Type I interferon shapes the quantity and quality of the anti-Zika virus antibody response.

OBJECTIVES: Zika virus (ZIKV) is a mosquito-borne flavivirus that re-emerged in 2015. The association between ZIKV and neurological complications initiated the development of relevant animal models to understand the mechanisms underlying ZIKV-induced pathologies. Transient inhibition of the type I interferon (IFN) pathway through the use of an IFNAR1-blocking antibody, MAR1-5A3, could efficiently permit active virus replication in immunocompetent animals. Type I IFN signalling is involved in the regulation of humoral responses, and thus, it is crucial to investigate the potential effects of type I IFN blockade towards B-cell responses. METHODS: In this study, comparative analysis was conducted using serum samples collected from ZIKV-infected wild-type (WT) animals either administered with or without MAR1-5A3. RESULTS: Serological assays revealed a more robust ZIKV-specific IgG response and subtype switching upon inhibition of type I IFN due to the abundance of antigen availability. This observation was corroborated by an increase in germinal centres, plasma cells and germinal centre B cells. Interestingly, although both groups of animals recognised different B-cell linear epitopes in the E and NS1 regions, there was no difference in neutralising capacity. Further characterisation of these epitopes in the E protein revealed a detrimental role of antibodies that were generated in the absence of type I IFN. CONCLUSION: This study highlights the role of type I IFN in shaping the anti-ZIKV antibody response to generate beneficial antibodies and will help guide development of better vaccine candidates triggering efficient neutralising antibodies and avoiding detrimental ones.

Erratum: Novel differential linear B-cell epitopes to identify Zika and dengue virus infections in patients.

[This corrects the article DOI: 10.1002/cti2.1066.].

Novel differential linear B-cell epitopes to identify Zika and dengue virus infections in patients.

OBJECTIVES: Recent Zika virus (ZIKV) outbreaks challenged existing laboratory diagnostic standards, especially for serology-based methods. Because of the genetic and structural similarity of ZIKV with other flaviviruses, this results in cross-reactive antibodies, which confounds serological interpretations. METHODS: Plasma from Singapore ZIKV patients was screened longitudinally for antibody responses and neutralising capacities against ZIKV. Samples from healthy controls, ZIKV patients and DENV patients were further assessed using ZIKV and DENV peptides of precursor membrane (prM), envelope (E) or non-structural 1 (NS1) viral proteins in a peptide-based ELISA for epitope identification. Identified epitopes were re-validated and diagnostically evaluated using sera of patients with DENV, bacteria or unknown infections from Thailand. RESULTS: Long-lasting ZIKV-neutralising antibodies were elicited during ZIKV infection. Thirteen potential linear B-cell epitopes were identified, and of these, four common flavivirus, three ZIKV-specific and one DENV-specific differential epitopes had more than 50% sensitivity and specificity. Notably, ZIKV-specific peptide 26 on domain I/II of E protein (amino acid residues 271-288) presented 80% sensitivity and 85.7% specificity. Importantly, the differential epitopes also showed significance in differentiating non-flavivirus patient samples. CONCLUSION: Linear B-cell epitope candidates to differentiate between ZIKV and DENV infections were identified, providing the first step towards the design of a much-needed serology-based assay.

Mutating chikungunya virus non-structural protein produces potent live-attenuated vaccine candidate.

Currently, there are no commercially available live-attenuated vaccines against chikungunya virus (CHIKV). Here, CHIKVs with mutations in non-structural proteins (nsPs) were investigated for their suitability as attenuated CHIKV vaccines. R532H mutation in nsP1 caused reduced infectivity in mouse tail fibroblasts but an enhanced type-I IFN response compared to WT-CHIKV Adult mice infected with this nsP-mutant exhibited a mild joint phenotype with low-level viremia that rapidly cleared. Mechanistically, ingenuity pathway analyses revealed a tilt in the anti-inflammatory IL-10 versus pro-inflammatory IL-1ß and IL-18 balance during CHIKV nsP-mutant infection that modified acute antiviral and cell signaling canonical pathways. Challenging CHIKV nsP-mutant-infected mice with WT-CHIKV or the closely related O'nyong-nyong virus resulted in no detectable viremia, observable joint inflammation, or damage. Challenged mice showed high antibody titers with efficient neutralizing capacity, indicative of immunological memory. Manipulating molecular processes that govern CHIKV replication could lead to plausible vaccine candidates against alphavirus infection.

Nonstructural Proteins of Alphavirus-Potential Targets for Drug Development.

