mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g. RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. To define how these factors control Dbp5 and mRNA export, an analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT ~ 4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD ~ 0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors.

Endogenous viral elements (EVEs), accounting for 15% of our genome, serve as a genetic reservoir from which new genes can emerge. Nematode EVEs are particularly diverse and informative of virus evolution. We identify Atlas virus—an intact retrovirus-like EVE in the human hookworm Ancylostoma ceylanicum , with an envelope protein genetically related to G N -G C glycoproteins from the family Phenuiviridae. A cryo-EM structure of Atlas G C reveals a class II viral membrane fusion protein fold not previously seen in retroviruses. Atlas G C has the structural hallmarks of an active fusogen. Atlas G C trimers insert into membranes with endosomal lipid compositions and low pH. When expressed on the plasma membrane, Atlas G C has cell-cell fusion activity. With its preserved biological activities, Atlas G C has the potential to acquire a cellular function. Our work reveals structural plasticity in reverse-transcribing RNA viruses.

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Endogenous viral elements (EVEs), accounting for 15% of our genome, serve as a genetic reservoir from which new genes can emerge. Nematode EVEs are particularly diverse and informative of virus evolution. We identify Atlas virus – an intact retrovirus-like EVE in the human hookworm Ancylostoma ceylanicum, with an envelope protein genetically related to GN- GC glycoproteins from phleboviruses. A cryo-EM structure of Atlas GC reveals a class II viral membrane fusion protein fold not previously seen in retroviruses. Atlas GC has the structural hallmarks of an active fusogen. Atlas GC trimers insert into membranes with endosomal lipid compositions and low pH. When expressed on the plasma membrane, Atlas GC has cell-cell fusion activity. RNA-Seq data analysis detected transcripts mapping to Atlas virus at different stages of hookworm development. With its preserved biological activities, Atlas GC has the potential to acquire a cellular function. Our work reveals structural plasticity in reverse-transcribing RNA viruses.

Within the parasitic nematode Ancylostoma ceylanicum, a ~20 million-year-old Bel/Pao LTR retrotransposon encodes an ancient viral class II envelope fusion protein termed Atlas Gc. Typically, retroviruses and related degenerate retrotransposons encode a hemagglutinin-like class I envelope fusion protein. A subset of Bel/Pao LTR retrotransposons within the phylum Nematoda have acquired a phlebovirus-like envelope gene and utilized the encoded fusion machinery to escape the genome as intact exogenous retroviruses. This includes C. elegans retroelement 7 virus which was recently reclassified as a member of the genus Semotivirus. A 3.76 Å cryoEM reconstruction confirms Atlas Gc as a closely related phleboviral homologue and class II fusion protein in a novel case of gene exaptation. Preliminary biophysical and biochemical characterization indicate Atlas Gc functions under specific physiological conditions targeting late-endosomal membranes, much like modern viral class II envelope fusion proteins. Phylogenetic analyses support the reclassification of the Atlas endogenous retrovirus and five other A. ceylanicum ERVs as novel semotiviruses of Belpaoviridae of the new viral order of reverse-transcribing viruses Ortervirales.

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