ADP-ribosylation is a ubiquitous post-translational addition of either monomers or polymers of ADP-ribose to target proteins by ADP-ribosyltransferases, usually by interferon-inducible diphtheria toxin-like enzymes known as PARPs. While several PARPs have known antiviral activities, these activities are mostly independent of ADP-ribosylation. Consequently, less is known about the antiviral effects of ADP-ribosylation. Several viral families, including Coronaviridae, Togaviridae, and Hepeviridae, encode for macrodomain proteins that bind to and hydrolyze ADP-ribose from proteins and are critical for optimal replication and virulence. These results suggest that macrodomains counter cellular ADP-ribosylation, but whether PARPs or, alternatively, other ADP-ribosyltransferases cause this modification is not clear. Here we show that pan-PARP inhibition enhanced replication and inhibited interferon production in primary macrophages infected with macrodomain-mutant but not wild-type coronavirus. Specifically, knockdown of two abundantly expressed PARPs, PARP12 and PARP14, led to increased replication of mutant but did not significantly affect wild-type virus. PARP14 was also important for the induction of interferon in mouse and human cells, indicating a critical role for this PARP in the regulation of innate immunity. In summary, these data demonstrate that the macrodomain is required to prevent PARP-mediated inhibition of coronavirus replication and enhancement of interferon production.

The UL97 protein of human cytomegalovirus (HCMV, or HHV-5 (human herpesvirus 5)), is a kinase that phosphorylates the cellular retinoblastoma (Rb) tumor suppressor and lamin A/C proteins that are also substrates of cellular cyclin-dependent kinases (Cdks). A functional complementation assay has further shown that UL97 has authentic Cdk-like activity. The other seven human herpesviruses each encode a kinase with sequence and positional homology to UL97. These UL97-homologous proteins have been termed the conserved herpesvirus protein kinases (CHPKs) to distinguish them from other human herpesvirus-encoded kinases. To determine if the Cdk-like activities of UL97 were shared by all of the CHPKs, we individually expressed epitope-tagged alleles of each protein in human Saos-2 cells to test for Rb phosphorylation, human U-2 OS cells to monitor nuclear lamina disruption and lamin A phosphorylation, or S. cerevisiae cdc28-13 mutant cells to directly assay for Cdk function. We found that the ability to phosphorylate Rb and lamin A, and to disrupt the nuclear lamina, was shared by all CHPKs from the beta- and gamma-herpesvirus families, but not by their alpha-herpesvirus homologs. Similarly, all but one of the beta and gamma CHPKs displayed bona fide Cdk activity in S. cerevisiae, while the alpha proteins did not. Thus, we have identified novel virally-encoded Cdk-like kinases, a nomenclature we abbreviate as v-Cdks. Interestingly, we found that other, non-Cdk-related activities reported for UL97 (dispersion of promyelocytic leukemia protein nuclear bodies (PML-NBs) and disruption of cytoplasmic or nuclear aggresomes) showed weak conservation among the CHPKs that, in general, did not segregate to specific viral families. Therefore, the genomic and evolutionary conservation of these kinases has not been fully maintained at the functional level. Our data indicate that these related kinases, some of which are targets of approved or developmental antiviral drugs, are likely to serve both overlapping and non-overlapping functions during viral infections.