Alphaviruses are enveloped, positive single-stranded RNA viruses, typically transmitted by arthropods. They often cause arthralgia or encephalitic diseases in infected humans and there is currently no targeted antiviral treatment available. The re-emergence of alphaviruses in Asia, Europe, and the Americas over the last decade, including chikungunya and o'nyong'nyong viruses, have intensified the search for selective inhibitors. In this review, we highlight key molecular determinants within the alphavirus replication complex that have been identified as viral targets, focusing on their structure and functionality in viral dissemination. We also summarize recent structural data of these viral targets and discuss how these could serve as templates to facilitate structure-based drug design and development of small molecule inhibitors.

p130Cas-dependent actin remodelling regulates myogenic differentiation.

Actin dynamics are implicated in various cellular processes, not only through the regulation of cytoskeletal organization, but also via the control of gene expression. In the present study we show that the Src family kinase substrate p130Cas (Cas is Crk-associated substrate) influences actin remodelling and concomitant muscle-specific gene expression, thereby regulating myogenic differentiation. In C2C12 myoblasts, silencing of p130Cas expression by RNA interference impaired F-actin (filamentous actin) formation and nuclear localization of the SRF (serum-response factor) co-activator MAL (megakaryocytic acute leukaemia) following the induction of myogenic differentiation. Consequently, formation of multinucleated myotubes was abolished. Re-introduction of wild-type p130Cas, but not its phosphorylation-defective mutant, into p130Cas-knockdown myoblasts restored F-actin assembly, MAL nuclear localization and myotube formation. Depletion of the adhesion molecule integrin ß3, a key regulator of myogenic differentiation as well as actin cytoskeletal organization, attenuated p130Cas phosphorylation and MAL nuclear localization during C2C12 differentiation. Moreover, knockdown of p130Cas led to the activation of the F-actin-severing protein cofilin. The introduction of a dominant-negative mutant of cofilin into p130Cas-knockdown myoblasts restored muscle-specific gene expression and myotube formation. The results of the present study suggest that p130Cas phosphorylation, mediated by integrin ß3, facilitates cofilin inactivation and promotes myogenic differentiation through modulating actin cytoskeleton remodelling.

Expanding the chemical biologist's tool kit: chemical labelling strategies and its applications.

Methods that allow visualisation of proteins in living systems, in real time have been key to our understanding of the molecular underpinnings of life. Although the use of genetically encoded fusions to fluorescent proteins have greatly advanced such studies, the large size of these tags and their ability to perturb protein activity has been major limitations. Attempts to circumvent these issues have seen the genesis of complementary strategies to chemically label/modify proteins. Thus, chemical labelling approaches seek to "decorate" biomolecules in live cells through the site-specific introduction of a small, non-native chemical tag (or reporter group). The introduced tag is minimally invasive such that the activity and/or function of the target molecule in not perturbed/compromised by its inclusion. In most cases, this modification is brought about by fusing target biomolecules to protein domains/peptide tags or via the incorporation of reactive "handles" by either exploiting the cell's biosynthetic machinery or during protein synthesis. Selective tagging of the biomolecule then proceeds via a bioorthogonal chemical reaction following exogenous addition of probe(s). Depending on the nature of the probe, the method can be applied to either visualise/track the dynamics of target molecule(s) in their native cellular milieu or for affinity enrichment for further downstream applications. The versatility of these approaches has been demonstrated by their ability to tag not just proteins but also intractable biomolecules like lipids and glycans. In this review, we summarise the various strategies available to "chemically" tag proteins and provide a comparative analysis their advantages and disadvantages. We also highlight the many creative applications of such methodologies and discuss their future prospects.

Use of intein-mediated protein ligation strategies for the fabrication of functional protein arrays.

This section introduces a simple, rapid, high-throughput methodology for the site-specific biotinylation of proteins for the purpose of fabricating functional protein arrays. Step-by-step protocols are provided to generate biotinylated proteins using in vitro, in vivo, or cell-free systems, together with useful hints for troubleshooting. In vitro and in vivo biotinylation rely on the chemoselective native chemical ligation (NCL) reaction between the reactive alpha-thioester group at the C-terminus of target proteins, generated via intein-mediated cleavage, and the added cysteine biotin. The cell-free system uses a low concentration of biotin-conjugated puromycin. The biotinylated proteins can be either purified or directly captured from crude cellular lysates onto an avidin-functionalized slide to afford the corresponding protein array. The methods were designed to preserve the activity of the immobilized protein such that the arrays provide a highly miniaturized platform to simultaneously interrogate the functional activities of thousands of proteins. This is of paramount significance, as new applications of microarray technologies continue to emerge, fueling their growth as an essential tool for high-throughput proteomic studies.