A Structural Perspective of Reps from CRESS-DNA Viruses and Their Bacterial Plasmid Homologues

Rolling circle replication (RCR) is ubiquitously used by cellular and viral systems for genome and plasmid replication. While the molecular mechanism of RCR has been described, the structural mechanism is desperately lacking. Circular-rep encoded single stranded DNA (CRESS-DNA) viruses employ a viral encoded replicase (Rep) to initiate RCR. The recently identified prokaryotic homologues of Reps may also be responsible for initiating RCR. Reps are composed of an endonuclease, oligomerization, and ATPase domain. Recent structural studies have provided structures for all these domains such that an overall mechanism of RCR initiation can begin to be synthesized. However, structures of Rep in complex with its various DNA substrates and/or ligands are lacking. Here we provide a 3D bioinformatic review of the current structural information available for Reps. We combine an excess of 1590 sequences with experimental and predicted structural data from 22 CRESS-DNA groups to identify similarities and differences between Reps that lead to potentially important functional sites. Experimental studies of these sites may shed light on how Reps execute their functions. Furthermore, we identify Rep-substrate or Rep-ligand structures that are urgently needed to better understand the structural mechanism of RCR.

Monitoring Drug-Target Interactions through Target Engagement-Mediated Amplification on Arrays and in situ

Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to a panel of fixed adherent cells agreed with expectations from expression profiles of the cells. These findings were corroborated by competition experiments using kinase inhibitors with overlapping and non-overlapping target specificities, and translated to pathology tissue sections. We also introduce a proximity ligation variant of TEMA in which these drug-DNA conjugates are combined with antibody-DNA conjugates to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug-target interactions at spatial resolution in protein arrays, cells and tissues.

Novel circular DNA virus identified in Opuntia discolor (Cactaceae) that codes for proteins with similarity to those of geminiviruses

Viral metagenomic studies have enabled the discovery of many unknown viruses and revealed that viral communities are much more diverse and ubiquitous than previously thought. Some viruses have multiple genome components that are encapsidated either in separate virions (multipartite viruses) or in the same virion (segmented viruses). In this study, we identify what is possibly a novel bipartite plant-associated circular single-stranded DNA virus in a wild prickly pear cactus, Opuntia discolor, that is endemic to the Chaco ecoregion in South America. Two ~1.8 kb virus-like circular DNA components were recovered, one encoding a replication-associated protein (Rep) and the other a capsid protein (CP). Both of the inferred protein sequences of the Rep and CP are homologous to those encoded by members of the family Geminiviridae. These two putatively cognate components each have a nonanucleotide sequence within a likely hairpin structure that is homologous to the origins of rolling-circle replication (RCR), found in diverse circular single-stranded DNA viruses. In addition, the two components share similar putative replication-associated iterative sequences (iterons), which in circular single-stranded DNA viruses are important for Rep binding during the initiation of RCR. Such molecular features provide support for the possible bipartite nature of this virus, which we named utkilio virus (common name of the Opuntia discolor in South America) components A and B. In the infectivity assays conducted in Nicotiana benthamiana plants, only the A component of utkilio virus, which encodes the Rep protein, was found to move and replicate systemically in N. benthamiana. This was not true for component B, for which we did not detect replication, which may have been due to this being a defective molecule or because of the model plants (N. benthamiana) used for the infection assays. Future experiments need to be conducted with other plants, including O. discolor, to understand more about the biology of these viral components.

Clinical Validation of Fetal cfDNA Analysis Using Rolling-Circle-Replication and Imaging Technology in Osaka (CRITO Study)

Background: Noninvasive prenatal genetic testing (NIPT) has been adopted as the first choice for aneuploidy screening. The purposes of this study were to investigate the accuracy of Vanadis® NIPT (hereafter CRITO-NIPT) in order to gain a deeper insight into the reasons for discrepancies, as well as to discuss the role of fetal ultrasound. Methods: Between 2019 and 2020, CRITO-NIPT was performed in 1218 cases of patients who underwent CVS or amniocentesis after a detailed fetal ultrasound exam and genetic counseling. The CRITO-NIPT results were compared with the genetic results. In cases of test discrepancies, the placentae were collected for detailed genetic research, and the pre-procedure fetal ultrasound findings were referred to. Results: The positive predictive value of T21, T18, and T13 was 93.55%, 88.46%, and 100%, respectively. In 90% of the of false positive (FP) cases, the placentae were examined. In 75% of the CRITO FP-T21 cases, placental mosaicism, or a demised twin’s T21, were confirmed. There were complicated mosaic cases, including tetrasomy 21/trisomy7 and monosomy 21/trisomy21 cases. In one of three no-call cases, an intermediate deletion of chromosome 13 was detected. Conclusions: The CRITO study investigated the mechanism of false positives, and the detailed mechanisms in mosaic and no-call cases. There have hitherto been no reports that have provided insight by partitioning the placenta to compare the NIPT and invasive test results, nor that have provided detailed ultrasound findings in the cases of discordant results, revealing the demonstrated importance of, and necessity for, detailed ultrasonography. This article describes the potential of rolling-circle replication as a powerful biosensing platform, as well as the importance of examining the fetus in detail with ultrasound. However, we should remember that the potential applications raise ethical and social concerns that go beyond aneuploidy and its methodology.

