On the stability of sequences inserted into viral genomes

Abstract

Viruses are widely used as vectors for heterologous gene expression in cultured cells or natural hosts, and therefore a large number of viruses with exogenous sequences inserted into their genomes have been engineered. Many of these engineered viruses are viable and express heterologous proteins at high levels, but the inserted sequences often prove to be unstable over time and are rapidly lost, limiting heterologous protein expression. Although virologists are aware that inserted sequences can be unstable, processes leading to insert instability are rarely considered from an evolutionary perspective. Here, we review experimental work on the stability of inserted sequences over a broad range of viruses, and we present some theoretical considerations concerning insert stability. Different virus genome organizations strongly impact insert stability, and factors such as the position of insertion can have a strong effect. In addition, we argue that insert stability not only depends on the characteristics of a particular genome, but that it will also depend on the host environment and the demography of a virus population. The interplay between all factors affecting stability is complex, which makes it challenging to develop a general model to predict the stability of genomic insertions. We highlight key questions and future directions, finding that insert stability is a surprisingly complex problem and that there is need for mechanism-based, predictive models. Combining theoretical models with experimental tests for stability under varying conditions can lead to improved engineering of viral modified genomes, which is a valuable tool for understanding genome evolution as well as for biotechnological applications, such as gene therapy.

A cassava common mosaic virus vector for virus-induced gene silencing in cassava

“…However, the loss of PDS inserts still occurred when this FoMV vector was used to silence PDS in maize [ 34 ]. Therefore, the stability of sequences inserted into viral genomes is regard as a surprisingly complex problem involved in the genome characteristics, the host environment and the demography of a virus population [ 41 ].…”

The Expression of Hemagglutinin by a Recombinant Newcastle Disease Virus Causes Structural Changes and Alters Innate Immune Sensing

“…The present study confirmed these results and found significant structural differences between rNDV-H5 and the parental LaSota NDV. Because the H5 gene is inserted upstream of the 3’ end of the genome of the rNDV-H5 construct, between phosphoprotein and matrix genes, the level of expression of downstream F and HN could be impacted and reduced [ 46 , 47 ]. However, the investigation of both surface glycoproteins distribution demonstrated a higher expression of HN and a lower expression of F by rNDV-H5 when compared to parental NDV LaSota, although HN is positioned more distally than F in the NDV genome.…”

Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis

“…In contrast, PCR analysis of the three successive passages (P1-P3) and the OBs obtained after injection of BVs at P0 in larvae revealed that only the product corresponding to Bac-polh-Ø was present, which implies the complete loss of these genes. It has been shown that genes inserted in the genomes of recombinant viruses, to express heterologous proteins at high levels, often prove to be unstable over time and can be rapidly lost [40]. This loss might be particularly rapid when the inserted gene encodes a protein that is deleterious for the virus.…”