Structurally similar woodchuck and human hepadnavirus core proteins have distinctly different temperature dependences of assembly.

UNLABELLED: Woodchuck hepatitis virus (WHV), a close relative of human hepatitis B virus (HBV), has been a key model for disease progression and clinical studies. Sequences of the assembly domain of WHV and HBV core proteins (wCp149 and hCp149, respectively) have 65% identity, suggesting similar assembly behaviors. We report a cryo-electron microscopy (cryo-EM) structure of the WHV capsid at nanometer resolution and characterization of wCp149 assembly. At this resolution, the T=4 capsid structures of WHV and HBV are practically identical. In contrast to their structural similarity, wCp149 demonstrates enhanced assembly kinetics and stronger dimer-dimer interactions than hCp149: at 23 °C and at 100 mM ionic strength, the pseudocritical concentrations of assembly of wCp149 and hCp149 are 1.8 µM and 43.3 µM, respectively. Transmission electron microscopy reveals that wCp149 assembles into predominantly T=4 capsids with a sizeable population of larger, nonicosahedral structures. Charge detection mass spectrometry indicates that T=3 particles are extremely rare compared to the ∼ 5% observed in hCp149 reactions. Unlike hCp149, wCp149 capsid assembly is favorable over a temperature range of 4 °C to 37 °C; van't Hoff analyses relate the differences in temperature dependence to the high positive values for heat capacity, enthalpy, and entropy of wCp149 assembly. Because the final capsids are so similar, these findings suggest that free wCp149 and hCp149 undergo different structural transitions leading to assembly. The difference in the temperature dependence of wCp149 assembly may be related to the temperature range of its hibernating host. IMPORTANCE: In this paper, we present a cryo-EM structure of a WHV capsid showing its similarity to HBV. We then observe that the assembly properties of the two homologous proteins are very different. Unlike human HBV, the capsid protein of WHV has evolved to function in a nonhomeostatic environment. These studies yield insight into the interplay between core protein self-assembly and the host environment, which may be particularly relevant to plant viruses and viruses with zoonotic cycles involving insect vectors.

Microtubule Attachment and Centromeric Tension Shape the Protein Architecture of the Human Kinetochore.

The nanoscale protein architecture of the kinetochore plays an integral role in specifying the mechanisms underlying its functions in chromosome segregation. However, defining this architecture in human cells remains challenging because of the large size and compositional complexity of the kinetochore. Here, we use Förster resonance energy transfer to reveal the architecture of individual kinetochore-microtubule attachments in human cells. We find that the microtubule-binding domains of the Ndc80 complex cluster at the microtubule plus end. This clustering occurs only after microtubule attachment, and it increases proportionally with centromeric tension. Surprisingly, Ndc80 complex clustering is independent of the organization and number of its centromeric receptors. Moreover, this clustering is similar in yeast and human kinetochores despite significant differences in their centromeric organizations. These and other data suggest that the microtubule-binding interface of the human kinetochore behaves like a flexible "lawn" despite being nucleated by repeating biochemical subunits.

How Kinetochore Architecture Shapes the Mechanisms of Its Function.

The eukaryotic kinetochore is a sophisticated multi-protein machine that segregates chromosomes during cell division. To ensure accurate chromosome segregation, it performs three major functions using disparate molecular mechanisms. It operates a mechanosensitive signaling cascade known as the spindle assembly checkpoint (SAC) to detect and signal the lack of attachment to spindle microtubules, and delay anaphase onset in response. In addition, after attaching to spindle microtubules, the kinetochore generates the force necessary to move chromosomes. Finally, if the two sister kinetochores on a chromosome are both attached to microtubules emanating from the same spindle pole, they activate another mechanosensitive mechanism to correct the monopolar attachments. All three of these functions maintain genome stability during cell division. The outlines of the biochemical activities responsible for these functions are now available. How the kinetochore integrates the underlying molecular mechanisms is still being elucidated. In this Review, we discuss how the nanoscale protein organization in the kinetochore, which we refer to as kinetochore 'architecture', organizes its biochemical activities to facilitate the realization and integration of emergent mechanisms underlying its three major functions. For this discussion, we will use the relatively simple budding yeast kinetochore as a model, and extrapolate insights gained from this model to elucidate functional roles of the architecture of the much more complex human kinetochore.

Charge Detection Mass Spectrometry Identifies Preferred Non-Icosahedral Polymorphs in the Self-Assembly of Woodchuck Hepatitis Virus Capsids.

Woodchuck hepatitis virus (WHV) is prone to aberrant assembly in vitro and can form a broad distribution of oversized particles. Characterizing aberrant assembly products is challenging because they are both large and heterogeneous. In this work, charge detection mass spectrometry (CDMS) is used to measure the distribution of WHV assembly products. CDMS is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of each ion's charge and m/z (mass-to-charge) ratio. Under relatively aggressive, assembly promoting conditions, roughly half of the WHV assembly products are T=4 capsids composed of exactly 120 dimers while the other half are a broad distribution of larger species that extends to beyond 210 dimers. There are prominent peaks at around 132 dimers and at 150 dimers. In part, the 150 dimer complex can be attributed to elongating a T=4 capsid along its 5-fold axis by adding a ring of hexamers. However, most of the other features cannot be explained by existing models for hexameric defects. Cryo-electron microscopy provides evidence of elongated capsids. However, image analysis reveals that many of them are not closed but have "spiral-like" morphologies. The CDMS data indicate that oversized capsids have a preference for growth by addition of 3 or 4 dimers, probably by completion of hexameric vertices.