Leveraging Marburg virus model systems to elucidate filovirus gene expression illuminates VP30 interplay with genomic regulatory sequences
Embargo Date
2027-02-06
OA Version
Citation
Abstract
Viruses of the family Filoviridae, including Marburg virus (MARV) and Ebola virus (EBOV), carry out a plethora of complex processes in the course of infection using only a few, multifunctional proteins. Critical among these processes is the expression of viral genes and regulation thereof, which is not yet thoroughly understood. The phosphoprotein VP30, specific to the filoviruses, performs necessary functions in gene expression, partially through interactions with genomic regulatory sequences. While indispensable for rescue of infectious virus from cDNA of any filovirus genus, the requirement for VP30 in other facets of gene expression varies between filovirus genera. Minigenome systems modeling transcription and replication of MARV minigenomes function independently of VP30, whereas EBOV systems require VP30 for transcriptional activation at the start of the first gene, a dependence also present in filoviruses of the cuevavirus genus. The inability of filovirus polymerases to initiate transcription at sites internal to the genome has restricted the extent to which VP30 function can be assessed in EBOV model systems, due to the impact of the immediate transcriptional defect in the absence of VP30 on all potential subsequent transcriptional events. To elucidate further transcriptional roles of VP30 in filovirus gene expression with potential application across genera, we leveraged the VP30-independence of MARV transcriptional activation by generating MARV systems modeling transcriptional re-initiation and viral particle production, as well as a novel application of targeted protein degradation for inducible depletion of MARV VP30 at different stages of infection. Furthermore, by dissecting the discrepancy between VP30 interactions with a regulatory sequence in the untranslated region of the respective nucleoprotein genes of EBOV and MARV, we built a VP30-independent EBOV minigenome system using MARV sequence elements and identified two nucleotides primarily responsible for the dependence of EBOV transcription on VP30. Surprisingly, this effect appeared more specific to the sequence than to the stem-loop structure already implicated in VP30 regulation of EBOV transcription.
966BIn addition to filovirus gene expression, this work describes the application of multiple approaches to visualize and quantify viral infection in the investigation of viruses for which reagents are not yet available, including detection of a novel filovirus and antiviral drug testing in specialized cell types early in the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Description
2025
License
Attribution-NonCommercial-NoDerivatives 4.0 International