Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome.

During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model, which enables exploring the parameter space of the WT and DI competition. We also experimentally examined virus and DI replication kinetics during co-infection, and used these data to infer model parameters. Our model identifies, and our experimental measurements confirm, that the efficiencies of DI genome replication and encapsidation are two most critical parameters determining the outcome of WT replication. However, an equilibrium can be established which enables WT to replicate, albeit to reduced levels.

Characterization of Viral Populations by Using Circular Sequencing.

With the enormous sizes viral populations reach, many variants are at too low a frequency to be detected by conventional next-generation sequencing (NGS) methods. Circular sequencing (CirSeq) is a method by which the error rate of next-generation sequencing is decreased so that even low-frequency viral variants can be accurately detected. The ability to visualize almost the entire genetic makeup of a viral swarm has implications for epidemiology, viral evolution, and vaccine design. Here we discuss experimental planning, analysis, and recent insights using CirSeq.

Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition.

The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, we previously described proteome remodeling during Drosophila egg activation. Here, we describe our quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey, to our knowledge, of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. We show that Muskelin, found to be up-regulated during oocyte maturation, is required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. We find down-regulation of this class of proteins to be mediated partially by APC/C(CORT), a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C).

A meiosis-specific form of the APC/C promotes the oocyte-to-embryo transition by decreasing levels of the Polo kinase inhibitor matrimony.

Oocytes are stockpiled with proteins and mRNA that are required to drive the initial mitotic divisions of embryogenesis. But are there proteins specific to meiosis whose levels must be decreased to begin embryogenesis properly? The Drosophila protein Cortex (Cort) is a female, meiosis-specific activator of the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. We performed immunoprecipitation of Cortex followed by mass spectrometry, and identified the Polo kinase inhibitor Matrimony (Mtrm) as a potential interactor with Cort. In vitro binding assays showed Mtrm and Cort can bind directly. We found Mtrm protein levels to be reduced dramatically during the oocyte-to-embryo transition, and this downregulation did not take place in cort mutant eggs, consistent with Mtrm being a substrate of APC(Cort). We showed that Mtrm is subject to APC(Cort)-mediated proteasomal degradation and have identified a putative APC/C recognition motif in Mtrm that when mutated partially stabilized the protein in the embryo. Furthermore, overexpression of Mtrm in the early embryo caused aberrant nuclear divisions and developmental defects, and these were enhanced by decreasing levels of active Polo. These data indicate APC(Cort) ubiquitylates Mtrm at the oocyte-to-embryo transition, thus preventing excessive inhibition of Polo kinase activity due to Mtrm's presence.

Species-Specific Evolution of Ebola Virus during Replication in Human and Bat Cells.

Ebola virus (EBOV) causes a severe, often fatal disease in humans and nonhuman primates. Within the past decade, EBOV has caused two large and difficult-to-control outbreaks, one of which recently ended in the Democratic Republic of the Congo. Bats are the likely reservoir of EBOV, but little is known of their relationship with the virus. We perform serial passages of EBOV in human and bat cells and use circular sequencing to compare the short-term evolution of the virus. Virus populations passaged in bat cells have sequence markers indicative of host RNA editing enzyme activity, including evidence for ADAR editing of the EBOV glycoprotein. Multiple regions in the EBOV genome appear to have undergone adaptive evolution when passaged in bat and human cells. Individual mutated viruses are rescued and characterized. Our results provide insight into the host species-specific evolution of EBOV and highlight the adaptive flexibility of the virus.

Control of RNA viruses in mosquito cells through the acquisition of vDNA and endogenous viral elements.

