Donohue, Ryan C.

Genome diversity is key to viral fitness and adaptability, especially for RNA viruses, which generate it through both mutation and recombination. Recombination occurs frequently in positive strand RNA virus infections, where the RNA‐dependent RNA polymerase can jump from one genomic template to another. On the other hand, template exchange events have been rarely detected in infections of negative strand RNA viruses even when sensitive functional assays were used. However, while characterizing defective interfering RNAs (DI RNA) generated by an innate immunity‐stimulating measles virus (MeV) mutant, we documented an unexpectedly high number of recombination events ( J. Virol. 89, 7735‐47, 2015 ). We asked here how frequently these events occur in the 15,894 bases genome of a MeV strain with intact innate immunity control functions. Towards this we purified MeV ribonucleocapsids and performed unbiased deep sequencing analyses. We detected more than 3000 recombination events while covering 32,000 genomes, or about one event every ten genomes. Mapping of breakpoints and re‐initiation sites indicated that recombination occurred along the entire genome, rather than being restricted to either end, where most DI RNAs were previously mapped. Recombination was phased neither by encapsidation (6‐base periodicity) nor by ribonucleocapsid helical turns (approximately 75‐base periodicity). Rather, most recombination events joined sequences located more than 1000 bases apart. Jump‐back events were prominent, occurring about 1.5 times more frequently than deletion or copy‐back events. Most events were unique, but a few short jump‐back events retaining 6‐base periodicity were detected up to 40 times, suggesting that the corresponding genomes were amplified. At new junctions, deletions or insertions were rare (<5%). Analyses of 10 nucleotides on each side of the new junctions suggested that base pairing guided about one third of the re‐initiation events. In addition to the recombination events, we detected clusters of adenosine deaminase acting on RNA 1 (ADAR1)‐like hypermutations in about 4% of the genomes. ADAR1 de‐stabilizes base pairing on double‐strand RNA, catalyzing C‐6 deamination of adenosine to produce inosine until double‐strand regions are unwound. Indeed, we document that in an infection with a MeV mutant known to generate many DI RNAs, the frequency of ADAR1‐like hypermutations was even higher than in standard infections. In summary, we show here that significant levels of RNA recombination occur even in standard lytic infections of a prototypic negative strand RNA virus. Even if some recombinant genomes may form double‐strand RNA, infections remain productive, probably due to the unwinding/modification activity of ADAR1. We discuss our data in the context of published observations indicating that recombination events, as well as ADAR1‐like hypermutations, contribute to the adaptation of other medically relevant negative strand RNA viruses to new hosts and environments. Support or Funding Information R21 AI 128037; Mayo Clinic Graduate School of Biomedical Sciences This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Replication of negative-strand RNA viruses relies on two components: a helical ribonucleocapsid and an RNA-dependent RNA polymerase composed of a catalytic subunit, the L protein, and a cofactor, the P protein. We show that the measles virus (MeV) C protein is an additional component of the replication complex. We provide evidence that the C protein is recruited to the ribonucleocapsid by the P protein and map the interacting segments of both C and P proteins. We conclude that the primary function of MeV C is to improve polymerase processivity and accuracy, rather than uniquely to antagonize the type I interferon response. Since most viruses of the Paramyxoviridae family express C proteins, their primary function may be conserved.

ABSTRACT Here, we show that cells expressing the adherens junction protein nectin-1 capture nectin-4-containing membranes from the surface of adjacent cells in a trans-endocytosis process. We find that internalized nectin-1–nectin-4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents trans-endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1-expressing cells acquire dye-labeled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as a receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related trans-endocytosis processes may be exploited by pathogens to extend tropism. This article has an associated First Person interview with the first author of the paper.