Polyadenylation of genomic RNA and initiation of antigenomic RNA in a positive-strand RNA virus are controlled by the same cis-element

Genomes and antigenomes of many positive-strand RNA viruses contain 3′-poly(A) and 5′-poly(U) tracts, respectively, serving as mutual templates. Mechanism(s) controlling the length of these homopolymeric stretches are not well understood. Here, we show that in coxsackievirus B3 (CVB3) and three other enteroviruses the poly(A) tract is ∼80–90 and the poly(U) tract is ∼20 nt-long. Mutagenesis analysis indicate that the length of the CVB3 3′-poly(A) is determined by the oriR, a cis-element in the 3′-noncoding region of viral RNA. In contrast, while mutations of the oriR inhibit initiation of (−) RNA synthesis, they do not affect the 5′-poly(U) length. Poly(A)-lacking genomes are able to acquire genetically unstable AU-rich poly(A)-terminated 3′-tails, which may be generated by a mechanism distinct from the cognate viral RNA polyadenylation. The aberrant tails ensure only inefficient replication. The possibility of RNA replication independent of oriR and poly(A) demonstrate that highly debilitated viruses are able to survive by utilizing ‘emergence’, perhaps atavistic, mechanisms

Biological Significance of a Human Enterovirus B-Specific RNA Element in the 3′ Nontranslated Region

The secondary structures predicted for the enteroviral 3′ nontranslated region (3′NTR) all seem to indicate a conformation consisting of two (X and Y) hairpin structures. The higher-order RNA structure of the 3′NTR appears to exist as an intramolecular kissing interaction between the loops of these two hairpin structures. The enterovirus B-like subgroup possesses an additional stem-loop structure, domain Z, which is not present in the poliovirus-like enteroviruses. It has been suggested that the Z domain originated from a burst of short sequence repetitions (E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V. I. Agol, Nucleic Acids Res. 20:1739-1745, 1992). However, no functional features have yet been ascribed to this enterovirus B-like-specific RNA element in the 3′NTR. In this study, we tested the functional characteristics and biological significance of domain Z. A mutant of the cardiovirulent coxsackievirus group B3 strain Nancy which completely lacked the Z domain and which therefore acquired enterovirus C-like secondary structures exhibited a wild-type growth phenotype, as determined by single-cycle growth analysis with BGM cells. This result proves that the Z domain is virtually dispensable for viral growth in tissue cultures. Partial distortion of the Z domain structure resulted in a disabled virus with reduced growth kinetics, probably due to alternative conformations of the overall structure of the domain. Infection of mice showed that the recombinant coxsackievirus group B3 mutant which completely lacked the Z domain was less virulent. Pancreatic tissues from mice infected with wild-type virus and recombinant virus were equally affected. However, the heart tissue from mice infected with the recombinant virus showed only slight signs of myocarditis. These results suggest that the enterovirus B-like-specific Z domain plays a role in coxsackievirus-induced pathogenesis

Characterization of CVB3 3′NCR deletion mutants using single-cycle growth curves (), plaque assay () and replication assay ()

<p><b>Copyright information:</b></p><p>Taken from "Polyadenylation of genomic RNA and initiation of antigenomic RNA in a positive-strand RNA virus are controlled by the same element"</p><p>Nucleic Acids Research 2006;34(10):2953-2965.</p><p>Published online 31 May 2006</p><p>PMCID:PMC1474053.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Δ indicates a deletion. () Luciferase activity of wt or mutant RNA determined 8 and 24 h post transfection in BGM (see legend of for details). (), 3′-Poly(A) size. * and ** correspond to < 0.05 and < 0.01, respectively. The columns represent the means ± SD of three independent experiments

