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Freie Universität Berlin
Freie Universität Berlin
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Marek’s disease virus (MDV) is a strictly cell-associated herpesvirus that is deadly and oncogenic
in its natural host with up to 100% mortalities in unvaccinated flocks. In addition to the economic
and agriculture importance, it is a natural virus-host model for the investigation of virus-induced
lymphomas. Lymphomas are the main cause of MDV-induced tremendous suffering and death in
chickens. MDV harbours a linear double-stranded genome of about 180-kilobase pairs composed
of two unique regions, unique long (UL) and unique short (US). Unique regions are flanked by
further repeats that have a set of genes that contributes to MDV pathogenicity and oncogenicity.
These genes are the major oncogene meq, the open reading frames (ORF) RLORF5a and
RLORF4, and the chemokine homolog vIL-8. Moreover, these genes encode a set of splice
variants that are thought to contribute to MDV pathogenesis and carcinogenesis. Yet it is unclear
if all or some of these splice variants encode an expressed protein. Interestingly, these variants
are spliced to the vIL-8 gene that consists of three exons and two introns.
Introns are non-coding sequences and yet their significance is underappreciated or poorly
understood, despite they include important junctions that mediate splicing. In this study, one or
all introns of vIL-8 were removed to abrogate the splicing of vIL-8 and characterize the importance
of introns for virus replication and protein secretion. Interestingly, vIL-8 introns were non-essential
for virus replication, but they were indispensable for protein secretion. In addition, intron II was
found to harbour a novel exon (E3`) that is spliced to express novel vIL-8 protein variants that
were non-essential for virus replication or protein secretion. This study is the first to report that
alphaherpesviruses express novel chemokine variants and lay the foundation for future studies
to dissect the role of vIL-8 splice variants in MDV pathogenesis and tumour formation.
CRISPR/Cas was discovered in bacteria and archaea as an adaptive antiviral immune
mechanism. Streptococcus pyogenes have evolved CRISPR/Cas9, which is the best-studied
example of the CRISPR/Cas systems until now. CRISPR/Cas9 has been recently used in various
applications that revolutionized the basic molecular biology. In principle, guide RNAs recruit Cas9
to cleave the targets in a very precise manner.
In this study, CRISPR/Cas9 was used to target MDV essential genes to inhibit virus replication.
Interestingly, while single gRNAs significantly impaired virus replication to different degrees,
multiple gRNAs completely inhibited MDV replication. Moreover, CRISPR/Cas9 was tested for
the long-term elimination of MDV from infected cultures that were serially passaged. Intriguingly,
the single guides showed MDV escape mutants that appeared very quickly, yet the combined
gRNAs did not produce MDV that is able to evade CRISPR/Cas9. Sequencing of the escape
mutants revealed specific mutations that were seen in the recognition sites of two MDV single
gRNAs, which would eliminate their ability to functionally cleave the mutated MDV targets. In all,
this is the first demonstration that CRISPR/Cas9 can inhibit MDV replication and evasion,
especially when two or more gRNAs are used. This study provides the basis for future studies to
protect chickens from this deadly oncogenic herpesvirus.
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