Charge-changing point mutations in the E protein of tick-borne encephalitis virus.

Introduction of point mutations is one of the forces enabling arboviruses to rapidly adapt in a changing environment. The influence of these mutations on the properties of the virus is not always obvious. In this study, we attempted to clarify this influence using an in silico approach. Using molecular dynamics (MD) simulations, we investigated how the position of charge-changing point mutations influences the structure and conformational stability of the E protein for a set of variants of a single TBEV strain. The computational findings were supported by experimental evaluation of relevant properties of virions, such as binding to heparan sulfate, thermostability, and susceptibility of the viral hemagglutinating activity to detergents. Our results also point to relationships between E protein dynamics and viral neuroinvasiveness.

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression.

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type-specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III ß-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.

Intracellular degradation and localization of NS1 of tick-borne encephalitis virus affect its protective properties.

Currently, many DNA vaccines against infectious diseases are in clinical trials; however, their efficacy needs to be improved. The potency of DNA immunogen can be optimized by targeting technologies. In the current study, to increase the efficacy of NS1 encoded by plasmid, proteasome targeting was applied. NS1 variants with or without translocation sequence and with ornithine decarboxylase as a signal of proteasomal degradation were tested for expression, localization, protein turnover, proteasomal degradation and protection properties. Deletion of translocation signal abrogated presentation of NS1 on the cell surface and increased proteasomal processing of NS1. Fusion with ornithine decarboxylase led to an increase of protein turnover and the proteasome degradation rate of NS1. Immunization with NS1 variants with increased proteasome processing protected mice from viral challenge only partially; however, the survival time of infected mice was prolonged in these groups. These data can give a presupposition for formulation of specific immune therapy for infected individuals.

Lethal Experimental Tick-Borne Encephalitis Infection: Influence of Two Strains with Similar Virulence on the Immune Response.

Tick-borne encephalitis virus (TBEV) is a tick-transmitted arbovirus that causes serious diseases in humans in Europe and Northern Asia. About 6000-10,000 cases are registered annually, and one-third of them lead to sequela with different degrees of severity. Two TBEV strains (Absettarov and EK-328) similar in virulence rate in laboratory mice were used to study pathogenesis and immune response upon lethal infection in mice. The strains differed in the dynamics of appearance of virus, IFNs and other cytokines in blood of mice, and ability to induce a cytokine storm in the terminal stages of disease and a non-sterile immunity. Moreover, the TBEV strains differed in characteristics of their interactions with DCs: level of reproduction in these cells, virus dose triggering IFN-α production, and impact on DCs' maturation. Infection of DCs with Absettarov strain led to IFN-α induction only at high multiplicity of infection (MOI), and an increased amount of the mature DCs with high adhesion activity and low-level of MHCII positive cells. While reproduction of the EK-328 strain in DCs was less efficient, a low dose of the virus induced IFN-α production and stimulated maturation of DCs with relatively low adhesive capacity, but with the high percentage of cells expressing MHCII molecules. Thus, the studied strains differed significantly in the impact on DCs' maturation and antigen presentation to CD4+ lymphocytes. Injection of low (103 PFU) and high (106 PFU) doses of both TBEV strains caused a lethal infection in mice. At the same time, the dose of the virus in the inoculum, regardless of the strain properties, affected the following virulence characteristics: the time of virus appearance in brain (day 4-5 vs. day 1 p.i.), time of IFN-α appearance in blood (10 h vs. 5 h p.i.), concentration of IFN-α in blood, and induction of IFN-α during infection of DCs. Therefore, virulent TBEV strains during lethal infection can interact differently with the host immune system, and the infectious dose has an impact on both: virus spread in the infected organism and immune response activation.

Minimal features of efficient incorporation of the hemagglutinin-neuraminidase protein into sendai virus particles.

Two transmembrane glycoproteins form spikes on the surface of Sendai virus, a member of the Respirovirus genus of the Paramyxovirinae subfamily of the Paramyxoviridae family: the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. HN, in contrast to F, is dispensable for viral particle production, as normal amounts of particles can be produced with highly reduced levels of HN. This HN reduction can result from mutation of an SYWST motif in its cytoplasmic tail to AFYKD. HNAFYKD accumulates at the infected cell surface but does not get incorporated into particles. In this work, we derived experimental tools to rescue HNAFYKD incorporation. We found that coexpression of a truncated HN harboring the wild-type cytoplasmic tail, the transmembrane domain, and at most 80 amino acids of the ectodomain was sufficient to complement defective HNAFYKD incorporation into particles. This relied on formation of disulfide-bound heterodimers carried out by the two cysteines present in the HN 80-amino-acid (aa) ectodomain. Finally, the replacement of the measles virus H cytoplasmic and transmembrane domains with the corresponding HN domains promoted measles virus H incorporation in Sendai virus particles.

Mutation of the TYTLE motif in the cytoplasmic tail of the sendai virus fusion protein deeply affects viral assembly and particle production.

Enveloped viruses contain glycoproteins protruding from the viral membrane. These proteins play a crucial role in the extra-cellular steps of the virus life cycle, namely attachment to and entry into cells. Their role during the intracellular late phase of virus multiplication has been less appreciated, overlooked by the documented central organizer role of the matrix M protein. Sendai virus, a member of the Paramyxoviridae family, expresses two trans-membrane proteins on its surface, HN and F. In previous work, we have shown that suppression of F in the context of an infection, results in about 70% reduction of virus particle production, a reduction similar to that observed upon suppression of the matrix M protein. Moreover, a TYTLE motif present in F cytoplasmic tail has been proposed essential for virus particle production. In the present work, using original alternate conditional siRNA suppression systems, we generated a double F gene recombinant Sendai virus expressing wt-F and a nonviable mutated TYTLE/5A F protein (F5A). Suppression of the wild type F gene expression in cells infected with this virus allowed the analysis of F5A properties in the context of the infection. Coupling confocal imaging analysis to biochemical characterization, we found that F5A i) was not expressed at the cell surface but restricted to the endoplasmic reticulum, ii) was still capable of interaction with M and iii) had profound effect on M and HN cellular distribution. On the basis of these data, we propose a model for SeV particle formation based on an M/F complex that would serve as nucleation site for virus particle assembly at the cell surface.