Human parainfluenza virus evolution during lung infection of immunocompromised individuals promotes viral persistence.

The capacity of respiratory viruses to undergo evolution within the respiratory tract raises the possibility of evolution under the selective pressure of the host environment or drug treatment. Long-term infections in immunocompromised hosts are potential drivers of viral evolution and development of infectious variants. We showed that intrahost evolution in chronic human parainfluenza virus 3 (HPIV3) infection in immunocompromised individuals elicited mutations that favored viral entry and persistence, suggesting that similar processes may operate across enveloped respiratory viruses. We profiled longitudinal HPIV3 infections from 2 immunocompromised individuals that persisted for 278 and 98 days. Mutations accrued in the HPIV3 attachment protein hemagglutinin-neuraminidase (HN), including the first in vivo mutation in HN's receptor binding site responsible for activating the viral fusion process. Fixation of this mutation was associated with exposure to a drug that cleaves host-cell sialic acid moieties. Longitudinal adaptation of HN was associated with features that promote viral entry and persistence in cells, including greater avidity for sialic acid and more active fusion activity in vitro, but not with antibody escape. Long-term infection thus led to mutations promoting viral persistence, suggesting that host-directed therapeutics may support the evolution of viruses that alter their biophysical characteristics to persist in the face of these agents in vivo.

Structures reveal opening of the store-operated calcium channel Orai.

The store-operated calcium (Ca2+) channel Orai governs Ca2+ influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to the depletion of Ca2+ stored in the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Our previous work revealed the structure of Orai with a closed pore. Here, using a gain-of-function mutation that constitutively activates the channel, we present an X-ray structure of Drosophila melanogaster Orai in an open conformation. Well-defined electron density maps reveal that the pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and Ca2+ selectivity. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed conformation, we propose a sequence for store-operated activation.

Cyanylated Cysteine Reports Site-Specific Changes at Protein-Protein-Binding Interfaces Without Perturbation.

To investigate the cyanylated cysteine vibrational probe group's ability to report on binding-induced changes along a protein-protein interface, the probe group was incorporated at several sites in a peptide of the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase. Isothermal titration calorimetry was used to determine the binding thermodynamics between calmodulin and each peptide. For all probe positions, the binding affinity was nearly identical to that of the unlabeled peptide. The CN stretching infrared band was collected for each peptide free in solution and bound to calmodulin. Binding-induced shifts in the IR spectral frequencies were correlated with estimated solvent accessibility based on molecular dynamics simulations. This work generally suggests (1) that site-specific incorporation of this vibrational probe group does not cause major perturbations to its local structural environment and (2) that this small probe group might be used quite broadly to map dynamic protein-binding interfaces. However, site-specific perturbations due to artificial labeling groups can be somewhat unpredictable and should be evaluated on a site-by-site basis through complementary measurements. A fully quantitative, simulation-based interpretation of the rich probe IR spectra is still needed but appears to be possible given recent advances in simulation techniques.