On the Effect of Thermodynamic Equilibrium on the Assembly Efficiency of Complex Multi-Layered Virus-Like Particles (VLP): the Case of Rotavirus VLP

Previous studies have reported the production of malformed virus-like-particles (VLP) in recombinant host systems. Here we computationally investigate the case of a large triple-layered rotavirus VLP (RLP). In vitro assembly, disassembly and reassembly data provides strong evidence of microscopic reversibility of RLP assembly. Light scattering experimental data also evidences a slow and reversible assembly untypical of kinetic traps, thus further strengthening the fidelity of a thermodynamically controlled assembly. In silico analysis further reveals that under favourable conditions particles distribution is dominated by structural subunits and completely built icosahedra, while other intermediates are present only at residual concentrations. Except for harshly unfavourable conditions, assembly yield is maximised when proteins are provided in the same VLP protein mass composition. The assembly yield decreases abruptly due to thermodynamic equilibrium when the VLP protein mass composition is not obeyed. The latter effect is more pronounced the higher the Gibbs free energy of subunit association is and the more complex the particle is. Overall this study shows that the correct formation of complex multi-layered VLPs is restricted to a narrow range of association energies and protein concentrations, thus the choice of the host system is critical for successful assembly. Likewise, the dynamic control of intracellular protein expression rates becomes very important to minimize wasted proteins

Formation of Trans-Activation Competent HIV-1 Rev:RRE Complexes Requires the Recruitment of Multiple Protein Activation Domains

The HIV-1 Rev trans-activator is a nucleocytoplasmic shuttle protein that is essential for virus replication. Rev directly binds to unspliced and incompletely spliced viral RNA via the cis-acting Rev Response Element (RRE) sequence. Subsequently, Rev oligomerizes cooperatively and interacts with the cellular nuclear export receptor CRM1. In addition to mediating nuclear RNA export, Rev also affects the stability, translation and packaging of Rev-bound viral transcripts. Although it is established that Rev function requires the multimeric assembly of Rev molecules on the RRE, relatively little is known about how many Rev monomers are sufficient to form a trans-activation competent Rev:RRE complex, or which specific activity of Rev is affected by its oligomerization. We here analyzed by functional studies how homooligomer formation of Rev affects the trans-activation capacity of this essential HIV-1 regulatory protein. In a gain-of-function approach, we fused various heterologous dimerization domains to an otherwise oligomerization-defective Rev mutant and were able to demonstrate that oligomerization of Rev is not required per se for the nuclear export of this viral trans-activator. In contrast, however, the formation of Rev oligomers on the RRE is a precondition to trans-activation by directly affecting the nuclear export of Rev-regulated mRNA. Moreover, experimental evidence is provided showing that at least two protein activation domains are required for the formation of trans-activation competent Rev:RRE complexes. The presented data further refine the model of Rev trans-activation by directly demonstrating that Rev oligomerization on the RRE, thereby recruiting at least two protein activation domains, is required for nuclear export of unspliced and incompletely spliced viral RNA

The expansion field: The value of H_0

Any calibration of the present value of the Hubble constant requires recession velocities and distances of galaxies. While the conversion of observed velocities into true recession velocities has only a small effect on the result, the derivation of unbiased distances which rest on a solid zero point and cover a useful range of about 4-30 Mpc is crucial. A list of 279 such galaxy distances within v<2000 km/s is given which are derived from the tip of the red-giant branch (TRGB), from Cepheids, and from supernovae of type Ia (SNe Ia). Their random errors are not more than 0.15 mag as shown by intercomparison. They trace a linear expansion field within narrow margins from v=250 to at least 2000 km/s. Additional 62 distant SNe Ia confirm the linearity to at least 20,000 km/s. The dispersion about the Hubble line is dominated by random peculiar velocities, amounting locally to <100 km/s but increasing outwards. Due to the linearity of the expansion field the Hubble constant H_0 can be found at any distance >4.5 Mpc. RR Lyr star-calibrated TRGB distances of 78 galaxies above this limit give H_0=63.0+/-1.6 at an effective distance of 6 Mpc. They compensate the effect of peculiar motions by their large number. Support for this result comes from 28 independently calibrated Cepheids that give H_0=63.4+/-1.7 at 15 Mpc. This agrees also with the large-scale value of H_0=61.2+/-0.5 from the distant, Cepheid-calibrated SNe Ia. A mean value of H_0=62.3+/-1.3 is adopted. Because the value depends on two independent zero points of the distance scale its systematic error is estimated to be 6%. Typical errors of H_0 come from the use of a universal, yet unjustified P-L relation of Cepheids, the neglect of selection bias in magnitude-limited samples, or they are inherent to the adopted models.Comment: 44 pages, 4 figures, 6 tables, accepted for publication in the Astronony and Astrophysics Review 15

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta