DNA-PK and ATM drive phosphorylation signatures that antagonistically regulate cytokine responses to herpesvirus infection or DNA damage.

The DNA-dependent protein kinase, DNA-PK, is an essential regulator of DNA damage repair. DNA-PK-driven phosphorylation events and the activated DNA damage response (DDR) pathways are also components of antiviral intrinsic and innate immune responses. Yet, it is not clear whether and how the DNA-PK response differs between these two forms of nucleic acid stress-DNA damage and DNA virus infection. Here, we define DNA-PK substrates and the signature cellular phosphoproteome response to DNA damage or infection with the nuclear-replicating DNA herpesvirus, HSV-1. We establish that DNA-PK negatively regulates the ataxia-telangiectasia-mutated (ATM) DDR kinase during viral infection. In turn, ATM blocks the binding of DNA-PK and the nuclear DNA sensor IFI16 to viral DNA, thereby inhibiting cytokine responses. However, following DNA damage, DNA-PK enhances ATM activity, which is required for IFN-ß expression. These findings demonstrate that the DDR autoregulates cytokine expression through the opposing modulation of DDR kinases.

Sirtuin 2 promotes human cytomegalovirus replication by regulating cell cycle progression.

IMPORTANCE: This study expands the growing understanding that protein acetylation is a highly regulated molecular toggle of protein function in both host anti-viral defense and viral replication. We describe a pro-viral role for the human enzyme SIRT2, showing that its deacetylase activity supports HCMV replication. By integrating quantitative proteomics, flow cytometry cell cycle assays, microscopy, and functional virology assays, we investigate the temporality of SIRT2 functions and substrates. We identify a pro-viral role for the SIRT2 deacetylase activity via regulation of CDK2 K6 acetylation and the G1-S cell cycle transition. These findings highlight a link between viral infection, protein acetylation, and cell cycle progression.

Intercellular communication within the virus microenvironment affects the susceptibility of cells to secondary viral infections.

Communication between infected cells and cells in the surrounding tissue is a determinant of viral spread. However, it remains unclear how cells in close or distant proximity to an infected cell respond to primary or secondary infections. We establish a cell-based system to characterize a virus microenvironment, distinguishing infected, neighboring, and distal cells. Cell sorting, microscopy, proteomics, and cell cycle assays allow resolving cellular features and functional consequences of proximity to infection. We show that human cytomegalovirus (HCMV) infection primes neighboring cells for both subsequent HCMV infections and secondary infections with herpes simplex virus 1 and influenza A. Neighboring cells exhibit mitotic arrest, dampened innate immunity, and altered extracellular matrix. Conversely, distal cells are poised to slow viral spread due to enhanced antiviral responses. These findings demonstrate how infection reshapes the microenvironment through intercellular signaling to facilitate spread and how spatial proximity to an infection guides cell fate.

Nuclear antiviral innate responses at the intersection of DNA sensing and DNA repair.

The coevolution of vertebrate and mammalian hosts with DNA viruses has driven the ability of host cells to distinguish viral from cellular DNA in the nucleus to induce intrinsic immune responses. Concomitant viral mechanisms have arisen to inhibit DNA sensing. At this virus-host interface, emerging evidence links cytokine responses and cellular homeostasis pathways, particularly the DNA damage response (DDR). Nuclear DNA sensors, such as the interferon (IFN)-γ inducible protein 16 (IFI16), functionally intersect with the DDR regulators ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK). Here, we discuss accumulating knowledge for the DDR-innate immunity signaling axis. Through the lens of this infection-driven signaling axis, we present host and viral molecular strategies acquired to regulate autoinflammation and antiviral responses.

BK Polyomavirus Requires the Mismatch Repair Pathway for DNA Damage Response Activation.

BK polyomavirus (PyV) infects the genitourinary tract of >90% of the adult population. Immunosuppression increases the risk of viral reactivation, making BKPyV a leading cause of graft failure in kidney transplant recipients. Polyomaviruses have a small double-stranded DNA (dsDNA) genome that requires host replication machinery to amplify the viral genome. Specifically, polyomaviruses promote S phase entry and delay S phase exit by activating the DNA damage response (DDR) pathway via an uncharacterized mechanism requiring viral replication. BKPyV infection elevates expression of MutSα, a mismatch repair (MMR) pathway protein complex that senses and repairs DNA mismatches and can activate the DDR. Thus, we investigated the role of the MMR pathway by silencing the MutSα component, Msh6, in BKPyV-infected primary cells. This resulted in severe DNA damage that correlated with weak DNA damage response activation and a failure to arrest the cell cycle to prevent mitotic entry during infection. Furthermore, silencing Msh6 expression resulted in significantly fewer infectious viral particles due to significantly lower levels of VP2, a minor capsid protein important for trafficking during subsequent infections. Since viral assembly occurs in the nucleus, our findings are consistent with a model in which entry into mitosis disrupts viral assembly due to nuclear envelope breakdown, which disperses VP2 throughout the cell, reducing its availability for encapsidation into viral particles. Thus, the MMR pathway may be required to activate the ATR (ATM-Rad3-related) pathway during infection to maintain a favorable environment for both viral replication and assembly. IMPORTANCE Since there are no therapeutics that target BKPyV reactivation in organ transplant patients, it is currently treated by decreasing immunosuppression to allow the natural immune system to fight the viral infection. Antivirals would significantly improve patient outcomes since reducing immunosuppression carries the risk of graft failure. PyVs activate the DDR, for which there are several promising inhibitors. However, a better understanding of how PyVs activate the DDR and what role the DDR plays during infection is needed. Here, we show that a component of the mismatch repair pathway is required for DDR activation during PyV infection. These findings show that the mismatch repair pathway is important for DDR activation during PyV infection and that inhibiting the DDR reduces viral titers by generating less infectious virions that lack the minor capsid protein VP2, which is important for viral trafficking.

