Emergence and spread of SARS-CoV-2 lineage B.1.620 with variant of concern-like mutations and deletions.

Distinct SARS-CoV-2 lineages, discovered through various genomic surveillance initiatives, have emerged during the pandemic following unprecedented reductions in worldwide human mobility. We here describe a SARS-CoV-2 lineage - designated B.1.620 - discovered in Lithuania and carrying many mutations and deletions in the spike protein shared with widespread variants of concern (VOCs), including E484K, S477N and deletions HV69Δ, Y144Δ, and LLA241/243Δ. As well as documenting the suite of mutations this lineage carries, we also describe its potential to be resistant to neutralising antibodies, accompanying travel histories for a subset of European cases, evidence of local B.1.620 transmission in Europe with a focus on Lithuania, and significance of its prevalence in Central Africa owing to recent genome sequencing efforts there. We make a case for its likely Central African origin using advanced phylogeographic inference methodologies incorporating recorded travel histories of infected travellers.

Mutations of Kluyveromyces lactis dolichol kinase enhances secretion of recombinant proteins.

Although there are similarities in the core steps of the secretion pathway from yeast to higher eukaryotes, significant functional differences exist even among diverse yeast species. Here, we used next-generation sequencing to identify two mutations in the Kluyveromyces lactis KlSEC59 gene, encoding dolichol kinase (DK), which are responsible for an enhanced secretion phenotype in a previously isolated mutant, MD2/1-9. Compared with the temperature-sensitive Saccharomyces cerevisiae sec59-1 mutant, which exhibits reduced N-glycosylation and decreased secretory efficacy, the identified K. lactis DK mutations had fewer effects on glycosylation, as well as on survival at high temperature and cell wall integrity. Moreover, despite some glycosylation defects, double DK mutations (G405S and I419S) in the K. lactis mutant strain demonstrated three times the level of recombinant α-amylase secretion as the wild-type strain. Overexpression of potential suppressors KlMNN10, KlSEL1, KlERG20, KlSRT1, KlRER2, KlCAX4, KlLPP1 and KlDPP1 in the DK-mutant strain restored carboxypeptidase Y glycosylation to different extents and, with the exception of KISRT1, reduced α-amylase secretion to levels observed in wild-type cells. Our results suggest that enhanced secretion related to reduced activity of mutant DK in K. lactis results from mild glycosylation changes that affect activity of other proteins in the secretory pathway.

Purification of recombinant trichodysplasia spinulosa-associated polyomavirus VP1-derived virus-like particles using chromatographic techniques.

Trichodysplasia spinulosa-associated polyomavirus (TSPyV) has been linked to a rare and recently characterized skin disease occurring in immunocompromised patients. In analogy with other polyomaviruses, the major capsid protein VP1 of TSPyV can self-assemble into virus-like particles (VLPs). VLPs are increasingly applied for the vaccination and diagnostics. Mostly, non-scalable and labor intensive ultracentrifugation-based techniques are used for the purification of VLPs. In this work, we developed a purification procedure for TSPyV VP1 VLPs based on two chromatographic steps, ion-exchange monolith and core bead chromatography. Prior to chromatography, ammonium sulfate precipitation was used for the initial purification of TSPyV VP1 VLPs from yeast lysate. The VLPs were further purified using CIMmultus QA ion-exchange monolith in bind-elute mode. Most of TSPyV VP1 VLPs bound to the monolith and were subsequently eluted by a linear NaCl gradient. After ion-exchange monolith chromatography, the purity of TSPyV VP1 protein was about 75%. The final purification step of TSPyV VP1 VLPs was core bead chromatography using Capto Core 700 resin in flow-through mode. After core bead chromatography, 42% of TSPyV VP1 protein was recovered with a purity of 93%. The assembly of purified TSPyV VP1 protein into VLPs approximately 45-50 nm in diameter was confirmed by electron microscopy analysis. The purification procedure for TSPyV VP1 VLPs described here could be a scalable alternative to the conventional ultracentrifugation-based purification methods.

Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes.

Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes.

Production of recombinant VP1-derived virus-like particles from novel human polyomaviruses in yeast.

BACKGROUND: Eleven new human polyomaviruses (HPyVs) have been identified in the last decade. Serological studies show that these novel HPyVs sub-clinically infect humans at an early age. The routes of infection, entry pathways, and cell tropism of new HPyVs remain unknown. VP1 proteins of polyomaviruses can assembly into virus-like particles (VLPs). As cell culturing systems for HPyV are currently not available, VP1-derived VLPs may be useful tools in basic research and biotechnological applications. RESULTS: Recombinant VP1-derived VLPs from 11 newly identified HPyVs were efficiently expressed in yeast. VP1 proteins derived from Merkel cell polyomavirus (MCPyV), trichodysplasia spinulosa-associated polyomavirus (TSPyV), and New Jersey polyomavirus (NJPyV) self-assembled into homogeneous similarly-sized VLPs. Karolinska Institutet polyomavirus (KIPyV), HPyV7, HPyV9, HPyV10, and St. Louis polyomavirus (STLPyV) VP1 proteins formed VLPs that varied in size with diameters ranging from 20 to 60 nm. Smaller-sized VLPs (25-35 nm in diameter) predominated in preparations from Washington University polyomavirus (WUPyV) and HPyV6. Attempts to express recombinant HPyV12 VP1-derived VLPs in yeast indicate that translation of VP1 might start at the second of two potential translation initiation sites in the VP1-encoding open reading frame (ORF). This translation resulted in a 364-amino acid-long VP1 protein, which efficiently self-assembled into typical PyV VLPs. MCPyV-, KIPyV-, TSPyV-, HPyV9-, HPyV10-, and HPyV12-derived VLPs showed hemagglutination (HA) assay activity in guinea pig erythrocytes, whereas WUPyV-, HPyV6-, HPyV7-, STLPyV- and NJPyV-derived VP1 VLPs did not. CONCLUSIONS: The yeast expression system was successfully utilized for high-throughput production of recombinant VP1-derived VLPs from 11 newly identified HPyVs. HPyV12 VP1-derived VLPs were generated from the second of two potential translation initiation sites in the VP1-encoding ORF. Recombinant VLPs produced in yeast originated from different HPyVs demonstrated distinct HA activities and may be useful in virus diagnostics, capsid structure studies, or investigation of entry pathways and cell tropism of HPyVs until cell culture systems for new HPyVs are developed.