Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization

West Nile virions incorporate 180 envelope (E) proteins that orchestrate the process of virus entry and are the primary target of neutralizing antibodies. The E proteins of newly synthesized West Nile virus (WNV) are organized into trimeric spikes composed of pre-membrane (prM) and E protein heterodimers. During egress, immature virions undergo a protease-mediated cleavage of prM that results in a reorganization of E protein into the pseudo-icosahedral arrangement characteristic of mature virions. While cleavage of prM is a required step in the virus life cycle, complete maturation is not required for infectivity and infectious virions may be heterogeneous with respect to the extent of prM cleavage. In this study, we demonstrate that virion maturation impacts the sensitivity of WNV to antibody-mediated neutralization. Complete maturation results in a significant reduction in sensitivity to neutralization by antibodies specific for poorly accessible epitopes that comprise a major component of the human antibody response following WNV infection or vaccination. This reduction in neutralization sensitivity reflects a decrease in the accessibility of epitopes on virions to levels that fall below a threshold required for neutralization. Thus, in addition to a role in facilitating viral entry, changes in E protein arrangement associated with maturation modulate neutralization sensitivity and introduce an additional layer of complexity into humoral immunity against WNV

Ups and Downs of Poised RNA Polymerase II in B-Cells.

Recent genome-wide analyses have uncovered a high accumulation of RNA polymerase II (Pol II) at the 5' end of genes. This elevated Pol II presence at promoters, referred to here as Poll II poising, is mainly (but not exclusively) attributed to temporal pausing of transcription during early elongation which, in turn, has been proposed to be a regulatory step for processes that need to be activated "on demand". Yet, the full genome-wide regulatory role of Pol II poising is yet to be delineated. To elucidate the role of Pol II poising in B cell activation, we compared Pol II profiles in resting and activated B cells. We found that while Pol II poised genes generally overlap functionally among different B cell states and correspond to the functional groups previously identified for other cell types, non-poised genes are B cell state specific. Focusing on the changes in transcription activity upon B cell activation, we found that the majority of such changes were from poised to non-poised state. The genes showing this type of transition were functionally enriched in translation, RNA processing and mRNA metabolic process. Interestingly, we also observed a transition from non-poised to poised state. Within this set of genes we identified several Immediate Early Genes (IEG), which were highly expressed in resting B cell and shifted from non-poised to poised state after B cell activation. Thus Pol II poising does not only mark genes for rapid expression in the future, but it is also associated with genes that are silenced after a burst of their expression. Finally, we performed comparative analysis of the presence of G4 motifs in the context of poised versus non-poised but active genes. Interestingly we observed a differential enrichment of these motifs upstream versus downstream of TSS depending on poising status. The enrichment of G4 sequence motifs upstream of TSS of non-poised active genes suggests a potential role of quadruplexes in expression regulation

Protonation of Individual Histidine Residues Is Not Required for the pH-Dependent Entry of West Nile Virus: Evaluation of the “Histidine Switch” Hypothesis▿

Histidine residues have been hypothesized to function as sensors of environmental pH that can trigger the activity of viral fusion proteins. We investigated a requirement for histidine residues in the envelope (E) protein of West Nile virus during pH-dependent entry into cells. Each histidine was individually replaced with a nonionizable amino acid and tested functionally. In each instance, mutants capable of orchestrating pH-dependent infection were identified. These results do not support a requirement for any single histidine as a pH-sensing “switch,” and they suggest that additional features of the E protein are involved in triggering pH-dependent steps in the flavivirus life cycle

Pol II density profiles of Fos and Jun in resting and activated B cells.

<p>Pol II density profiles in the genomic region along (a) Fos and (b) Jun genes in RESTB and ACTB cells. Data were normalized as reads per million reads mapped (TPM).</p

Biological processes enriched in poised with background of Pol II⁺ genes.

<p>Biological processes enriched in poised with background of Pol II⁺ genes.</p

Distributions of Pol II density and its poising index.

<p>Violin plots showing (a) the distributions of Pol II promoter density, (b) Pol II gene body density, and (c) poising index values for 9710 genes in ACTB and 9290 genes in RESTB cells with Pol II⁺ promoters. Pol II densities were normalized to reads per kilobase per million reads mapped (RPKM). The y-axes are in a logarithmic scale.</p

Changes in Pol II profile, gene expression, and poising index upon B cell activation.

<p>a) Violin plots comparing distributions of fold changes of Pol II promoter density (left) and Pol II gene body density (right) for two groups genes: genes that continued to be poised after B cell activation (6802 genes) and genes that switch from poised to non-poised class during B cell activation (1138 genes). The y-axes are in a logarithmic scale. The p-values from Mann Whitney U test and Cohen's d effect sizes that reflect the differences between two groups are reported on top. b) Violin plots comparing the distributions of fold changes of poising index and mRNA expression in two groups of genes based on their change in transcriptional activity along gene body upon B cell activation: genes with similar Pol II body density (2/3 < fold change < 1.5; 6069 genes) and genes with large increase of Pol II body density (fold change > 1.5; 1491 genes) between RESTB and ACTB. c) The Spearman and partial Spearman correlations between changes in gene expression and changes in Pol II promoter/gene body density during the transition from RESTB to ACTB for common 8769 Pol II⁺ genes.</p

Statistical differences in the number of G4 sequence motifs between poised and non-poised genes, and between high and low ratio of expression at 30m over 72h after cell activation.

<p>We plot p-values of the Mann-Whitney-Wilcoxon tests in log scale. The relative position of the bars with respect to the central line indicates enriched category for a given gene group.</p

Biological processes enriched in poised genes with background of all genes.

<p>Biological processes enriched in poised genes with background of all genes.</p

Biological processes enriched in non-poised genes with background of Pol II⁺ genes.

<p>Biological processes enriched in non-poised genes with background of Pol II⁺ genes.</p