Biology subjects

Achouri, E.

Publications and source records attributed to Achouri, E..

4 recordsLinked to original sources

bioRxiv2026

Detailed single-cell mapping of the transcriptional response to a virus infection driven by copy-back viral genomes

The antiviral response to several clinically significant viruses, including respiratory syncytial virus and parainfluenza virus, is driven by copy-back viral genomes (cbVGs) generated during virus replication. However, the broader impact of cbVGs on the functional states of host cells remains undefined. Here, we developed a single-cell RNA-sequencing and computational framework to map cbVG-driven host responses during Sendai virus infection. Unsupervised profiling identified distinct transcriptional states throughout the course of infection, highlighting a shift from early antiviral signaling to later inflammatory and remodeling programs. Stratifying infected cells by cbVG status demonstrated that cbVG-positive cells initiate interferon and chemokine programs, which later spread to cbVG-negative cells. At later stages, cbVG-positive cells acquire additional signaling, cytoskeletal, transcriptional, and stress-adaptation programs, which are absent in cbVG-clean infection. This work defines the broader cbVG-driven layered and dynamic host response and provides a valuable high-resolution resource of the temporal cellular response to a virus infection.

microbiology↗

bioRxiv2025

Validation of diverse and previously untraceable Sendai virus copyback viral genomes by Direct RNA Sequencing

Copyback viral genomes (cbVGs) are truncated viral genomes with complementary ends produced when the viral negative-sense RNA virus polymerase detaches from the replication template and resumes elongation from the nascent strand. Despite advances in methods to identify cbVGs based on the site of polymerase break and rejoin, PCR-based tools cannot provide full length sequences of most cbVGs and/or can introduce errors and artifacts during cbVG amplification. These limitations have painted a limited picture of the diverse population of cbVGs generated during infection. To improve our ability to obtain native full-length sequences of cbVGs, we optimized Direct RNA Sequencing (DRS) as a fast and simple tool to sequence full-length cbVGs and designed a BLAST-based analysis approach to identify cbVGs from long-read sequencing data. We analyzed the DRS outputs of multiple Sendai virus stocks to highlight both the utility and limitations of this tool. We found that to capture the dominant 546 nucleotide cbVG produced by Sendai virus strain Cantell, the length of complementarity between the virus trailer and the DRS oligonucleotide should optimally be increased to up to 32 nucleotides. We also demonstrate comparable quality of cbVG sequences by DRS from as little RNA as 17.6ng from the media fraction or 50ng of from the cellular fraction of cells infected with SeV, in contrast to the recommended 1000ng. Importantly, we validated different cbVG species from two recombinant Sendai virus stocks, including for the first time cbVGs whose break positions occurred at or near position one in the reference genome. ImportanceMost viruses of the order Mononegavirales has been demonstrated to naturally generate copyback viral genomes. These genomes are critical determinants of infection outcomes; they interfere with standard virus replication by competing for viral resources, activate antiviral responses, and inhibit protein translation. Despite their critical roles in infection, current tools to study copyback viral genomes rely either on preexisting knowledge of the sequence of a target RNA or require reverse-transcription and amplification of the target RNA, biasing toward short cbVGs and introducing relatively high rates of errors. Our lab has long advocated for RNA virologists to sequence their stocks to assess the role cbVGs may have in their infections. Toward this effort, we detail the optimization of Direct RNA Sequencing to sequence full-length cbVGs while maintaining the integrity of the native cbVG RNA and present the Long-Read cbVG Analysis (LoCA; https://github.com/lopezlab-washu/LOCA.git) script as a highly accessible BLAST-based tool for cbVG detection.

microbiology↗

bioRxiv2023

VODKA2: An accurate method to detect copy-back and deletion viral genomes from next-generation sequencing data

During viral replication, viruses carrying an RNA genome produce non-standard viral genomes (nsVGs), including copy-back viral genomes (cbVGs) and deletion viral genomes (delVGs), that play a crucial role in regulating viral replication and pathogenesis. Because of their critical roles in determining the outcome of RNA virus infections, the study of nsVGs has flourished in recent years exposing a need for bioinformatic tools that can accurately identify them within Next-Generation Sequencing data obtained from infected samples. Here, we present our data analysis pipeline, Viral Opensource DVG Key Algorithm2 (VODKA2), that is optimized to run on a High Performance Computing (HPC) environment for fast and accurate detection of nsVGs from large data sets. Availability and implementationVODKA2 is freely available at GitHub (https://github.com/lopezlab-washu/VODKA2)

bioinformatics↗

bioRxiv2022

Accumulation of copy-back viral genomes during respiratory syncytial virus infection is preceded by diversification of the copy-back viral genome population followed by selection

RNA viruses generate non-standard viral genomes during their replication, including viral genomes of the copy-back (cbVG) type that cannot replicate in the absence of a standard virus. cbVGs play a crucial role in shaping virus infection outcomes due to their ability to interfere with virus replication and induce strong immune responses. However, despite their critical role during infection, the principles that drive the selection and evolution of cbVGs within a virus population are poorly understood. As cbVGs are dependent on the virus replication machinery to be generated and replicated, we hypothesized that host factors that affect virus replication exert selective pressure on cbVGs and drive their evolution within a virus population. To test this hypothesis, we used respiratory syncytial virus (RSV) as model and took an experimental evolution approach by serially passaging RSV in immune competent A549 control and immune deficient A549 STAT1 KO cells which allow higher levels of virus replication. As predicted, we observed that virus populations accumulated higher amounts of cbVGs in the more permissive A549 STAT1 KO cells over time but, unexpectedly, the predominant cbVG species after passages in the two conditions were different. While A549 STAT1 KO cells accumulated relatively short cbVGs, A549 control cells mainly contained cbVGs of much longer predicted size that have not been described previously. These long cbVGs were predominant at first in both cell lines in vitro and the predominant ones observed in samples from RSV infected patients. Although sustained high replication levels are associated with cbVG generation and accumulation, our data show that sustained high levels of virus replication are critical for cbVG population diversification, a process that preceded the generation of shorter cbVGs that selectively accumulated over time. Taken together, we show that selection and evolution of cbVGs within a virus population is shaped by how resistant or permissive a host is to RSV.

microbiology↗