PLoS Pathogens最新文献

C-type lectin receptors (CLRs) are essential to execute host defense against fungal infection. Nevertheless, a comprehensive understanding of the molecular underpinnings of CLR signaling remains a work in progress. Here, we searched for yet-to-be-identified tyrosine-phosphorylated proteins in Dectin-1 signaling and linked the stress-response protein valosin containing protein (VCP)/p97 to Dectin-1 signaling. Knockdown of VCP expression or chemical inhibition of VCP's segregase activity dampened Dectin-1-elicited SYK activation in BMDMs and BMDCs, leading to attenuated expression of proinflammatory cytokines/chemokines such as TNF-α, IL-6 and CXCL1. Biochemical analyses demonstrated that VCP and its cofactor UFD1 form a complex with SYK and its phosphatase SHP-1 following Dectin-1 ligation, and knockdown of VCP led to a more prominent SYK and SHP-1 association. Further, SHP-1 became polyubiquitinated upon Dectin-1 activation, and VCP or UFD1 overexpression accelerated SHP-1 degradation. Conceivably, VCP may promote Dectin-1 signaling by pulling the ubiquitinated SHP-1 out of the SYK complex for degradation. Finally, genetic ablation of VCP in the neutrophil and macrophage compartment rendered the mice highly susceptible to infection by Candida albicans, an observation also phenocopied by administering the VCP inhibitor. These results collectively demonstrate that VCP is a previously unappreciated signal transducer of the Dectin-1 pathway and a crucial component of antifungal defense, and suggest a new mechanism regulating SYK activation.

Artemisinin-based combination therapy (ACT) is the mainstay of effective treatment of Plasmodium falciparum malaria. However, the long-term utility of ACTs is imperiled by widespread partial artemisinin resistance in Southeast Asia and its recent emergence in parts of East Africa. This underscores the need to identify chemotypes with new modes of action (MoAs) to circumvent resistance to ACTs. In this study, we characterized the asexual blood stage antiplasmodial activity and resistance mechanisms of LDT-623, a 4-aminoquinoline (4-AQ). We also detected LDT-623 activity against multiple stages (liver schizonts, stage IV-V gametocytes, and ookinetes) of Plasmodium's life cycle, a feature unlike other 4-AQs such as chloroquine (CQ) and piperaquine (PPQ). Using heme fractionation profiling and drug uptake studies in PfCRT-containing proteoliposomes, we observed inhibition of hemozoin formation and PfCRT-mediated transport, which constitute characteristic features of 4-AQs' MoA. We also found minimal cross-resistance to LDT-623 in a panel of mutant pfcrt or pfmdr1 lines, but not the PfCRT F145I mutant that is highly resistant to PPQ resistance yet is very unfit. No P. falciparum parasites were recovered in an in vitro resistance selection study, suggesting a high barrier for resistance to emerge. Finally, a competitive growth assay comprising >50 barcoded parasite lines with mutated resistance mediators or major drug targets found no evidence of cross-resistance. Our findings support further exploration of this promising 4-AQ.

Posttranslational modifications (PTMs) such as phosphorylation, ubiquitination, SUMOylation, and ISGylation are involved in various cellular pathways, including innate immunity and disease processes. Many viruses have developed sophisticated mechanisms to modulate these host PTMs, either by inhibiting the interferon pathway or by enhancing the stability and function of viral proteins essential for replication. In this Pearl, we review the literature on how flaviviruses are impacted by and exploit posttranslational modifications to their advantage.

Non-structural protein 2 (NSP2) of PRRSV is highly variable and plays crucial roles in the virus's life cycle. To elucidate the function of NSP2 during PRRSV infection, we identified SH3KBP1 as an NSP2-interacting host protein using mass spectrometry. Exogenous SH3KBP1 expression significantly inhibited PRRSV replication by enhancing IFN-I and related ISGs production. Conversely, SH3KBP1 knockdown promoted viral replication by downregulating IFN-I and ISGs levels. In vivo experiments revealed that Sh3kbp1-/- mice were more susceptible to VSV infection, exhibiting reduced serum IFN-β levels. Further investigation showed that SH3KBP1 enhances RIG-I signal transduction by increasing K63-linked polyubiquitination through interaction with the E3 ubiquitin ligase TRIM25. We also found that PRRSV infection and NSP2 overexpression induce the autophagic degradation of SH3KBP1, counteracting the host's innate immune response. A critical interaction site was identified within the third polyproline-arginine motif in NSP2 (453PVPAPR458). Recombinant PRRSV lacking this motif displayed reduced virulence and decreased SH3KBP1 degradation. This study advances our understanding of how PRRSV interferes with the host immune response and offers valuable insights for developing novel attenuated vaccines against PRRSV.

