A Novel Virus Discovered in the Yeast Pichia membranifaciens.

Mycoviruses are widely distributed across fungi, including yeasts of the Saccharomycotina subphylum. It was recently discovered that the yeast species Pichia membranifaciens contained double stranded RNAs (dsRNAs) that were predicted to be of viral origin. The fully sequenced dsRNA is 4,578 bp in length, with RNA secondary structures similar to the packaging, replication, and frameshift signals of totiviruses of the family Totiviridae. This novel virus has been named Pichia membranifaciens virus L-A (PmV-L-A) and is related to other totiviruses previously described within the Saccharomycotina yeasts. PmV-L-A is part of a monophyletic subgroup within the I-A totiviruses, implying a common ancestry between mycoviruses isolated from the Pichiaceae and Saccharomycetaceae yeasts. Energy minimized AlphaFold2 molecular models of the PmV-L-A Gag protein revealed structural conservation with the previously solved structure of the Saccharomyces cerevisiae virus L-A (ScV-L-A) Gag protein. The predicted tertiary structure of the PmV-L-A Pol and its homologs provide details of the potential mechanism of totivirus RNA-dependent RNA polymerases (RdRps) because of structural similarities to the RdRps of mammalian dsRNA viruses. Insights into the structure, function, and evolution of totiviruses gained from yeasts is important because of their parallels with mammalian viruses and the emerging role of totiviruses in animal disease.

Affinity and Specificity for Binding to Glycosaminoglycans Can Be Tuned by Adapting Peptide Length and Sequence

Although glycosaminoglycan (GAG)–protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound to cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs.

Transposons Hidden in Arabidopsis thaliana Genome Assembly Gaps and Mobilization of Non-Autonomous LTR Retrotransposons Unravelled by Nanotei Pipeline

Long-read data is a great tool to discover new active transposable elements (TEs). However, no ready-to-use tools were available to gather this information from low coverage ONT datasets. Here, we developed a novel pipeline, nanotei, that allows detection of TE-contained structural variants, including individual TE transpositions. We exploited this pipeline to identify TE insertion in the Arabidopsis thaliana genome. Using nanotei, we identified tens of TE copies, including ones for the well-characterized ONSEN retrotransposon family that were hidden in genome assembly gaps. The results demonstrate that some TEs are inaccessible for analysis with the current A. thaliana (TAIR10.1) genome assembly. We further explored the mobilome of the ddm1 mutant with elevated TE activity. Nanotei captured all TEs previously known to be active in ddm1 and also identified transposition of non-autonomous TEs. Of them, one non-autonomous TE derived from (AT5TE33540) belongs to TR-GAG retrotransposons with a single open reading frame (ORF) encoding the GAG protein. These results provide the first direct evidence that TR-GAGs and other non-autonomous LTR retrotransposons can transpose in the plant genome, albeit in the absence of most of the encoded proteins. In summary, nanotei is a useful tool to detect active TEs and their insertions in plant genomes using low-coverage data from Nanopore genome sequencing.

Biological and physiological properties of reverse ankyrin engineered for dimer construction to enhance HIV-1 capsid interaction

Assembly and budding in the late-stage of human immunodeficiency virus type 1 (HIV-1) production relies on the polymerization of Gag protein at the inner leaflet of the plasma membrane. We previously generated an ankyrin repeat protein (Ank1D4) that specifically interacts with the CAp24 protein. This study aimed to improve the binding activity of Ank1D4 by generating two platforms for the Ank1D4 dimer. The design of these constructs featured a distinct orientation of monomeric Ank1D4 connected by a linker peptide (G S) . The binding surfaces in either dimer generated from the C-terminus of the Ank1D4 monomer linked with the N-terminus of another monomer (Ank1D4 ) or its inverted form (Ank1D4 ), similar to monomeric Ank1D4. The interaction of Ank1D4 with CAp24 from capture ELISA was significantly greater than that of Ank1D4 and the parental Ank1D4. The bifunctional characteristic of Ank1D4 was further demonstrated using sandwich ELISA. The binding kinetics of these ankyrins were evaluated using bio-layer interferometry analysis. The K of Ank1D4 , Ank1D4 and monomeric Ank1D4 was 3.5 nM, 53.7 nM, and 126.2 nM, respectively. The dynamics of the interdomain linker and the behavior of ankyrin dimers were investigated in silico. Upon the binding distance calculation from the candidate structures, the achievement in obtaining double active sites is more possible in Ank1D4 . The CD spectroscopic data indicated that secondary structure of dimer forms resemble Ank1D4 monomer α-helical content. This finding confers the strategy to generate dimer from rigid scaffold for acquiring the binding avidity.

RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein

A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from Saccharomyces cerevisiae lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo.

Chondroitin Sulfate/Dermatan Sulfate-Protein Interactions and Their Biological Functions in Human Diseases: Implications and Analytical Tools

Chondroitin sulfate (CS) and dermatan sulfate (DS) are linear anionic polysaccharides that are widely present on the cell surface and in the cell matrix and connective tissue. CS and DS chains are usually attached to core proteins and are present in the form of proteoglycans (PGs). They not only are important structural substances but also bind to a variety of cytokines, growth factors, cell surface receptors, adhesion molecules, enzymes and fibrillary glycoproteins to execute series of important biological functions. CS and DS exhibit variable sulfation patterns and different sequence arrangements, and their molecular weights also vary within a large range, increasing the structural complexity and diversity of CS/DS. The structure-function relationship of CS/DS PGs directly and indirectly involves them in a variety of physiological and pathological processes. Accumulating evidence suggests that CS/DS serves as an important cofactor for many cell behaviors. Understanding the molecular basis of these interactions helps to elucidate the occurrence and development of various diseases and the development of new therapeutic approaches. The present article reviews the physiological and pathological processes in which CS and DS participate through their interactions with different proteins. Moreover, classic and emerging glycosaminoglycan (GAG)-protein interaction analysis tools and their applications in CS/DS-protein characterization are also discussed.

Insights into structure, affinity, specificity, and function of GAG-protein interactions through the chemoenzymatic preparation of defined sulfated oligohyaluronans

High amounts of glycosaminoglycans (GAG) such as hyaluronan (HA) occur in connective tissues. There is nowadays increasing evidence that a “sulfation code” exists which mediates numerous GAG functions. High molecular weight and inhomogeneity of GAG, however, aggravated detailed studies. Thus, synthetic oligosaccharides were urgently required. We will review here chemoenzymatic and analytic strategies to provide defined sulfated and anomerically modified GAG oligosaccharides of the HA type. Representative studies of protein/GAG interactions by (bio)chemical and biophysical methods are reported yielding novel insights into GAG-protein binding. Finally, the biological conclusions and in vivo applications of defined sulfated GAG oligosaccharides will be discussed.

Structural determinants of virion assembly and release in the C-terminus of the M-PMV capsid protein

The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid lattice is located downstream of the capsid (CA) protein in many retroviral Gags. The HIV-1 Gag contains a stretch of five amino acid residues termed the ‘clasp motif’, important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of the HIV-1 and Mason-Pfizer Monkey Virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide comparable function. The importance of the sequences spanning the CA-NC cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant protein in vitro , and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant virus in vivo . The mutants revealed major defects in virion assembly and release in 293T and HeLa cells, and even larger defects in infectivity. Our data identifies the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient viral infection. Importance The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short ‘clasp’ motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.