between R‐ and A‐brooded quail, nor between R and A adoptive mothers. Thus, the rhythmic phenotypes of quail mothers influence the rhythmic phenotypes of their young. Our results demonstrate that mothers of precocial birds influence epigenetically the ontogeny of endogenous rhythms of the young they raise.     

Daily temporal organization of laying in Japanese quail: variability and heritability

In birds, many behavioral and physiological processes that occur during reproduction show daily rhythms in response to environmental temporal constraints. In this study, the individual daily organization of laying and its genetic determinant in Japanese quail (Coturnix coturnix japonica) were analyzed. For this purpose, the oviposition time of 102 randomly chosen females, maintained in long-day photoperiodic conditions (LD 14h:10h) for 1 mon was observed and extreme phenotypes selected. Laying is characterized by two parameters: oviposition interval and laying hour.

The birds showed a specific time of laying during 24 h. All eggs were laid in the afternoon between 6.5 and 14 h after lights on (HALO). Two laying profiles were determined: 20% of females with an oviposition interval greater than 24 h (24.7±0.2 h) (the “delayed” profile) laid progressively later each day until a pause day. The remaining 80% of the females laid at the same time each day, with few pause days and an oviposition interval close to 24 h (24.0±0.2 h) (the “stable” profile). Among the females, showing this last profile, an intra-individual stability and an inter-individual variability of laying hour was established. Two extreme laying phenotypes were then determined: the “early” phenotype (E) for females laying on average between 7.5 and 9.5 HALO and the “late” phenotype (L) for females laying between 12.5 and 14 HALO.

In order to study the genetic basis of the laying hour, three females of each extreme phenotype were selected and crossed with two different males. The E and L females produced 57 F1E and 42 F1L daughters, respectively. F1 females displayed both laying profiles. However, the proportion of females displaying a “delayed” profile was higher in the L line (50%) than in the E line (29.8%). For the “stable” daughters, artificial selection induced an advance in laying hour of 4.7% for the E line and a delay of 4.7% for the L line. Realized heritability was estimated at 0.5. Moreover, the laying hour of the daughters was correlated positively to that of the mothers (N=61;r=0.45). These results support the notion of heritability of oviposition time in Japanese quail.     

The Measles Virus Nucleocapsid Protein Tail Domain Is Dispensable for Viral Polymerase Recruitment and Activity

Paramyxovirus genomes are ribonucleoprotein (RNP) complexes consisting of nucleoprotein (N)-encapsidated viral RNA. Measles virus (MeV) N features an amino-terminal RNA-binding core and a 125-residue tail domain, of which only the last 75 residues are considered fully mobile on the nucleocapsid surface. A molecular recognition element (MoRE) domain mediates binding of the viral phosphoprotein (P). This P N-tail interaction is considered instrumental for recruiting the polymerase complex to the template. We have engineered MeV N variants with tail truncations progressively eliminating the MoRE domain and upstream tail sections. Confirming previous reports, RNPs with N truncations lacking the carboxyl-terminal 43-residues harboring the MoRE domain cannot serve as polymerase template. Remarkably, further removal of all tail residues predicted to be surface-exposed significantly restores RNP bioactivity. Insertion of structurally dominant tags into the central N-tail section reduces bioactivity, but the negative regulatory effect of exposed N-tail stems is sequence-independent. Bioactive nucleocapsids lacking exposed N-tail sections are unable to sustain virus replication, because of weakened interaction of the advancing polymerase complex with the template. Deletion of the N-MoRE-binding domain in P abrogates polymerase recruitment to standard nucleocapsids, but polymerase activity is partially restored when N-tail truncated RNPs serve as template. Revising central elements of the current replication model, these data reveal that MeV polymerase is capable of productively docking directly to the nucleocapsid core. Dispensable for polymerase recruitment, N-MoRE binding to P-tail stabilizes the advancing polymerase-RNP complex and may rearrange unstructured central tail sections to facilitate polymerase access to the template.     

Designing a Soluble Near Full-length HIV-1 gp41 Trimer

The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors, whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses was tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intramolecular HR1-HR2 interactions but not intermolecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-amino acid foldon at the C terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into the mechanism of entry and development of gp41-based HIV-1 vaccines.     

The Ebola Virus Matrix Protein Penetrates into the Plasma Membrane: A KEY STEP IN VIRAL PROTEIN 40 (VP40) OLIGOMERIZATION AND VIRAL EGRESS*

Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu²¹³, Ile²⁹³, Leu²⁹⁵, and Val²⁹⁸ demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.     

Induction of Anti-influenza Immunity by Modified Green Fluorescent Protein (GFP) Carrying Hemagglutinin-derived Epitope Structure

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure. Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.     

Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus

Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.     

Molecular Mechanism by Which a Potent Hepatitis C Virus NS3-NS4A Protease Inhibitor Overcomes Emergence of Resistance

Although optimizing the resistance profile of an inhibitor can be challenging, it is potentially important for improving the long term effectiveness of antiviral therapy. This work describes our rational approach toward the identification of a macrocyclic acylsulfonamide that is a potent inhibitor of the NS3-NS4A proteases of all hepatitis C virus genotypes and of a panel of genotype 1-resistant variants. The enhanced potency of this compound versus variants D168V and R155K facilitated x-ray determination of the inhibitor-variant complexes. In turn, these structural studies revealed a complex molecular basis of resistance and rationalized how such compounds are able to circumvent these mechanisms.     

Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency

New viral strains can be evolved to recognize different host glycans through mutagenesis and experimental adaptation. However, such mutants generally harbor amino acid changes that affect viral binding to a single class of carbohydrate receptors. We describe the rational design and synthesis of novel, chimeric adeno-associated virus (AAV) strains that exploit an orthogonal glycan receptor for transduction. A dual glycan-binding AAV strain was first engineered as proof of concept by grafting a galactose (Gal)-binding footprint from AAV serotype 9 onto the heparan sulfate-binding AAV serotype 2. The resulting chimera, AAV2G9, continues to bind heparin affinity columns but interchangeably exploits Gal and heparan sulfate receptors for infection, as evidenced by competitive inhibition assays with lectins, glycans, and parental AAV strains. Although remaining hepatotropic like AAV2, the AAV2G9 chimera mediates rapid onset and higher transgene expression in mice. Similarly, engraftment of the Gal footprint onto the laboratory-derived strain AAV2i8 yielded an enhanced AAV2i8G9 chimera. This new strain remains liver-detargeted like AAV2i8 while selectively transducing muscle tissues at high efficiency, comparable with AAV9. The AAV2i8G9 chimera is a promising vector candidate for targeted gene therapy of cardiac and musculoskeletal diseases. In addition to demonstrating the modularity of glycan receptor footprints on viral capsids, our approach provides design strategies to expand the AAV vector toolkit.