Assessment and Clinical Utility of a Non-Next-Generation Sequencing-Based Non-Invasive Prenatal Testing Technology

Background: Rolling-circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus, and 25 synthetic samples, were analyzed for the fetal risk of trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), by three laboratories on three continents. All the screen-positive pregnancies were provided post-test genetic counseling and confirmatory diagnostic invasive testing (e.g., amniocentesis). The screen-negative pregnancies were routinely evaluated at birth for fetal aneuploidies, using newborn examinations, and any suspected aneuploidies would have been offered diagnostic testing or confirmed with karyotyping. Results: The study found rolling-circle replication to be a highly viable technology for the clinical assessment of fetal aneuploidies, with 100% sensitivity for T21 (95% CI: 82.35–100.00%); 100.00% sensitivity for T18 (71.51–100.00%); and 100.00% sensitivity for T13 analyses (66.37–100.00%). The specificities were >99% for each trisomy (99.7% (99.01–99.97%) for T21; 99.5% (98.62–99.85%) for T18; 99.7% (99.03–99.97%) for T13), along with a first-pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling-circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening, and it would provide comparable results to other reported cfDNA methodologies.

A phage parasite deploys a nicking nuclease effector to inhibit replication of its viral host

PLEs are phage parasites integrated into the chromosome of epidemic Vibrio cholerae. In response to infection by its viral host ICP1, PLE excises, replicates and hijacks ICP1 structural components for transduction. Through an unknown mechanism PLE prevents ICP1 from transitioning to rolling circle replication (RCR), a prerequisite for efficient packaging of the viral genome. Here, we characterize a PLE-encoded nuclease, NixI, that blocks phage development likely by nicking ICP1s genome as it transitions to RCR. NixI-dependent cleavage sites appear in ICP1s genome during infection of PLE(+) V. cholerae. Purified NixI demonstrates in vitro specificity for sites in ICP1s genome and NixI activity is enhanced by a putative specificity determinant co-expressed with NixI during phage infection. Importantly, NixI is sufficient to limit ICP1 genome replication and eliminate progeny production. We identify distant NixI homologs in an expanded family of putative phage satellites in Vibrios that lack nucleotide homology to PLEs but nonetheless share genomic synteny with PLEs. More generally, our results reveal a previously unknown mechanism deployed by phage parasites to limit packaging of their viral hosts genome and highlight the prominent role of nuclease effectors as weapons in the arms race between antagonizing genomes.

Assessment and clinical utility of a non-Next Generation Sequencing based Non-Invasive Prenatal Testing technology

Background: Rolling circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus and 25 synthetic samples were analyzed for the fetal risk of Trisomy 21, Trisomy 18 and Trisomy 13 by three laboratories on three continents. All women who provided the samples were followed to birth, where evaluation for fetal aneuploidies was performed using newborn examinations and any suspected aneuploidies were confirmed with karyotyping. Results: The study found rolling circle replication to be a highly viable technology for clinical assessment of fetal aneuploidies with 100% sensitivity for T21 (95% CI:82.35% - 100.00%); 100.00% sensitivity for T18 (71.51% - 100.00%) and 100.00% sensitivity for T13 analyses (66.37% - 100.00%). The specificities were >99% for each trisomies [99.7% (99.01% - 99.97%) for T21; 99.5% (98.62% - 99.85%) for T18; 99.7% (99.03% - 99.97%) for T13], along with a first pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening and comparable results to other reported cfDNA methodologies.

Herpes simplex virus co-infection facilitates rolling circle replication of the adeno-associated virus genome

Adeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin replication (RHR) mechanism initiated at the AAV 3’ inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. Using Southern analysis and nanopore sequencing as a novel, high-throughput approach to study viral genome replication we demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in the presence of HSV-1, thus providing evidence for an unequivocal rolling circle replication (RCR) mechanism. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus.

Rolling-Circle Replication in Mitochondrial DNA Inheritance: Scientific Evidence and Significance from Yeast to Human Cells

Studies of mitochondrial (mt)DNA replication, which forms the basis of mitochondrial inheritance, have demonstrated that a rolling-circle replication mode exists in yeasts and human cells. In yeast, rolling-circle mtDNA replication mediated by homologous recombination is the predominant pathway for replication of wild-type mtDNA. In human cells, reactive oxygen species (ROS) induce rolling-circle replication to produce concatemers, linear tandem multimers linked by head-to-tail unit-sized mtDNA that promote restoration of homoplasmy from heteroplasmy. The event occurs ahead of mtDNA replication mechanisms observed in mammalian cells, especially under higher ROS load, as newly synthesized mtDNA is concatemeric in hydrogen peroxide-treated human cells. Rolling-circle replication holds promise for treatment of mtDNA heteroplasmy-attributed diseases, which are regarded as incurable. This review highlights the potential therapeutic value of rolling-circle mtDNA replication.