Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family that, unlike other herpesviruses, triggers a strong innate immune response in infected cells that includes transcription of the beta interferon gene via activation of interferon regulatory factor 3 (IRF3). IRF3 activation requires signaling from pattern recognition receptors that is initiated by their interaction with specific pathogen-associated molecules. However, while IRF3-activating pathways are increasingly well characterized, the cellular molecules involved in HCMV-mediated IRF3-dependent beta interferon transcription are virtually unknown. We undertook a systematic examination of new and established IRF3-terminal pathway components to identify those that are essential to HCMV-triggered IRF3 activation. We show here that IRF3 activation induced by HCMV infection involves the newly identified protein STING but, in contrast to infections with other herpesviruses, occurs independently of the adaptor molecule IPS-1. We also show that the protein DDX3 contributes to HCMV-triggered expression of beta interferon. Moreover, we identify Z-DNA binding protein 1 (ZBP1) as being essential for IRF3 activation and interferon beta expression triggered by HCMV, as well as being sufficient to enhance HCMV-stimulated beta interferon transcription and secretion. ZBP1 transcription was also found to be induced following exposure to HCMV in a JAK/STAT-dependent manner, thus perhaps also contributing to a positive feedback signal. Finally, we show that constitutive overexpression of ZBP1 inhibits HCMV replication. ZBP1 was recently identified as a cytosolic pattern recognition receptor of double-stranded DNA, and thus, we propose a model for HCMV-mediated IRF3 activation that involves HCMV-associated DNA as the principal innate immune-activating pathogen-associated molecular pattern.

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is induced as part of the IFN response in mammals and acts to shut down protein synthesis by the phosphorylation of eukaryotic initiation factor 2α (eIF2α). In fish, a PKR-like kinase activity has been detected, but the enzyme responsible has eluded characterization. Here, we describe a PKR-like kinase from zebrafish. Phylogenetic analysis shows that the C-terminal kinase domain is more closely related to the kinase domain of PKR than to any of the other three known eIF2α kinases. Surprisingly, instead of the two dsRNA binding domains found at the N terminus of PKR, there are two Zα domains. Zα domains specifically bind dsDNA and RNA in the left-handed Z conformation, often with high affinity. They have been found previously in two other IFN-inducible proteins, the dsRNA editing enzyme, ADAR1, and Z-DNA binding protein 1 (ZBP1), as well as in the poxvirus virulence factor, E3L. This previously undescribed kinase, designated PKZ (protein kinase containing Z-DNA binding domains), is transcribed constitutively at low levels and is highly induced after injection of poly(inosinic)–poly(cytidylic) acid, which simulates viral infection. Binding of Z-DNA by the Zα domain of PKZ was demonstrated by circular dichroism. PKZ inhibits translation in transfected cells; site-directed mutagenesis indicates that this inhibition depends on its catalytic activity. Identification of a gene combining Zα domains with a PKR-like kinase domain strengthens the hypothesis that the ability to bind left-handed nucleic acid plays a role in the host response to viruses.

PKR responds to viral infection and shuts down translation through phosphorylation of eIF2α. PKR is found to have rapidly evolved in comparison to other kinases targeting the same substrate across a broad range of vertebrate lineages. Some positively selected residues are found to confer resistance to poxviral inhibitors that mimic substrate. In addition, substituting a single residue in mouse PKR with the corresponding residue under positive selection in human PKR renders mouse PKR more resistant to K3L and vice versa, providing evidence for species-specific selection driven by beneficial mutations. Protein kinase PKR (also known as EIF2AK2) is activated during viral infection and phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2), leading to inhibition of translation and viral replication. We report fast evolution of the PKR kinase domain in vertebrates, coupled with positive selection of specific sites. Substitution of positively selected residues in human PKR with residues found in related species altered sensitivity to PKR inhibitors from different poxviruses. Species-specific differences in sensitivity to poxviral pseudosubstrate inhibitors were identified between human and mouse PKR, and these differences were traced to positively selected residues near the eIF2α binding site. Our findings indicate how an antiviral protein evolved to evade viral inhibition while maintaining its primary function. Moreover, the identified species-specific differences in the susceptibility to viral inhibitors have important implications for studying human infections in nonhuman model systems.

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