Biology subjects

Kalnciema, I.

Publications and source records attributed to Kalnciema, I..

4 recordsLinked to original sources

bioRxiv2026

A plant virus-based virus like-particle platform for therapeutic vaccination targeting IL-23A

Interleukin-23 (IL-23) is a pivotal cytokine in the pathogenesis of immune-mediated inflammatory diseases, making it an attractive therapeutic target. Vaccination against self-antigens offers an alternative to monoclonal antibody (mAb) therapies by eliciting endogenous Ab responses. Here, we report the development of a virus-like particle (VLP)-based vaccine targeting murine IL-23A (mIL23A) using the eggplant mosaic virus (EMV) coat protein as a carrier. mIL23A was incorporated either as a direct fusion or within a mosaic system, with the latter supporting efficient VLP assembly. Alternative antigen presentation strategies employed synthetic coiled-coil and zipper pairs. All constructs were expressed in E. coli, purified, and characterized by SDS-PAGE, TEM, and DLS. Immunization of BALB/c mice elicited robust mIL23A-specific Abs, with mosaic VLPs inducing high-avidity, Th1-biased responses. Encapsidated RNA within VLPs was double-stranded and stimulated TLR3, enhancing immunogenicity. Scale-up in bioreactors preserved VLP integrity and antigen incorporation, yielding 1.72 mg per gram of cells. These results establish EMV VLPs as a flexible and scalable platform for presenting challenging antigens and provide a promising approach for therapeutic vaccination against self-antigens.

immunology↗

bioRxiv2024

Investigation of plant virus-like particle formation in bacterial and yeast expression systems

Virus-like particles (VLPs) have garnered significant attention due to their potential applications in various fields, particularly in biomedicine, where they are used for drug delivery, vaccine development, and as diagnostic tools. Plant virus-derived VLPs have been especially successful, leading to a surge in research aimed at developing new VLPs. Despite this growing interest, not all attempts at VLP formation are successful. Understanding the factors that contribute to successful VLP assembly is crucial for advancing this field. In this study, we focus on the coat proteins (CPs) of three plant viruses belonging to the Sobemovirus genus as a model to investigate the process of VLP formation. Our findings demonstrated that the strong binding of CPs to ssDNA can be a major reason for unsuccessful VLP production. This issue can potentially be overcome by transitioning from an episomal to a chromosome-integrated expression system. By doing so, we aim to elucidate the mechanisms that facilitate successful VLP assembly. Our findings provide valuable insights into the properties of sobemovirus CPs and highlight their potential for encapsulating foreign nucleic acids, thereby providing additional applications as nanocontainers or vaccine platforms with interior modification capabilities for enhanced immune response. Moreover, our RNA-Seq data indicates that sobemovirus-derived VLPs predominantly package their CP mRNA, irrespective of the expression system used. This characteristic can be utilized for the functional encapsulation of nucleic acids, enhancing the versatility and utility of these VLPs in various biotechnological applications.

molecular biology↗

bioRxiv2023

VPg impact on Ryegrass mottle virus serine-like 3C protease proteolysis and structure

Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). NMR studies show a Pro-VPg complex interaction and VPg tertiary structure; however, information regarding structural changes of the Pro-VPg complex during interaction is lacking. Here, we solved a full Pro-VPg 3D structure of ryegrass mottle virus (RGMoV) that demonstrates the structural changes in three different conformations due to VPg interaction with Pro. We identified a unique site of VPg interaction with Pro that was not observed in other sobemoviruses and observed different conformations of the Pro {beta}2 barrel. This is the first report of a full plant Pro crystal structure with its VPg cofactor. We also confirmed the existence of an unusual previously unmapped cleavage site for sobemovirus Pro in the transmembrane domain: E/A. We demonstrated that RGMoV Pro in cis activity is not regulated by VPg and that in trans, VPg can also mediate Pro in free form. Additionally, we observed Ca2+ and Zn2+ inhibitory activities on the Pro cleavage activity. Author summaryThe gRNA of sobemoviruses encodes two polyproteins that are processed by a serine protease. We found that in the bacterial expression system, Pro is active in cis and in trans, where only in trans activity is mediated by VPg not only in the fusion form with Pro but also in the free form. Here, we present structural changes in the catalytic and substrate-binding sites of Pro caused by VPg, which can explain the in trans activity and structure of sobemovirus VPg C-terminal peptide. In addition, we confirmed a new cleavage site not previously characterized in sobemoviruses. Additionally, Ca2+ and Zn2+ decreased Pro cleavage activity. This information could provide a better understanding of a serine protease and their proteolytic mechanisms during viral protein maturation.

microbiology↗

bioRxiv2022

Identification and full genome analysis of the first putative virus of sea buckthorn (Hippophae rhamnoides L.)

The agricultural importance of sea buckthorn (Hippophae rhamnoides L.) is rapidly increasing. Several bacterial and fungal pathogens infecting sea buckthorn have been identified and characterized; however, the viral pathogens are not yet known. In this study, we identified, isolated, and sequenced a virus from a wild plantation of sea buckthorn for the first time. Sequence analysis of the obtained viral genome revealed high similarity with sequences of several viruses belonging to the genus Marafivirus, especially olive latent virus 3 (OLV-3). The genome of the new virus is 6,989 nucleotides (nt) in length according to 5' and 3' rapid amplification of cDNA ends (RACE) (without polyA-tail), with 5' and 3' untranslated regions being 133 and 109 nt long, respectively. The viral genome encoded two open reading frames (ORFs). ORF1 encoded a polyprotein of 1,954 amino acids (aa) with the characteristic marafivirus non-structural protein domains--methyltransferase, Salyut domain, papain-like cysteine protease, helicase, and RNA-dependent RNA polymerase. ORF1 was separated from ORF2 by a six nt and encoded the coat protein (CP). CP had typical signatures of minor (30.96 kDa) and major (21.18 kDa) forms. Both CP forms were cloned and expressed in a bacterial expression system, and only the major CP was able to self-assemble into 30 nm virus-like particles that resembled the native virus, thus demonstrating that minor CP is not essential for virion assembly. We suggest the newly discovered virus to be named as "Sea buckthorn marafivirus", abbreviated as "SBuMV". Author summarySea buckthorn is an exceptionally valuable plant that is currently widely cultivated as multipurpose horticultural species for food, pharmacology, cosmetics, and landscape conservation. Diseases and pests directly affect the cultivation of SBT. To date, several pests and diseases, mainly fungal and bacterial, but no viral, sea buckthorn have been reported. Identification of new pathogens would assist in the development of control strategies, and quarantine purposes and ensure sustainable sea buckthorn cultivation. Here, for the first time, we present a virus putatively infecting sea buckthorn. We had characterized its full genome, cloned and expressed minor and major forms of coat protein either individually or co-expressed. We also showed that major coat protein-derived virus-like particles self- assembled directly in the bacterial cells, and the majority of the expressed CPs were soluble. Our study suggests that the minor CP is not essential for the assembly of seemingly structurally intact viral particles, meaning that it can have other functions.

molecular biology↗