A functional interleukin-4 homolog is encoded in the genome of infectious laryngotracheitis virus: unveiling a novel virulence factor
Herpesviruses have evolved numerous immune evasion tactics, persisting within their hosts through self-perpetuating strategies. One such tactic involves acquiring functional copies of host genes encoding cytokines such as IL-6 (HHV-8), IL-10 (HHV-4, HHV-5), and IL-17 (SaHV-2). These viral mimics, or virokines, can bind to cellular receptors, modulating the natural cytokine signaling to manipulate the immune response in favor of the virus or stimulate target cell growth to enhance virus replication. In the course of full-length cDNA sequencing of infectious laryngotracheitis virus (ILTV) transcripts, a previously unknown highly-spliced gene was discovered in the viral genome predicted to encode a 147 amino acid protein with similarity to vertebrate interleukin-4. The three-intron gene structure was precisely conserved with chicken and other vertebrate IL-4 homologs, and the amino acid sequence displayed structural conservation with vertebrate homologs at the primary, secondary, and tertiary levels based on computational modeling. The viral IL-4 gene was subsequently identified in all sequenced ILTV genomes. The mature transcript was highly expressed both in vitro and in vivo, and protein expression in infected cells was confirmed using LC-MS/MS. Phylogenetic analyses, along with the conserved gene structure, suggested direct capture from a Galliformes host. Functionally, an LPS-stimulation assay showed that the expressed viral IL-4 homolog stimulated nitric oxide production in a macrophage cell line at comparable levels to recombinant chicken IL-4. A recombinant virus lacking vIL-4 exhibited slightly higher titers in cell culture compared to the parental strain. In vivo bird studies demonstrated reduced pathogenicity of the vIL-4 knockout compared to wildtype. These results represent the first report of a previously unknown virokine encoded in the ILTV genome expressing a functional IL-4 homolog and virulence factor. Author SummaryHerpesviruses are large DNA viruses, several of which express proteins that are homologous to host cytokines (termed virokines) and are thought to modulate the host immune response in favor of the virus. We report the identification of a novel virokine in the genome of infectious laryngotracheitis virus (ILTV) that is structurally and functionally similar to avian interleukin-4. The significance of this finding is threefold: O_LINovel mechanism: The identification of vIL-4 expands our understanding of the strategies employed by viruses to evade the host immune response, including through the acquisition, adaptation, and expression of cellular genes for their own advantage. C_LIO_LIImplications for disease pathogenesis: We demonstrate that vIL-4 plays a functional role in ILTV virulence. Understanding the mechanisms by which this happens could lead to the development of novel therapeutic strategies. C_LIO_LIEvolutionary insights: The presence of the highly spliced vIL-4 gene in the ILTV genome suggests direct genomic capture of a host gene, providing insights into the evolutionary history of ILTV and its interactions with avian theropods. C_LI Overall, this research represents a significant contribution to our understanding of viral immunology and has potential implications for the development of improved vaccines for ILTV infection.