The orf virus (ORFV) protein OV20.0 interacts with the microprocessor complex subunit DGCR8 to regulate miRNA biogenesis and ORFV infection.

Cellular double-stranded RNA-binding proteins (DRBPs) play important roles in the regulation of innate immune responses and microRNA (miRNA) biogenesis. The current study aimed to understand whether OV20.0, a DRBP of orf virus (ORFV), is involved in cellular RNA biogenesis via association with host DRBPs. We found that OV20.0 interacts with DiGeorge syndrome critical region 8 (DGCR8), a subunit of the miRNA processor complex, and binds to primary- and precursor-miRNA. Additionally, OV20.0 regulates DGCR8 expression in multiple ways, including through interaction with the DGCR8 protein and binding to DGCR8 mRNA. Lastly, our data show that DGCR8 plays an antiviral role against ORFV infection, whereas it is beneficial for influenza virus propagation, indicating that the underlying mechanisms could be diverse among different viruses.

Interleukin-17 mediates inflammatory tissue injury during orf development in goats.

Orf is an epithelial zoonotic infectious disease caused by orf virus (ORFV). Mounting studies have shown that IL-17-driven neutrophil inflammation plays a central role in inflammatory skin diseases. However, whether IL-17 plays a similar role and how does it work in the pathogenesis of orf is unclear. In this study, we found that during orf development, numerous inflammatory cells, especially neutrophils, infiltrated in the damaged lip tissue. Meanwhile, the production of IL-17 was increased in the lesion site. Further evidence showed that IL-17 potently stimulated the production of several chemokines that are crucial for neutrophil migration. In addition, IL-17 was mostly produced by CD4+ T cells and gamma delta T (γδ T) cells of the skin. In conclusion, the present study highlighted a critical role of IL-17-driven inflammation in the pathogenesis of orf and suggested that this cytokine may be a potential therapeutic target of this disease in goats.

Orf virus (ORFV) infection in a three-dimensional human skin model: Characteristic cellular alterations and interference with keratinocyte differentiation.

ORF virus (ORFV) is the causative agent of contagious ecthyma, a pustular dermatitis of small ruminants and humans. Even though the development of lesions caused by ORFV was extensively studied in animals, only limited knowledge exists about the lesion development in human skin. The aim of the present study was to evaluate a three-dimensional (3D) organotypic culture (OTC) as a human skin model for ORFV infection considering lesion development, replication of the virus, viral gene transcription and modulation of differentiation of human keratinocytes by ORFV. ORFV infection of OTC was performed using the ORFV isolate B029 derived from a human patient. The OTC sections showed a similar structure of stratified epidermal keratinocytes as human foreskin and a similar expression profile of the differentiation markers keratin 1 (K1), K10, and loricrin. Upon ORFV infection, OTCs exhibited histological cytopathic changes including hyperkeratosis and ballooning degeneration of the keratinocytes. ORFV persisted for 10 days and was located in keratinocytes of the outer epidermal layers. ORFV-specific early, intermediate and late genes were transcribed, but limited viral spread and restricted cell infection were noticed. ORFV infection resulted in downregulation of K1, K10, and loricrin at the transcriptional level without affecting proliferation as shown by PCNA or Ki-67 expression. In conclusion, OTC provides a suitable model to study the interaction of virus with human keratinocytes in a similar structural setting as human skin and reveals that ORFV infection downregulates several differentiation markers in the epidermis of the human skin, a hitherto unknown feature of dermal ORFV infection in man.

Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.

Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.

The ribosome binding site of a mini-ORF protects a T3SS mRNA from degradation by RNase E.

Enterohaemorrhagic Escherichia coli harbours a pathogenicity island encoding a type 3 secretion system used to translocate effector proteins into the cytosol of intestinal epithelial cells and subvert their function. The structural proteins of the translocon are encoded in a major espADB mRNA processed from a precursor. The translocon mRNA should be highly susceptible to RNase E cleavage because of its AU-rich leader region and monophosphorylated 5'-terminus, yet it manages to avoid rapid degradation. Here, we report that the espADB leader region contains a strong Shine-Dalgarno element (SD2) and a translatable mini-ORF of six codons. Disruption of SD2 so as to weaken ribosome binding significantly reduces the concentration and stability of esp mRNA, whereas codon substitutions that impair translation of the mini-ORF have no such effect. These findings suggest that occupancy of SD2 by ribosomes, but not mini-ORF translation, helps to protect espADB mRNA from degradation, likely by hindering RNase E access to the AU-rich leader region.

Orf virus ORFV121 encodes a novel inhibitor of NF-kappaB that contributes to virus virulence.

Orf virus (ORFV), the type member of the genus Parapoxvirus of the Poxviridae, has evolved novel strategies (proteins and/or mechanisms of action) to modulate host cell responses regulated by the nuclear factor-κB (NF-κB) signaling pathway. Here, we present data indicating that ORFV ORFV121, a gene unique to parapoxviruses, encodes a novel viral NF-κB inhibitor that binds to and inhibits the phosphorylation and nuclear translocation of NF-κB-p65. The infection of cells with an ORFV121 deletion mutant virus (OV-IA82Δ121) resulted in increased NF-κB-mediated gene transcription, and the expression of ORFV121 in cell cultures significantly suppressed NF-κB-regulated reporter gene expression. ORFV ORFV121 physically interacts with NF-κB-p65 in the cell cytoplasm, thus providing a mechanism for the inhibition of NF-κB-p65 phosphorylation and nuclear translocation. Notably, the deletion of ORFV121 from the viral genome markedly decreased ORFV virulence and disease pathogenesis in sheep, indicating that ORFV121 is a virulence determinant for ORFV in the natural host.

A novel Bcl-2-like inhibitor of apoptosis is encoded by the parapoxvirus ORF virus.

Apoptotic cell death forms part of the host defense against virus infection. We tested orf virus, a member of the poxvirus family, for the ability to inhibit apoptosis and found that orf virus-infected cells were fully resistant to UV-induced changes in cell morphology, caspase activation, and DNA fragmentation. By using a library of vaccinia virus-orf virus recombinants, we identified an orf virus gene (ORFV125) whose presence was linked with the inhibition of apoptosis. The 173-amino-acid predicted protein had no clear homologs in public databases other than those encoded by other parapoxviruses. However, ORFV125 possessed a distinctive C-terminal domain which was necessary and sufficient to direct the protein to the mitochondria. We determined that ORFV125 alone could fully inhibit UV-induced DNA fragmentation, caspase activation, and cytochrome c release and that its mitochondrial localization was required for its antiapoptotic function. In contrast, ORFV125 did not prevent UV-induced activation of c-Jun NH2-terminal kinase, an event occurring upstream of the mitochondria. These features are comparable to the antiapoptotic properties of the mitochondrial regulator Bcl-2. Furthermore, bioinformatic analyses revealed sequence and secondary-structure similarities to Bcl-2 family members, including characteristic residues of all four Bcl-2 homology domains. Consistent with this, the viral protein inhibited the UV-induced activation of the proapoptotic Bcl-2 family members Bax and Bak. ORFV125 is the first parapoxvirus apoptosis inhibitor to be identified, and we propose that it is a new antiapoptotic member of the Bcl-2 family.