RESUMEN
Nuclear import of the hepatitis B virus (HBV) nucleocapsid is essential for replication that occurs in the nucleus. The ~360-angstrom HBV capsid translocates to the nuclear pore complex (NPC) as an intact particle, hijacking human importins in a reaction stimulated by host kinases. This paper describes the mechanisms of HBV capsid recognition by importins. We found that importin α1 binds a nuclear localization signal (NLS) at the far end of the HBV coat protein Cp183 carboxyl-terminal domain (CTD). This NLS is exposed to the capsid surface through a pore at the icosahedral quasi-sixfold vertex. Phosphorylation at serine-155, serine-162, and serine-170 promotes CTD compaction but does not affect the affinity for importin α1. The binding of 30 importin α1/ß1 augments HBV capsid diameter to ~620 angstroms, close to the maximum size trafficable through the NPC. We propose that phosphorylation favors CTD externalization and prompts its compaction at the capsid surface, exposing the NLS to importins.
Asunto(s)
Virus de la Hepatitis B , Nucleocápside , Humanos , Transporte Activo de Núcleo Celular , Carioferinas , Proteínas de la Cápside , Factores Inmunológicos , SerinaRESUMEN
Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/ß complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein's C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impß as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T = 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0 Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impß-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid.
Asunto(s)
Cápside , Virus de la Hepatitis B , Humanos , beta Carioferinas , Cápside/química , Proteínas de la Cápside/química , Microscopía por Crioelectrón , Hepatitis B/virología , Virus de la Hepatitis B/metabolismo , Ensamble de VirusRESUMEN
In certain conditions, dye-conjugated icosahedral virus shells exhibit suppression of concentration quenching. The recently observed radiation brightening at high fluorophore densities has been attributed to coherent emission, i.e., to a cooperative process occurring within a subset of the virus-supported fluorophores. Until now, the distribution of fluorophores among potential conjugation sites and the nature of the active subset remained unknown. With the help of mass spectrometry and molecular dynamics simulations, we found which conjugation sites in the brome mosaic virus capsid are accessible to fluorophores. Reactive external surface lysines but also those at the lumenal interface where the coat protein N-termini are located showed virtually unrestricted access to dyes. The third type of labeled lysines was situated at the intercapsomeric interfaces. Through limited proteolysis of flexible N-termini, it was determined that dyes bound to them are unlikely to be involved in the radiation brightening effect. At the same time, specific labeling of genetically inserted cysteines on the exterior capsid surface alone did not lead to radiation brightening. The results suggest that lysines situated within the more rigid structural part of the coat protein provide the chemical environments conducive to radiation brightening, and we discuss some of the characteristics of these environments.
Asunto(s)
Bromovirus , Virus , Cápside , Proteínas de la Cápside , Colorantes FluorescentesRESUMEN
Molecular dynamics (MD) simulations across spatiotemporal resolutions are widely applied to study viruses and represent the central technique uniting the field of computational virology. We discuss the progress of MD in elucidating the dynamics of the viral life cycle, including the status of modeling intact extracellular virions and leveraging advanced simulations to mimic active life cycle processes. We further remark on the prospects of MD for continued contributions to the basic science characterization of viruses, especially given the increasing availability of high-quality experimental data and supercomputing power. Overall, integrative computational methods that are closely guided by experiments are unmatched in the level of detail they provide, enabling-now and in the future-new discoveries relevant to thwarting viral infection.
Asunto(s)
Virosis , Virus , Humanos , Simulación de Dinámica Molecular , ViriónRESUMEN
The hepatitis B virus (HBV) capsid is an attractive drug target, relevant to combating viral hepatitis as a major public health concern. Among small molecules known to interfere with capsid assembly, the phenylpropenamides, including AT130, represent an important antiviral paradigm based on disrupting the timing of genome packaging. Here, all-atom molecular dynamics simulations of an intact AT130-bound HBV capsid reveal that the compound increases spike flexibility and improves recovery of helical secondary structure in the spike tips. Regions of the capsid-incorporated dimer that undergo correlated motion correspond to established sub-domains that pivot around the central chassis. AT130 alters patterns of correlated motion and other essential dynamics. A new conformational state of the dimer is identified, which can lead to dramatic opening of the intradimer interface and disruption of communication within the spike tip. A novel salt bridge is also discovered, which can mediate contact between the spike tip and fulcrum even in closed conformations, revealing a mechanism of direct communication across these sub-domains. Altogether, results describe a dynamical connection between the intra- and interdimer interfaces and enable mapping of allostery traversing the entire core protein dimer.
Asunto(s)
Benzamidas/metabolismo , Proteínas de la Cápside/metabolismo , Cápside/química , Virus de la Hepatitis B/química , Simulación de Dinámica Molecular , Sitio Alostérico , Antivirales/farmacología , Benzamidas/farmacología , Cápside/efectos de los fármacos , Cápside/metabolismo , Biología Computacional/métodos , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Humanos , Multimerización de Proteína , Ensamble de VirusRESUMEN
Protein plasticity and dynamics are important aspects of their function. Here we use solid-state NMR to experimentally characterize the dynamics of the 3.5 MDa hepatitis B virus (HBV) capsid, assembled from 240 copies of the Cp149 core protein. We measure both T 1 and T 1ρ relaxation times, which we use to establish detectors on the nanosecond and microsecond timescale. We compare our results to those from a 1 microsecond all-atom Molecular Dynamics (MD) simulation trajectory for the capsid. We show that, for the constituent residues, nanosecond dynamics are faithfully captured by the MD simulation. The calculated values can be used in good approximation for the NMR-non-detected residues, as well as to extrapolate into the range between the nanosecond and microsecond dynamics, where NMR has a blind spot at the current state of technology. Slower motions on the microsecond timescale are difficult to characterize by all-atom MD simulations owing to computational expense, but are readily accessed by NMR. The two methods are, thus, complementary, and a combination thereof can reliably characterize motions covering correlation times up to a few microseconds.