Viral gene drive in herpesviruses

Abstract

Gene drives are genetic modifications designed to propagate in a population with high efficiency. Current gene drive strategies rely on sexual reproduction and are thought to be restricted to sexual organisms. Here, we report on a gene drive system that allows the spread of an engineered trait in populations of DNA viruses and, in particular, herpesviruses. We describe the successful transmission of a gene drive sequence between distinct strains of human cytomegalovirus (human herpesvirus 5) and show that gene drive viruses can efficiently target and replace wildtype populations in cell culture experiments. Moreover, by targeting sequences necessary for viral replication, our results indicate that a viral gene drive can be used as a strategy to suppress a viral infection. Taken together, this work offers a proof of principle for the design of a gene drive in viruses.

Targeting conserved sequences circumvents the evolution of resistance in a viral gene drive against human cytomegalovirus

“…We first attempted to model the evolution of gene drive resistance using numerical simulations. Our initial study indicated that after co-infection with a wildtype and a gene drive viruses, around 5-10% of viruses remained unconverted and had a mutated target site that rendered them resistant to the drive (7). Using a 10% estimate and considering in this scenario that resistant viruses would replicate at the same level as wildtype, we modelled the evolution of the viral population over time ( Figure 1B, Supplementary Figure S1).…”

“…We next evaluated experimentally the evolution of resistance in cell culture. Our initial experimental system involved an unmodified hCMV virus expressing GFP (strain Towne, referred hereafter as Towne-GFP) and an mCherry-expressing gene drive virus (GD-UL23) targeting UL23 (7) (Figure 2A). UL23 is dispensable in normal cell culture conditions (8, 9), but the GD-UL23 virus was built in a different viral strain (TB40/E) and replicated significantly slower than Towne-GFP (t-test: p=0.0065, Figure 2B).…”

“…The difficulty lies in designing gene drive viruses with replicative defects that spread efficiently. Our previous study focused on a gene drive against UL23 , a viral gene that is dispensable in normal cell culture conditions (7, 8). UL23 is involved in immunity evasion, and the replication of UL23 mutant viruses is severely impaired by interferon γ.…”

“…Our initial study targeted UL23, a viral gene that encodes a tegument protein involved in immune evasion. UL23 is dispensable in normal cell culture conditions, but necessary in presence of interferon γ (7)(8)(9). The tegument of herpesvirus is a layer of protein that lies between the genome-containing capsid and the viral envelope (1,10).…”

“…Most gene drives rely on CRISPR-mediated homologous recombination and have been restricted to sexually reproducing organisms. Using hCMV as a model, we recently developed a gene drive system in herpesviruses that rely on co-infection of cells by a wildtype and an engineered virus (7). Cleavage by Cas9 and repair by homologous recombination leads to the conversion of wildtype viruses into new recombinant viruses ( Figure 1A).…”