Torsins: not your typical AAA+ ATPases.

Torsin ATPases (Torsins) belong to the widespread AAA+ (ATPases associated with a variety of cellular activities) family of ATPases, which share structural similarity but have diverse cellular functions. Torsins are outliers in this family because they lack many characteristics of typical AAA+ proteins, and they are the only members of the AAA+ family located in the endoplasmic reticulum and contiguous perinuclear space. While it is clear that Torsins have essential roles in many, if not all metazoans, their precise cellular functions remain elusive. Studying Torsins has significant medical relevance since mutations in Torsins or Torsin-associated proteins result in a variety of congenital human disorders, the most frequent of which is early-onset torsion (DYT1) dystonia, a severe movement disorder. A better understanding of the Torsin system is needed to define the molecular etiology of these diseases, potentially enabling corrective therapy. Here, we provide a comprehensive overview of the Torsin system in metazoans, discuss functional clues obtained from various model systems and organisms and provide a phylogenetic and structural analysis of Torsins and their regulatory cofactors in relation to disease-causative mutations. Moreover, we review recent data that have led to a dramatically improved understanding of these machines at a molecular level, providing a foundation for investigating the molecular defects underlying the associated movement disorders. Lastly, we discuss our ideas on how recent progress may be utilized to inform future studies aimed at determining the cellular role(s) of these atypical molecular machines and their implications for dystonia treatment options.

Arresting a Torsin ATPase reshapes the endoplasmic reticulum.

Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures.

Factors that determine the efficiency of HIV-1 strand transfer initiated at a specific site.

Human immunodeficiency virus-1 employs strand transfer for recombination between two viral genomes. We have previously provided evidence that strand transfer proceeds by an invasion-mediated mechanism in which a DNA segment on the original RNA template is invaded by a second RNA template at a gap site. The initial RNA-DNA hybrid then expands until the DNA is fully transferred. Ribonuclease H (RNase H) cleavages and nucleocapsid protein (NC) were required for long-distance propagation of the hybrid. Evaluation was performed on a unique substrate, with a short gap serving as a precreated invasion site. In our current work, this substrate provided an opportunity for us to test what factors influence a specific invasion site to support transfer, and to distinguish factors that influence invasion site creation from those that impact later steps. RNase H can act in a polymerization-dependent or polymerization-independent mode. Polymerization-dependent and polymerization-independent RNase H were found to be important in creating efficiently used invasion sites in the primer-donor complex, with or without NC. Propagation and terminus transfer steps, emanating from a precreated invasion site in the presence of NC, were stimulated by polymerization-dependent, but not polymerization-independent, RNase H. RNase H can carry out primary and secondary cleavages during synthesis. While both modes of cleavage promoted invasion, only primary cleavage promoted propagation in the presence of NC in our system. These observations suggest that once invasion is initiated at a short gap, it can propagate through an adjacent region interrupted only by nicks, with help by NC. We considered the possibility that propagation solely by strand exchange was a significant contributor to transfers. However, it did not promote transfer even if synthetic progress of reverse transcriptase was intentionally slowed, consistent with strand exchange by random walk in which rate declines precipitously with distance.

Mechanism analysis indicates that recombination events in HIV-1 initiate and complete over short distances, explaining why recombination frequencies are similar in different sections of the genome.

Strand transfer drives recombination between the co-packaged genomes of HIV-1, a process that allows rapid viral evolution. The proposed invasion-mediated mechanism of strand transfer during HIV-1 reverse transcription has three steps: (1) invasion of the initial or donor primer template by the second or acceptor template; (2) propagation of the primer-acceptor hybrid; and (3) primer terminus transfer. Invasion occurs at a site at which the reverse transcriptase ribonuclease H (RNase H) has created a nick or short gap in the donor template. We used biochemical reconstitution to determine the distance over which a single invasion site can promote transfer. The DNA-primed RNA donor template used had a single-stranded pre-created invasion site (PCIS). Results showed that the PCIS could influence transfer by 20 or more nucleotides in the direction of synthesis. This influence was augmented by viral nucleocapsid protein and additional reverse transcriptase-RNase H cleavage. Strand-exchange assays were performed specifically to assess the distance over which a hybrid interaction initiated at the PCIS could propagate to achieve transfer. Propagation by simple branch migration of strands was limited to 24-32 nt. Additional RNase H cuts in the donor RNA allowed propagation to a maximum distance of 32-64 nt. Overall, results indicate that a specific invasion site has a limited range of influence on strand transfer. Evidently, a series of invasion sites cannot collaborate over a long distance to promote transfer. This result explains why the frequency of recombination events does not increase with increasing distance from the start of synthesis, a characteristic that supports effective mixing of viral mutations.