Plasmodium falciparum infections elicit strong humoral immune responses to two main groups of antigens expressed by blood-stage parasites: merozoite antigens that are involved in the erythrocyte invasion process and variant surface antigens that mediate endothelial sequestration of infected erythrocytes. Long-lived B cells against both antigen classes can be detected in the circulation for years after exposure, but have not been directly compared. Here, we studied the phenotype of long-lived memory and atypical B cells to merozoite antigens (MSP1 and AMA1) and variant surface antigens (the CIDRα1 domain of PfEMP1) in ten Ugandan adults before and after local reduction of P. falciparum transmission. After a median of 1.7 years without P. falciparum infections, the percentage of antigen-specific activated B cells declined, but long-lived antigen-specific B cells were still detectable in all individuals. The majority of MSP1/AMA1-specific B cells were CD95+CD11c+ memory B cells, which are primed for rapid differentiation into antibody-secreting cells, and FcRL5-T-bet- atypical B cells. On the other hand, most CIDRα1-specific B cells were CD95-CD11c- memory B cells. CIDRα1-specific B cells were also enriched among a subset of atypical B cells that seem poised for antigen presentation. These results point to differences in how these antigens are recognized or processed by the immune system and how P. falciparum-specific B cells will respond upon re-infection.

Antibody-dependent cellular phagocytosis (ADCP) has been implicated in protection against HIV-1. However, methods for measuring ADCP currently rely on the phagocytosis of gp120- or gp41-coated beads that do not reflect physiologically relevant conformations of the viral envelope glycoprotein or the size of a virus-infected cell. We therefore developed a novel approach for measuring ADCP of HIV-infected cells expressing natural conformations of Env. A monocytic cell line (THP-1 cells) or primary human monocytes were incubated with a CD4+ T cell line that expresses eGFP upon HIV-1 infection in the presence of antibodies and ADCP was measured as the accumulation of eGFP+ material by flow cytometry. The internalization of HIV-infected cells by monocytes was confirmed visually by image-capture flow cytometry. Cytoskeletal remodeling, pseudopod formation and phagocytosis were also observed by confocal microscopy. We found that potent broadly neutralizing antibodies (bnAbs), but not non-neutralizing antibodies (nnAbs), mediate efficient phagocytosis of cells infected with either primary or lab-adapted HIV-1. A nnAb to a CD4-inducible epitope of gp120 (A32) failed to enable ADCP of HIV-infected cells but mediated efficient phagocytosis of gp120-coated beads. Conversely, a bnAb specific to intact Env trimers (PGT145) mediated potent ADCP of HIV-infected cells but did not facilitate the uptake of gp120-coated beads. These results underscore the importance of measuring ADCP of HIV-infected cells expressing physiologically relevant conformations of Env and show that most antibodies that are capable of binding to Env trimers on virions to neutralize virus infectivity are also capable of binding to Env on the surface of virus-infected cells to mediate ADCP.

The recurring spillover of pathogenic coronaviruses and demonstrated capacity of sarbecoviruses, such SARS-CoV-2, to rapidly evolve in humans underscores the need to better understand immune responses to this virus family. For this purpose, we characterized the functional breadth and potency of antibodies targeting the receptor binding domain (RBD) of the spike glycoprotein that exhibited cross-reactivity against SARS-CoV-2 variants, SARS-CoV-1 and sarbecoviruses from diverse clades and animal origins with spillover potential. One neutralizing antibody, C68.61, showed remarkable neutralization breadth against both SARS-CoV-2 variants and viruses from different sarbecovirus clades. C68.61, which targets a conserved RBD class 5 epitope, did not select for escape variants of SARS-CoV-2 or SARS-CoV-1 in culture nor have predicted escape variants among circulating SARS-CoV-2 strains, suggesting this epitope is functionally constrained. We identified 11 additional SARS-CoV-2/SARS-CoV-1 cross-reactive antibodies that target the more sequence conserved class 4 and class 5 epitopes within RBD that show activity against a subset of diverse sarbecoviruses with one antibody binding every single sarbecovirus RBD tested. A subset of these antibodies exhibited Fc-mediated effector functions as potent as antibodies that impact infection outcome in animal models. Thus, our study identified antibodies targeting conserved regions across SARS-CoV-2 variants and sarbecoviruses that may serve as therapeutics for pandemic preparedness as well as blueprints for the design of immunogens capable of eliciting cross-neutralizing responses.

