Calreticulin is a calcium-binding chaperone that is normally localized in the endoplasmic reticulum (ER). Calreticulin is detectable on the surface of apoptotic cell under some apoptosis-inducing conditions, where it promotes phagocytosis and immunogenicity of dying cells. However, the precise mechanism by which calreticulin, a soluble protein, localizes to the outer surface of the plasma membrane of dying cells is unknown, as are the molecular mechanisms that are relevant to calreticulin-induced cellular phagocytosis. Calreticulin comprises three distinct structural domains; a globular domain, an extended arm-like P-domain and a C-terminal acidic region containing multiple low affinity calcium binding sites. We show here that calreticulin, via its C-terminal acidic region, preferentially interacts with phosphatidylserine (PS) compared to other phospholipids, and that this interaction is calcium-dependent. Additionally, exogenous calreticulin binds apoptotic cells via a higher affinity calcium-dependent mode that is acidic-region-dependent. Exogenous calreticulin also binds live cells, including macrophages, via a second, lower affinity P-and globular domain-dependent, but calcium-independent binding mode that likely involves its generic polypeptide-binding site. Truncation constructs lacking the acidic region or arm-like P-domain of calreticulin are impaired in their abilities to induce apoptotic cell phagocytosis by murine peritoneal macrophages. Taken together, the results of this investigation provide the first molecular insights into the phospholipid-binding site of calreticulin as a key anchor point for the cell surface expression of calreticulin on apoptotic cells. These findings also support a role for calreticulin as a PS-bridging molecule that co-operates with other PS-binding factors to promote the phagocytosis of apoptotic cells.
Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites. IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.
Influenza A Virus (IAV) is a respiratory virus that causes seasonal outbreaks annually and pandemics occasionally. The main targets of the virus are epithelial cells in the respiratory tract. Like many other viruses, IAV employs the host cell’s machinery to enter cells, synthesize new genomes and viral proteins, and assemble new virus particles. The cytoskeletal system is a major cellular machinery, which IAV exploits for its entry to and exit from the cell. However, in some cases, the cytoskeleton has a negative impact on efficient IAV growth. In this review, we highlight the role of cytoskeletal elements in cellular processes that are utilized by IAV in the host cell. We further provide an in-depth summary of the current literature on the roles the cytoskeleton plays in regulating specific steps during the assembly of progeny IAV particles.
Identification of host cell determinants promoting or suppressing replication of viruses has been aided by analyses of host cells that impose inherent blocks on viral replication. In this study, we show that primary human MDM, which are not permissive to IAV replication, fail to support virus particle formation. This defect is specific to primary human macrophages, since a human monocytic cell line differentiated to macrophage-like cells supports IAV particle formation. We further identified association between two viral transmembrane proteins, HA and M2, on the cell surface as a discrete assembly step, which is defective in MDM. Defective HA-M2 association and particle budding, but not virus release, in MDM are rescued by disruption of actin cytoskeleton, revealing a previously unknown, negative role for actin, which specifically targets an early step in the multistep IAV production. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly.
18The primary target of Influenza A virus (IAV) is epithelial cells in the respiratory tract. In 19 contrast to epithelial cells, productive virus infection of most IAV strains is either 20 completely blocked or highly inefficient in macrophages. The exact nature of the defect 21 in IAV replication that leads to inefficient production of progeny virus particles in human 22 macrophages remains to be determined. In this study, we showed that primary human 42show that primary human MDM are not permissive to IAV replication due to a defect at 43 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/213447 doi: bioRxiv preprint first posted online Nov. 2, 2017; the virus particle formation step. This defect is specific to primary human macrophages, 44 since a human monocyte-derived cell line differentiated into macrophage-like 45 morphology supports IAV particle formation. Further, comparison of these cell types 46suggests that the defect in virus particle assembly in MDM is at least partly due to 47 inefficient association between two viral glycoproteins, HA and M2, on the surface of 48 these cells. Overall, by identifying the viral protein interaction susceptible to cell-type-49 specific differences, our study suggests the possibility that the interactions between viral 50 components can be targeted to block IAV assembly in infected cells. 52 53peer-reviewed)
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