Many chronic viral infections are marked by pathogen persistence and a generalized immunosuppression. The exact mechanisms by which this occurs are still unknown. Using a mouse model of persistent lymphocytic choriomeningitis virus (LCMV) infection, we demonstrate viral targeting of fibroblastic reticular cells (FRC) in the lymphoid organs. The FRC stromal networks are critical for proper lymphoid architecture and function. High numbers of FRC were infected by LCMV clone 13, which causes a chronic infection, whereas few were infected by the acute strain, LCMV Armstrong. The function of the FRC conduit network was altered after clone 13 infection by the action of CD8 ؉ T cells. Importantly, expression of the inhibitory programmed death ligand 1, which was up-regulated on FRC after infection, reduced early CD8 ؉ T cell-mediated immunopathology and prevented destruction of the FRC architecture in the spleen. Together, this reveals an important tropism during a persistent viral infection. These data also suggest that the inhibitory PD-1 pathway, which likely evolved to prevent excessive immunopathology, may contribute to viral persistence in FRC during chronic infection.immunopathology ͉ stromal cells ͉ viral infection M any acute and chronic viral infections induce a generalized immunosuppression (1, 2). This suppression of immunity is often transient, occurring during the acute phase of infection; however, prolonged suppression can also occur during certain chronic viral infections. The mechanisms of virus-induced immunosuppression are complex and varied (1-3). Infection of mice with lymphocytic choriomeningitis virus (LCMV) is a useful model to dissect the mechanisms of viral persistence and immunosuppression. The clone 13 (CL-13) strain of LCMV results in a chronic infection in adult mice, marked by persisting virus and a generalized immunosuppression. Viral load rises rapidly within days and remains high over a prolonged period, suppressing specific immunity by inducing an hierarchical loss of CD8 ϩ T cell function (4, 5). This CL-13 chronic infection is also associated with increased susceptibility to opportunistic secondary infections (6-8).Chronic LCMV infection results in reduced cellularity and altered splenic architecture (9). Enhanced infection of dendritic cells (DC), resulting in reduced T cell stimulatory capacity and destruction of the DC, has been proposed as one mechanism by which CL-13 initiates immunosuppression within the host (8, 10). Although DC in the spleen can be infected by LCMV CL-13, this represents a relatively small proportion of total infected cells. It follows that other cell types are infected by CL-13 and likely contribute to the pronounced immunosuppression and architectural disruption of lymphoid organs that are observed after infection.Through a detailed kinetic examination of the cell types infected during LCMV CL-13 infection we discovered that fibroblastic reticular cells (FRC) are an important target of infection by this virus. Secondary lymphoid tissues are supported by a c...

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.

Twik-related K + channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K + channel (TRAAK) form the TREK subfamily of two-pore-domain K + (K 2P ) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K 2P channels.potassium channels | single-molecule fluorescence | leak current | combinatorial diversity | heteromerization

These results suggest that Aqp0a is the primary water channel of the lens and that Aqp0b, though possibly a secondary water channel, has an unidentified function in the lens.