Cottontail rabbit papillomavirus (CRPV), the first papillomavirus associated with tumor development, has been used as a powerful model to study papillomavirus pathogenesis for more than 90 years. However, lack of a comprehensive analysis of the CRPV transcriptome has impeded the understanding of CRPV biology and molecular pathogenesis. Here, we report the construction of a complete CRPV transcription map from Hershey CRPV-induced skin tumor tissues. By using RNA-seq in combination with long-reads PacBio Iso-seq, 5' and 3' RACE, primer-walking RT-PCR, Northern blot, and RNA in situ hybridization, we demonstrated that the CRPV genome transcribes its early and late RNA transcripts unidirectionally from at least five distinct major promoters (P) and polyadenylates its transcripts at two major polyadenylation (pA) sites. The viral early transcripts are primarily transcribed from three "early" promoters, P90, P156, and P907 and polyadenylated at nt 4368 by using an early polyadenylation signal (PAS) at nt 4351. Like other low-risk human papillomaviruses and animal papillomaviruses, CRPV E6 and E7 transcripts are transcribed from three separate early promoters. Transcripts from two "late" promoters, P7525, and P1225, utilize either an early PAS for E1^E4 or a late PAS at 7399 for L2 and L1 RNA polyadenylation at nt 7415 to express capsid L2 and L1 proteins respectively. By using the mapped four 5' splice sites and three 3' splice sites, CRPV RNA transcripts undergo extensive alternative splicing to produce more than 33 viral RNA isoforms for production of at least 12 viral proteins, some of which without codon optimization are expressible in rabbit RK13 and human HEK293T cells. The constructed full CRPV transcription map in this study for the first time will enhance our understanding of the structures and expressions of CRPV genes and their contribution to molecular pathogenesis and tumorigenesis.

Inflammasome activation is a robust innate immune mechanism that promotes inflammatory responses through the release of alarmins and leaderless cytokines, including IL-1α, IL-1β, and IL-18. Various stimuli, including infectious agents and cellular stress, cause inflammasomes to assemble and activate caspase-1. Then, caspase-1 cleaves targets that lead to pore formation and leaderless cytokine activation and release. Toxoplasma gondii has been shown to promote inflammasome formation, but the cell types utilizing caspase-1 and the downstream effects on immunological outcomes during acute in vivo infection have not been explored. Here, using knockout mice, we examine the role of caspase-1 responses during acute T. gondii infection globally and in Cx3cr1-positive populations. We provide in vivo evidence that caspase-1 expression is critical for, IL-18 release, optimal interferon-γ (IFN-γ) production, monocyte and neutrophil recruitment to the site of infection, and parasite control. Specifically, we find that caspase-1 expression in Cx3cr1-positive cells drives IL-18 release, which potentiates CD4+ T cell IFN-γ production and parasite control. Notably, our Cx3cr1-Casp1 knockouts exhibited a selective T cell defect, mirroring the phenotype observed in Il18 knockouts. In further support of this finding, treatment of Cx3cr1-Casp1 knockout mice with recombinant IL-18 restored CD4+ T cell IFN-γ responses and parasite control. Additionally, we show that neutrophil recruitment is dependent on IL-1 receptor accessory protein (IL-1RAP) signaling but is dispensable for parasite control. Overall, these experiments highlight the multifaceted role of caspase-1 in multiple cell populations contributing to specific pathways that collectively contribute to caspase-1 dependent immunity to T. gondii.

Viral infection leads to heterogeneous cellular outcomes ranging from refractory to abortive and fully productive states. Single cell transcriptomics enables a high resolution view of these distinct post-infection states. Here, we have interrogated the host-pathogen dynamics following reactivation of Epstein-Barr virus (EBV). While benign in most people, EBV is responsible for infectious mononucleosis, up to 2% of human cancers, and is a trigger for the development of multiple sclerosis. Following latency establishment in B cells, EBV reactivates and is shed in saliva to enable infection of new hosts. Beyond its importance for transmission, the lytic cycle is also implicated in EBV-associated oncogenesis. Conversely, induction of lytic reactivation in latent EBV-positive tumors presents a novel therapeutic opportunity. Therefore, defining the dynamics and heterogeneity of EBV lytic reactivation is a high priority to better understand pathogenesis and therapeutic potential. In this study, we applied single-cell techniques to analyze diverse fate trajectories during lytic reactivation in three B cell models. Consistent with prior work, we find that cell cycle and MYC expression correlate with cells refractory to lytic reactivation. We further found that lytic induction yields a continuum from abortive to complete reactivation. Abortive lytic cells upregulate NFκB and IRF3 pathway target genes, while cells that proceed through the full lytic cycle exhibit unexpected expression of genes associated with cellular reprogramming. Distinct subpopulations of lytic cells further displayed variable profiles for transcripts known to escape virus-mediated host shutoff. These data reveal previously unknown and promiscuous outcomes of lytic reactivation with broad implications for viral replication and EBV-associated oncogenesis.