A functional interleukin-4 homolog is encoded in the genome of infectious laryngotracheitis virus: unveiling a novel virulence factor
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Abstract
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 Summary
Herpesviruses 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:
Novel 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.
Implications 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.
Evolutionary 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.
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.
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Computational methods at the time likely failed to identify potential protein-coding reading frames within the region, hindering its recognition as a functional transcript.
This is such an interesting and important point. Do you know what annotation tool was used to do the original annotation? Should this motivate annotation of viral genomes now that we have more advanced tools, as well as additional RNA sequencing datasets?
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espite the difference in viral TCID50/ml, viral genome load for both viruses showed an almost equivalent rate of increase by 72 and 96 hours post-absorption
Can you comment on where this discrepancy comes from, or what it might mean? As a non-subject matter expert, I wasn't sure how to interpret this result in the context of viral biology.
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Viral IL-4 evolved within Galliformes
Have you tried adding other vIL-4s to this tree to see if this is a single acquisition event? The vIL-4 tree below is really informative, and made me wonder what it would look like to see those sequences on the larger IL-4 tree
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The novel transcript (named vIL-4 as discussed below) was found to be highly abundant both in vitro and in vivo. In the in vitro semi-quantitative IsoSeq data, the vIL-4 transcript was present at 3247 and 2606 viral transcripts per million (vTPM) in 1874C5 and CEO/LaryngoVac, respectively. By rank, these equated to the 18th and 19th most abundant transcript in each strain. Short-read RNA-Seq revealed the vIL-4 transcript to be even more abundant in vivo, representing the fifth-most abundant viral transcript in birds inoculated with either 1874C5 or CEO/LaryngoVac (Figure 2).
Sequencing data are inherently compositional because they are constrained by the total number of reads obtained, which affects the interpretation of the data. Each read count is dependent not only on its own abundance but also on the abundance of other transcripts …
The novel transcript (named vIL-4 as discussed below) was found to be highly abundant both in vitro and in vivo. In the in vitro semi-quantitative IsoSeq data, the vIL-4 transcript was present at 3247 and 2606 viral transcripts per million (vTPM) in 1874C5 and CEO/LaryngoVac, respectively. By rank, these equated to the 18th and 19th most abundant transcript in each strain. Short-read RNA-Seq revealed the vIL-4 transcript to be even more abundant in vivo, representing the fifth-most abundant viral transcript in birds inoculated with either 1874C5 or CEO/LaryngoVac (Figure 2).
Sequencing data are inherently compositional because they are constrained by the total number of reads obtained, which affects the interpretation of the data. Each read count is dependent not only on its own abundance but also on the abundance of other transcripts in the sample. This means the data are subject to a constant sum constraint, which can lead to misleading conclusions about relative transcript abundances. Methods that do not account for the compositional structure of the data can lead to biased interpretations. It might be inappropriate to interpret the transcript per million (TPM) values and rankings directly. There are some packages for compositional analysis (CoDA) and some transformations in DESeq2 and edgeR designed to help with this. It might be worth exploring these and seeing if this observation still holds.
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Initial BLASTP and TBLASTN [66] searches of the chicken IL-4 sequence (NP_001385388) against the nr and nt databases, respectively, were used to generate a candidate list of published homologs.
Did you consider using your vIL-4 structure(s) and using the FoldSeek web interface/server t(https://search.foldseek.com/search) o search for structural homologs? Because there is relatively low sequence identity and viruses evolve so quickly, I would be curious if you uncover more homologs via a structural search. I think the results from the BFVD (viral) and AFDB50 databases would be particularly interesting to see.
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