A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene

Reporter genes for RNA viruses are well-known to be unstable due to putative RNA recombination events that excise inserted nucleic acids. RNA recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. Therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. Short-read RNA sequencing using ClickSeq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. A rapid removal of the GFP gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. Eventually, these small deletions merged to fully remove the GFP gene. The removal was significantly enhanced during the passaging of low-fidelity TC-83, suggesting that increased levels of recombination are a defining characteristic of this mutant.

Download Full-text

The transcriptional landscape of Venezuelan equine encephalitis virus (TC-83) infection

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009306 ◽

2021 ◽

Vol 15 (3) ◽

pp. e0009306

Author(s):

Zhiyuan Yao ◽

Fabio Zanini ◽

Sathish Kumar ◽

Marwah Karim ◽

Sirle Saul ◽

...

Keyword(s):

Single Cell ◽

Membrane Trafficking ◽

Single Cells ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Western Hemisphere ◽

Venezuelan Equine Encephalitis ◽

Live Attenuated Vaccine ◽

Time Points ◽

Equine Encephalitis Virus

Venezuelan Equine Encephalitis Virus (VEEV) is a major biothreat agent that naturally causes outbreaks in humans and horses particularly in tropical areas of the western hemisphere, for which no antiviral therapy is currently available. The host response to VEEV and the cellular factors this alphavirus hijacks to support its effective replication or evade cellular immune responses are largely uncharacterized. We have previously demonstrated tremendous cell-to-cell heterogeneity in viral RNA (vRNA) and cellular transcript levels during flaviviral infection using a novel virus-inclusive single-cell RNA-Seq approach. Here, we used this unbiased, genome-wide approach to simultaneously profile the host transcriptome and vRNA in thousands of single cells during infection of human astrocytes with the live-attenuated vaccine strain of VEEV (TC-83). Host transcription was profoundly suppressed, yet “superproducer cells” with extremely high vRNA abundance emerged during the first viral life cycle and demonstrated an altered transcriptome relative to both uninfected cells and cells with high vRNA abundance harvested at later time points. Additionally, cells with increased structural-to-nonstructural transcript ratio exhibited upregulation of intracellular membrane trafficking genes at later time points. Loss- and gain-of-function experiments confirmed pro- and antiviral activities in both vaccine and virulent VEEV infections among the products of transcripts that positively or negatively correlated with vRNA abundance, respectively. Lastly, comparison with single cell transcriptomic data from other viruses highlighted common and unique pathways perturbed by infection across evolutionary scales. This study provides a high-resolution characterization of the VEEV (TC-83)-host interplay, identifies candidate targets for antivirals, and establishes a comparative single-cell approach to study the evolution of virus-host interactions.

Download Full-text

Low-fidelity Venezuelan equine encephalitis virus polymerase mutants to improve live-attenuated vaccine safety and efficacy

Virus Evolution ◽

10.1093/ve/vey004 ◽

2018 ◽

Vol 4 (1) ◽

Cited By ~ 11

Author(s):

Tiffany F Kautz ◽

Mathilde Guerbois ◽

Kamil Khanipov ◽

Edward I Patterson ◽

Rose M Langsjoen ◽

...

Keyword(s):

Vaccine Safety ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Venezuelan Equine Encephalitis ◽

Live Attenuated Vaccine ◽

Safety And Efficacy ◽

Equine Encephalitis Virus ◽

Attenuated Vaccine ◽

Polymerase Mutants

Download Full-text

Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles

Viruses ◽

10.3390/v12050546 ◽

2020 ◽

Vol 12 (5) ◽

pp. 546 ◽

Cited By ~ 2

Author(s):

Edward I. Patterson ◽

Kamil Khanipov ◽

Daniele M. Swetnam ◽

Samantha Walsdorf ◽

Tiffany F. Kautz ◽

...

Keyword(s):

Cleavage Site ◽

Mutation Frequency ◽

Infectious Virus ◽

Sequencing Analysis ◽

Venezuelan Equine Encephalitis ◽

Virus Particles ◽

Equine Encephalitis Virus ◽

Next Generation Sequencing Analysis ◽

Multiple Replication ◽

Selection Of

Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3Δ56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine strain, non-infectious virus particles were used to assess the effect of single mutations on mutation frequency without the interference of selection that results from multiple replication cycles. Next-generation sequencing analysis revealed a significantly lower frequency of transversion mutations and overall mutation frequency for the fidelity mutants compared to VEEV TC-83 E3Δ56-59. We demonstrate that deletion of the PE2 cleavage site halts virus infection while making the virus particles available for downstream sequencing. The conservation of the site will allow the evaluation of suspected fidelity mutants across alphaviruses of medical importance.

Download Full-text

Toll-like receptor 4 mediates blood-brain barrier permeability and disease in C3H mice during Venezuelan equine encephalitis virus infection

Virulence ◽

10.1080/21505594.2020.1870834 ◽

2021 ◽

Vol 12 (1) ◽

pp. 430-443

Author(s):

Bradley S. Hollidge ◽

Courtney A. Cohen ◽

Justice Akuoku Frimpong ◽

Catherine V. Badger ◽

John M. Dye ◽

...

Keyword(s):

Virus Infection ◽

Blood Brain Barrier ◽

Venezuelan Equine Encephalitis Virus ◽

Toll Like Receptor ◽

Venezuelan Equine Encephalitis ◽

Toll Like Receptor 4 ◽

Barrier Permeability ◽

Equine Encephalitis Virus ◽

Brain Barrier Permeability ◽

C3h Mice

Download Full-text

Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway

Plants ◽

10.3390/plants10020346 ◽

2021 ◽

Vol 10 (2) ◽

pp. 346

Author(s):

Caitlin W. Lehman ◽

Kylene Kehn-Hall ◽

Megha Aggarwal ◽

Nicole R. Bracci ◽

Han-Chi Pan ◽

...

Keyword(s):

Antiviral Activity ◽

Fluorescent Protein ◽

Glycogen Synthase ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Host Cells ◽

Dynamics Simulation ◽

Luciferase Reporter ◽

Venezuelan Equine Encephalitis ◽

Equine Encephalitis Virus

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

Download Full-text