Secondary and tertiary structures of wild type and mutant CVB3 3′NCRs

<p><b>Copyright information:</b></p><p>Taken from "Polyadenylation of genomic RNA and initiation of antigenomic RNA in a positive-strand RNA virus are controlled by the same element"</p><p>Nucleic Acids Research 2006;34(10):2953-2965.</p><p>Published online 31 May 2006</p><p>PMCID:PMC1474053.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Secondary () and tertiary () structure of the wt CVB3 3′NCR. The 3′NCR consists of three hairpin domains, X, Y and Z. The structure can be closed by an interaction between the poly(A) with a 4 nt U-stretch overlapping the R and the 3D-coding region. The X domain can be stacked to the tertiary ‘kissing’ (K) interaction to form a coaxial helical element, which is connected by single-stranded nucleotide stretches to a second coaxial helical domain Z-Y. () Schematic representation of the kissing distortion mutants, KD1 and KD2, in which 6 and 4 bp, respectively, were mutated (underlined). () Partial 3′NCR sequence of a virus recovered after transfection with the KD1 mutant. The inserted cytosine residue in the recovered mutant is underlined. () The entire 3′NCR sequence of a virus recovered after transfection with the KD2 mutant

Structural and functional integrity of the coxsackievirus B3 oriR: spacing between coaxial RNA helices.

Contains fulltext : 35365.pdf (publisher's version ) (Closed access)The enterovirus oriR is composed of two helices, X and Y, anchored by a kissing (K) interaction. For proper oriR function, certain areas of these helices should be specifically oriented towards each other. It was hypothesized that the single-stranded nucleotides bridging the coaxial helices (Y-X and K-Y linkers) are important to determine this orientation. Spatial changes were introduced by altering the linker length between the helices of the coxsackievirus B3 oriR. Changing the linker lengths resulted in defective RNA replication, probably because of an altered oriR geometry. The identity of the linker residues also played a role, possibly because of sequence-specific ligand recognition. Although each point mutation altering the primary sequence of the Y-X spacer resulted in defective growth at 36 degrees C, the mutations had a wild-type phenotype at 39 degrees C, indicating a cold-sensitive phenotype. The results show that the intrinsic connection between oriR structure and function is fine-tuned by the spacing between the coaxial RNA helices

BYON4228 is a pan-allelic antagonistic SIRPα antibody that potentiates destruction of antibody-opsonized tumor cells and lacks binding to SIRPγ on T cells

Background Preclinical studies have firmly established the CD47-signal-regulatory protein (SIRP)α axis as a myeloid immune checkpoint in cancer, and this is corroborated by available evidence from the first clinical studies with CD47 blockers. However, CD47 is ubiquitously expressed and mediates functional interactions with other ligands as well, and therefore targeting of the primarily myeloid cell-restricted inhibitory immunoreceptor SIRPα may represent a better strategy.Method We generated BYON4228, a novel SIRPα-directed antibody. An extensive preclinical characterization was performed, including direct comparisons to previously reported anti-SIRPα antibodies.Results BYON4228 is an antibody directed against SIRPα that recognizes both allelic variants of SIRPα in the human population, thereby maximizing its potential clinical applicability. Notably, BYON4228 does not recognize the closely related T-cell expressed SIRPγ that mediates interactions with CD47 as well, which are known to be instrumental in T-cell extravasation and activation. BYON4228 binds to the N-terminal Ig-like domain of SIRPα and its epitope largely overlaps with the CD47-binding site. BYON4228 blocks binding of CD47 to SIRPα and inhibits signaling through the CD47-SIRPα axis. Functional studies show that BYON4228 potentiates macrophage-mediated and neutrophil-mediated killing of hematologic and solid cancer cells in vitro in the presence of a variety of tumor-targeting antibodies, including trastuzumab, rituximab, daratumumab and cetuximab. The silenced Fc region of BYON4228 precludes immune cell-mediated elimination of SIRPα-positive myeloid cells, implying anticipated preservation of myeloid immune effector cells in patients. The unique profile of BYON4228 clearly distinguishes it from previously reported antibodies representative of agents in clinical development, which either lack recognition of one of the two SIRPα polymorphic variants (HEFLB), or cross-react with SIRPγ and inhibit CD47-SIRPγ interactions (SIRPAB-11-K322A, 1H9), and/or have functional Fc regions thereby displaying myeloid cell depletion activity (SIRPAB-11-K322A). In vivo, BYON4228 increases the antitumor activity of rituximab in a B-cell Raji xenograft model in human SIRPαBIT transgenic mice. Finally, BYON4228 shows a favorable safety profile in cynomolgus monkeys.Conclusions Collectively, this defines BYON4228 as a preclinically highly differentiating pan-allelic SIRPα antibody without T-cell SIRPγ recognition that promotes the destruction of antibody-opsonized cancer cells. Clinical studies are planned to start in 2023