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Neuron-virus interactions that occur during herpes simplex virus (HSV) infection are not fully understood. Neurons are the site of lifelong latency and are a crucial target for long-term suppressive therapy or viral clearance. A reproducible neuronal model of human origin would facilitate studies of HSV and other neurotropic viruses. Current neuronal models in the herpesvirus field vary widely and have caveats, including incomplete differentiation, nonhuman origins, or the use of dividing cells that have neuropotential but lack neuronal morphology. In this study, we used a robust approach to differentiate human SH-SY5Y neuroblastoma cells over 2.5 weeks, producing a uniform population of mature human neuronal cells. We demonstrate that terminally differentiated SH-SY5Y cells have neuronal morphology and express proteins with subcellular localization indicative of mature neurons. These neuronal cells are able to support a productive HSV-1 infection, with kinetics and overall titers similar to those seen in undifferentiated SH-SY5Y cells and the related SK-N-SH cell line. However, terminally differentiated, neuronal SH-SY5Y cells release significantly less extracellular HSV-1 by 24 h postinfection (hpi), suggesting a unique neuronal response to viral infection. With this model, we are able to distinguish differences in neuronal spread between two strains of HSV-1. We also show expression of the antiviral protein cyclic GMP-AMP synthase (cGAS) in neuronal SH-SY5Y cells, which is the first demonstration of the presence of this protein in nonepithelial cells. These data provide a model for studying neuron-virus interactions at the single-cell level as well as via bulk biochemistry and will be advantageous for the study of neurotropic viruses Herpes simplex virus (HSV) affects millions of people worldwide, causing painful oral and genital lesions, in addition to a multitude of more severe symptoms such as eye disease, neonatal infection, and, in rare cases, encephalitis. Presently, there is no cure available to treat those infected or prevent future transmission. Due to the ability of HSV to cause a persistent, lifelong infection in the peripheral nervous system, the virus remains within the host for life. To better understand the basis of virus-neuron interactions that allow HSV to persist within the host peripheral nervous system, improved neuronal models are required. Here we describe a cost-effective and scalable human neuronal model system that can be used to study many neurotropic viruses, such as HSV, Zika virus, dengue virus, and rabies virus.

The Epstein-Barr virus (EBV)-encoded viral protein kinase, EBV-PK (the BGLF4 gene product), is required for efficient nuclear viral egress in 293 cells. However, since EBV-PK phosphorylates a number of different viral and cellular proteins (including lamin A/C), the relative importance of each target during lytic viral replication remains unclear. We show here that an EBV PK mutant (PKmut; containing stop codons at residues 1 and 5 in EBV-PK) is highly defective for release of infectious virus from 293 cells but not 293T cells. Furthermore, the phenotype of the PKmut in 293 cells is substantially reversed by expression of the simian virus 40 (SV40) large (T) and small (t) T antigens. Efficient rescue requires the presence of both SV40 T/t proteins. We show that 293T cells have a much higher level of constitutive lamin A/C phosphorylation than do 293 cells over residues (S22 and S392) that promote phosphorylation-dependent nuclear disassembly and that both large T and small t contribute to enhanced lamin A/C phosphorylation. Finally, we demonstrate that knockdown of lamin A/C expression using small interfering RNA also rescues the PKmut phenotype in 293 cells. These results suggest that essential roles of EBV-PK during lytic viral replication include the phosphorylation and dispersion of lamin A/C.The Epstein-Barr virus (EBV) BGLF4 gene product, EBV-PK, is homologous to the cytomegalovirus (CMV) UL97 kinase and belongs to a conserved family of herpesviral protein kinases. EBV-PK is the only protein kinase encoded by the EBV genome (6) and is expressed as an early lytic viral protein during viral reactivation (11). EBV-PK is also packaged into the virion tegument and thus is delivered in an active form to cells immediately after virus penetration (3). In the context of the intact virus, several recent studies indicate that a major and essential role of EBV-PK is to promote viral egress from the nucleus during lytic infection (10,13,41). Although EBV-PK phosphorylates a number of different viral and cellular proteins in vitro and when overexpressed in cells, the relative importance of the potential EBV-PK cellular and viral targets in the context of the intact viral genome has not been well studied.The nuclear EBV-PK protein is a serine/threonine kinase that shares many targets with cyclin-dependent cellular kinases. Indeed, half of the EBV-PK targets identified by a protein array in a recent study were also found to be in vitro substrates for the cellular kinase CDK1/cyclin B (63). Overexpression of EBV-PK in cells has been shown to result in phosphorylation of a number of different cellular proteins, including the cell cycle regulatory proteins p27 (19) and pRB (C. V. Kuny and R. F. Kalejta, unpublished data); nuclear lamin A/C (32); interferon regulatory factor 3 (IRF3) (54); cellular translational elongation factor 1␦ (24, 26); MCM4 (part of the MCM4-MCM6-MCM7 cellular replication origin binding complex) (29); and histone protein H2AX (52). The ability of EBV-PK to phosphorylate and inactivate two key cell cycl...