PLoS Pathogens最新文献

Recent HIV-1 vaccine development has centered on "near native" soluble envelope glycoprotein (Env) trimers that are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their effects in this context are unclear. To address this question, we used virus-like particle (VLP) produced in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed post-expression gp120/gp41 processing, concomitantly increasing trimer stability. Gp41 N-helix mutations I559P and NT1-5 imparted lateral trimer stability, but also reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ human plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, and also reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common side effects of these mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. A second, minor species of high mannose gp160 was unaffected by any mutations and instead bypasses normal folding and glycan maturation. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and greatly increased the proportion of high mannose gp160. Remarkably, monoclonal antibodies were unable to bind to this high mannose gp160 in native protein gels. Overall, our findings suggest caution in leveraging stabilizing mutations in nucleic acid-based immunogens to ensure they impart valuable membrane trimer phenotypes for vaccine use.

Among the many oral streptococci, Streptococcus pneumoniae (Spn) stands out for the capacity of encapsulated strains to cause invasive infection. Spread beyond upper airways, however, is a biological dead end for the organism, raising the question of the benefits of expending energy to coat its surface in a thick layer of capsular polysaccharide (CPS). In this study, we compare mutants of two serotypes expressing different amounts of CPS and test these in murine models of colonization, invasion infection and transmission. Our analysis of the effect of CPS amount shows that Spn expresses a capsule of sufficient thickness to shield its surface from the deposition of complement and binding of antibody to underlying epitopes. While effective shielding is permissive for invasive infection, its primary contribution to the organism appears to be in the dynamics of colonization. A thicker capsule increases bacterial retention in the nasopharynx, the first event in colonization, and also impedes IL-17-dependent clearance during late colonization. Enhanced colonization is associated with increased opportunity for host-to-host transmission. Additionally, we document substantial differences in CPS amount among clinical isolates of three common serotypes. Together, our findings show that CPS amount is highly variable among Spn and could be an independent determinant affecting host interactions.

Through the association of protein complexes to DNA, the eukaryotic nuclear genome is broadly organized into open euchromatin that is accessible for enzymes acting on DNA and condensed heterochromatin that is inaccessible. Chemical and physical alterations to chromatin may impact its organization and functionality and are therefore important regulators of nuclear processes. Studies in various fungal plant pathogens have uncovered an association between chromatin organization and expression of in planta-induced genes that are important for pathogenicity. This review discusses chromatin-based regulation mechanisms as determined in the fungal plant pathogen Verticillium dahliae and relates the importance of epigenetic transcriptional regulation and other nuclear processes more broadly in fungal plant pathogens.

[This corrects the article DOI: 10.1371/journal.ppat.1011522.].

The Coronavirus envelope (E) protein is a small structural protein with ion channel activity that plays an important role in virus assembly, budding, immunopathogenesis and disease severity. The viroporin E is also located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we evaluated in vitro antiviral activity, mechanism of action and in vivo efficacy of BIT225 for the treatment of SARS-CoV-2 infection. BIT225 showed broad-spectrum direct-acting antiviral activity against SARS-CoV-2 in Calu3 and Vero cells with similar potency across 6 different virus strains. BIT225 inhibited ion channel activity of E protein but did not inhibit endogenous currents or calcium-induced ion channel activity of TMEM16A in Xenopus oocytes. BIT225 administered by oral gavage for 12 days starting 12 hours before infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (100% survival, n = 12), while all vehicle-dosed animals reached a mortality endpoint by Day 9 across two studies (n = 12). When treatment started at 24 hours after infection, body weight loss, and mortality were also prevented (100% survival, n = 5), while 4 of 5 mice maintained and increased body weight and survived when treatment started 48 hours after infection. Treatment efficacy was dependent on BIT225 dose and was associated with significant reductions in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. These results validate viroporin E as a viable antiviral target and support the clinical study of BIT225 for treatment and prophylaxis of SARS-CoV-2 infection.

