Cell Surface Expression and Function of the Macromolecular C1 Complex on the Surface of Human Monocytes

The synthesis of the subunits of the C1 complex (C1q, C1s, C1r), and its regulator C1 inhibitor (C1-Inh) by human monocytes has been previously established. However, surface expression of these molecules by monocytes has not been shown. Using flow cytometry and antigen-capture enzyme-linked immunosorbent assay, we show here for the first time that, in addition to C1q, peripheral blood monocytes, and the monocyte-derived U937 cells express C1s and C1r, as well as Factor B and C1-Inh on their surface. C1s and C1r immunoprecipitated with C1q, suggesting that at least some of the C1q on these cells is part of the C1 complex. Furthermore, the C1 complex on U937 cells was able to trigger complement activation via the classical pathway. The presence of C1-Inh may ensure that an unwarranted autoactivation of the C1 complex does not take place. Since C1-Inh closely monitors the activation of the C1 complex in a sterile or infectious inflammatory environment, further elucidation of the role of C1 complex is crucial to dissect its function in monocyte, dendritic cell, and T cell activities, and its implications in host defense and tolerance

An Interaction between Glutathione and the Capsid Is Required for the Morphogenesis of C-Cluster Enteroviruses

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles

Dendritic cell lineage commitment is instructed by distinct cytokine signals

AbstractDendritic cells (DC) develop from hematopoietic stem cells, which is guided by instructive signals through cytokines. DC development progresses from multipotent progenitors (MPP) via common DC progenitors (CDP) into DC. Flt3 ligand (Flt3L) signaling via the Flt3/Stat3 pathway is of pivotal importance for DC development under steady state conditions. Additional factors produced during steady state or inflammation, such as TGF-β1 or GM-CSF, also influence the differentiation potential of MPP and CDP. Here, we studied how gp130, GM-CSF and TGF-β1 signaling influence DC lineage commitment from MPP to CDP and further into DC. We observed that activation of gp130 signaling promotes expansion of MPP. Additionally, gp130 signaling inhibited Flt3L-driven DC differentiation, but had little effect on GM-CSF-driven DC development. The inflammatory cytokine GM-CSF induces differentiation of MPP into inflammatory DC and blocks steady state DC development. Global transcriptome analysis revealed a GM-CSF-driven gene expression repertoire that primes MPP for differentiation into inflammatory DC. Finally, TGF-β1 induces expression of DC-lineage affiliated genes in MPP, including Flt3, Irf-4 and Irf-8. Under inflammatory conditions, however, the effect of TGF-β1 is altered: Flt3 is not upregulated, indicating that an inflammatory environment inhibits steady state DC development. Altogether, our data indicate that distinct cytokine signals produced during steady state or inflammation have a different outcome on DC lineage commitment and differentiation

The effect of BSO on CAV20 protein translation, RNA replication and transencapsidation.

<p>(<b>A</b>) <i>In vivo </i>³⁵S labeling of CAV20-wt and CAV20-BSOr viral proteins in the absence and presence of BSO in HeLa H1 cells. Viral proteins were separated on SDS-polyacrylamide gels (12.5%), as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004052#s4" target="_blank">Materials and Methods</a>. (<b>B</b>) The genome structure of the CAV20 FLuc replicon used in the experiment is shown above. The FLuc coding sequence was used to replace the P1 domain of the polyprotein. RNA transcripts of the replicon were transfected into untreated or BSO-treated HeLa H1 cells either in the absence or presence of GnHCl (2 mM). Luciferase activity was measured at 9 hr post transfection. (<b>C</b>) CAV20 FLuc replicon RNA transcript was transfected into BSO-treated HeLa H1 cells either in the absence or presence of GnHCl. At 1 hr post-transfection the cells were superinfected either with wt or with CAV20-BSOr (VP3 Y97H) at a moi of 0.5. The cells were lysed at 6 hr post-infection and were then used to re-infect fresh HeLa H1 cells in the absence or presence of GnHCl.</p

Genome structure of PV1 RNA and polyprotein processing.

<p>The PV1 genomic RNA contains a 5′NTR, a single open reading frame, a 3′NTR and poly (A) tail. The polyprotein, translated from a single open reading frame, has one structural (P1) and two nonstructural domains (P2, P3). The P1 domain is released from the polyprotein by 2A^pro. Further processing of the P1 domain into VP0, VP3 and VP1 is by 3CD^pro, followed by the maturation cleavage of VP0 into VP4 and VP2 by an unknown mechanism. The P2/P3 domains are processed by 3C^pro/3CD^pro to generate different precursors and mature nonstructural proteins.</p

GSH protects PV1-wt and PV1-BSOr from heat inactivation.

<p>(<b>A</b>) Protection from heat inactivation of PV1-wt or PV1-BSOr by GSH. Purified PV1-wt or PV1-BSOr (VP3, Q178L) 3×10⁹ PFU was incubated <i>in vitro</i> in PBS either in the absence or presence of various amounts of GSH for 25 min at 48°C. The amount of virus remaining after the incubation was determined by plaque assays. The virus titers obtained after heating without GSH for both viruses were taken as “1”. (<b>B</b>) Protection from heat inactivation of PV1-wt or PV1-BSOr by reducing agents or GSSG. PV1-wt or PV1-BSOr 3×10⁹ PFU were incubated in PBS either in the absence or presence of various reducing agents (5 mM) or of GSSG (5 mM) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004052#s4" target="_blank">Materials and Methods</a>). The virus titer obtained after heating without GSH for both viruses were taken as “1”.</p

GSH directly interacts with capsid proteins of both PV1 and CAV20.

<p>(A) GSH-pull down assay of PV1-infected lysates. HeLa cells, untreated or BSO-treated, were infected with PV1-wt or PV1-BSOr (VP3 Q178L). (B) GSH-pull down assay of CAV20-infected lysates. HeLa cells, untreated or BSO-treated, were infected with CAV20-wt or CAV20-BSOr. The viral proteins were labeled with ³⁵S-Translabel from 4 to 5 hr post-infection and the cells were harvested. The viral proteins in an aliquot of the lysates were analysed on SDS-polyacrylamide (11.5%) gels (lane 1–4). Samples were loaded onto GSH Sepharose beads and the pulled down material was analyzed by SDS-PAGE (lane 5–8).</p