Aedes aegypti transmit pathogenic arboviruses while the mosquito itself tolerates the infection. We examine a piRNA-based immunity that relies on the acquisition of viral derived cDNA (vDNA) and how this pathway discriminates between self and non-self. The piRNAs derived from these vDNAs are essential for virus control and Piwi4 has a central role in the pathway. Piwi4 binds preferentially to virus-derived piRNAs but not to transposon-targeting piRNAs. Analysis of episomal vDNA from infected cells reveals that vDNA molecules are acquired through a discriminatory process of reverse-transcription and recombination directed by endogenous retrotransposons. Using a high-resolution Ae. aegypti genomic sequence, we found that vDNAs integrated in the host genome as endogenous viral elements (EVEs), produce antisense piRNAs that are preferentially loaded onto Piwi4. Importantly, EVE-derived piRNAs are specifically loaded onto Piwi4 to inhibit virus replication. Thus, Ae. aegypti employs a sophisticated antiviral mechanism that promotes viral persistence and generates long-lasting adaptive immunity.

Mechanisms and Concepts in RNA Virus Population Dynamics and Evolution.

RNA viruses are unique in their evolutionary capacity, exhibiting high mutation rates and frequent recombination. They rapidly adapt to environmental changes, such as shifts in immune pressure or pharmacological challenge. The evolution of RNA viruses has been brought into new focus with the recent developments of genetic and experimental tools to explore and manipulate the evolutionary dynamics of viral populations. These studies have uncovered new mechanisms that enable viruses to overcome evolutionary challenges in the environment and have emphasized the intimate relationship of viral populations with evolution. Here, we review some of the emerging viral and host mechanisms that underlie the evolution of RNA viruses. We also discuss new studies that demonstrate that the relationship between evolutionary dynamics and virus biology spans many spatial and temporal scales, affecting transmission dynamics within and between hosts as well as pathogenesis.

Mapping the Evolutionary Potential of RNA Viruses.

The deterministic force of natural selection and stochastic influence of drift shape RNA virus evolution. New deep-sequencing and microfluidics technologies allow us to quantify the effect of mutations and trace the evolution of viral populations with single-genome and single-nucleotide resolution. Such experiments can reveal the topography of the genotype-fitness landscapes that shape the path of viral evolution. By combining historical analyses, like phylogenetic approaches, with high-throughput and high-resolution evolutionary experiments, we can observe parallel patterns of evolution that drive important phenotypic transitions. These developments provide a framework for quantifying and anticipating potential evolutionary events. Here, we examine emerging technologies that can map the selective landscapes of viruses, focusing on their application to pathogenic viruses. We identify areas where these technologies can bolster our ability to study the evolution of viruses and to anticipate and possibly intervene in evolutionary events and prevent viral disease.

The Diversity, Structure, and Function of Heritable Adaptive Immunity Sequences in the Aedes aegypti Genome.

The Aedes aegypti mosquito transmits arboviruses, including dengue, chikungunya, and Zika virus. Understanding the mechanisms underlying mosquito immunity could provide new tools to control arbovirus spread. Insects exploit two different RNAi pathways to combat viral and transposon infection: short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) [1, 2]. Endogenous viral elements (EVEs) are sequences from non-retroviral viruses that are inserted into the mosquito genome and can act as templates for the production of piRNAs [3, 4]. EVEs therefore represent a record of past infections and a reservoir of potential immune memory [5]. The large-scale organization of EVEs has been difficult to resolve with short-read sequencing because they tend to integrate into repetitive regions of the genome. To define the diversity, organization, and function of EVEs, we took advantage of the contiguity associated with long-read sequencing to generate a high-quality assembly of the Ae. aegypti-derived Aag2 cell line genome, an important and widely used model system. We show EVEs are acquired through recombination with specific classes of long terminal repeat (LTR) retrotransposons and organize into large loci (>50 kbp) characterized by high LTR density. These EVE-containing loci have increased density of piRNAs compared to similar regions without EVEs. Furthermore, we detected EVE-derived piRNAs consistent with a targeted processing of persistently infecting virus genomes. We propose that comparisons of EVEs across mosquito populations may explain differences in vector competence, and further study of the structure and function of these elements in the genome of mosquitoes may lead to epidemiological interventions.