Plasticity of influenza virus hemagglutinin (HA) conformation increases an opportunity to generate conserved non-native epitopes with unknown functionality. Here, we have performed an in-depth analysis of human monoclonal antibodies against a stem-helix region that is occluded in native prefusion yet exposed in postfusion HA. A stem-helix antibody, LAH31, provided IgG Fc-dependent cross-group protection by targeting a stem-helix kinked loop epitope, with a unique structure emerging in the postfusion state. The structural analysis and molecular modeling revealed key contact sites responsible for the epitope specificity and cross-group breadth that relies on somatically mutated light chain. LAH31 was inaccessible to the native prefusion HA expressed on cell surface; however, it bound to the HA structure present on infected cells with functional linkage to the Fc-mediated clearance. Our study uncovers a novel non-native epitope that emerges in the postfusion HA state, highlighting the utility of this epitope for a broadly protective antigen design.

By applying dual proteome profiling to Salmonella enterica serovar Typhimurium (S. Typhimurium) encounters with its epithelial host (here, S. Typhimurium infected human HeLa cells), a detailed interdependent and holistic proteomic perspective on host-pathogen interactions over the time course of infection was obtained. Data-independent acquisition (DIA)-based proteomics was found to outperform data-dependent acquisition (DDA) workflows, especially in identifying the downregulated bacterial proteome response during infection progression by permitting quantification of low abundant bacterial proteins at early times of infection when bacterial infection load is low. S. Typhimurium invasion and replication specific proteomic signatures in epithelial cells revealed interdependent host/pathogen specific responses besides pointing to putative novel infection markers and signalling responses, including regulated host proteins associated with Salmonella-modified membranes.

New variants of SARS-CoV-2 are continually emerging with mutations in spike associated with increased transmissibility and immune escape. Phenotypic maps can inform the prediction of concerning mutations from genomic surveillance, however most of these maps currently derive from studies using monomeric RBD, while spike is trimeric, and contains additional domains. These maps may fail to reflect interdomain interactions in the prediction of phenotypes. To try to improve on this, we developed a platform for deep mutational scanning using whole trimeric spike. We confirmed a previously reported epistatic effect within the RBD affecting ACE2 binding, that highlights the importance of updating the base spike sequence for future mutational scanning studies. Using post vaccine sera, we found that the immune response of vaccinated individuals was highly focused on one or two epitopes in the RBD and that single point mutations at these positions can account for most of the immune escape mediated by the Omicron BA.1 RBD. However, unexpectedly we found that the BA.1 RBD alone does not account for the high level of antigenic escape by BA.1 spike. We show that the BA.1 NTD amplifies the immune evasion of its associated RBD. BA.1 NTD reduces neutralistion by RBD directed monoclonal antibodies, and impacts ACE2 interaction. NTD variation is thus an important mechanism of immune evasion by SARS-CoV-2. Such effects are not seen when pre-stabilized spike proteins are used, suggesting the interdomain effects require protein mobility to express their phenotype.

The Bunyavirales order is a large and diverse group of segmented negative-strand RNA viruses. Several virus families within this order contain important human pathogens, including Sin Nombre virus (SNV) of the Hantaviridae. Despite the high epidemic potential of bunyaviruses, specific medical countermeasures such as vaccines or antivirals are missing. The multifunctional ~250 kDa L protein of hantaviruses, amongst other functional domains, harbors the RNA-dependent RNA polymerase (RdRp) and an endonuclease and catalyzes transcription as well as replication of the viral RNA genome, making it a promising therapeutic target. The development of inhibitors targeting these key processes requires a profound understanding of the catalytic mechanisms. Here, we established expression and purification protocols of the full-length SNV L protein bearing the endonuclease mutation K124A. We applied different biochemical in vitro assays to provide an extensive characterization of the different enzymatic functions as well as the capacity of the hantavirus L protein to interact with the viral RNA. By using single-particle cryo-EM, we obtained a 3D model including the L protein core region containing the RdRp, in complex with the 5' promoter RNA. This first high-resolution model of a New World hantavirus L protein shows striking similarity to related bunyavirus L proteins. The interaction of the L protein with the 5' RNA observed in the structural model confirms our hypothesis of protein-RNA binding based on our biochemical data. Taken together, this study provides an excellent basis for future structural and functional studies on the hantavirus L protein and for the development of antiviral compounds.

[This corrects the article DOI: 10.1371/journal.ppat.1011473.].