Systematic profiling of protein complex dynamics reveals DNA-PK phosphorylation of IFI16 en route to herpesvirus immunity.

Dynamically shifting protein-protein interactions (PPIs) regulate cellular responses to viruses and the resulting immune signaling. Here, we use thermal proximity coaggregation (TPCA) mass spectrometry to characterize the on-off behavior of PPIs during infection with herpes simplex virus 1 (HSV-1), a virus with an ancient history of coevolution with hosts. Advancing the TPCA analysis to infer associations de novo, we build a time-resolved portrait of thousands of host-host, virus-host, and virus-virus PPIs. We demonstrate that, early in infection, the DNA sensor IFI16 recruits the active DNA damage response kinase, DNA-dependent protein kinase (DNA-PK), to incoming viral DNA at the nuclear periphery. We establish IFI16 T149 as a substrate of DNA-PK upon viral infection or DNA damage. This phosphorylation promotes IFI16-driven cytokine responses. Together, we characterize the global dynamics of PPIs during HSV-1 infection, uncovering the co-regulation of IFI16 and DNA-PK functions as a missing link in immunity to herpesvirus infection.

Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair.

The integrity and regulation of the nuclear lamina is essential for nuclear organization and chromatin stability, with its dysregulation being linked to laminopathy diseases and cancer. Although numerous posttranslational modifications have been identified on lamins, few have been ascribed a regulatory function. Here, we establish that lamin B1 (LMNB1) acetylation at K134 is a molecular toggle that controls nuclear periphery stability, cell cycle progression, and DNA repair. LMNB1 acetylation prevents lamina disruption during herpesvirus type 1 (HSV-1) infection, thereby inhibiting virus production. We also demonstrate the broad impact of this site on laminar processes in uninfected cells. LMNB1 acetylation negatively regulates canonical nonhomologous end joining by impairing the recruitment of 53BP1 to damaged DNA. This defect causes a delay in DNA damage resolution and a persistent activation of the G1/S checkpoint. Altogether, we reveal LMNB1 acetylation as a mechanism for controlling DNA repair pathway choice and stabilizing the nuclear periphery.

A high-stringency blueprint of the human proteome.

The Human Proteome Organization (HUPO) launched the Human Proteome Project (HPP) in 2010, creating an international framework for global collaboration, data sharing, quality assurance and enhancing accurate annotation of the genome-encoded proteome. During the subsequent decade, the HPP established collaborations, developed guidelines and metrics, and undertook reanalysis of previously deposited community data, continuously increasing the coverage of the human proteome. On the occasion of the HPP's tenth anniversary, we here report a 90.4% complete high-stringency human proteome blueprint. This knowledge is essential for discerning molecular processes in health and disease, as we demonstrate by highlighting potential roles the human proteome plays in our understanding, diagnosis and treatment of cancers, cardiovascular and infectious diseases.

BK Polyomavirus Activates the DNA Damage Response To Prolong S Phase.

BK polyomavirus (PyV) is a major source of kidney failure in transplant recipients. The standard treatment for patients with lytic BKPyV infection is to reduce immunosuppressive therapy, which increases the risk of graft rejection. PyVs are DNA viruses that rely upon host replication proteins for viral genome replication. A hallmark of PyV infection is activation of the DNA damage response (DDR) to prevent severe host and viral DNA damage that impairs viral production by an unknown mechanism. Therefore, we sought to better understand why BKPyV activates the DDR through the ATR and ATM pathways and how this prevents DNA damage and leads to increased viral production. When ATR was inhibited in BKPyV-infected primary kidney cells, severe DNA damage occurred due to premature Cdk1 activation, which resulted in mitosis of cells that were actively replicating host DNA in S phase. Conversely, ATM was required for efficient entry into S phase and to prevent normal mitotic entry after G2 phase. The synergistic activation of these DDR kinases promoted and maintained BKPyV-mediated S phase to enhance viral production. In contrast to BKPyV infection, DDR inhibition did not disrupt cell cycle control in uninfected cells. This suggests that DDR inhibitors may be used to specifically target BKPyV-infected cells.IMPORTANCE BK polyomavirus (BKPyV) is an emerging pathogen that reactivates in immunosuppressed organ transplant patients. We wanted to understand why BKPyV-induced activation of the DNA damage response (DDR) enhances viral titers and prevents host DNA damage. Here, we show that the virus activates the DNA damage response in order to keep the infected cells in S phase to replicate the viral DNA. The source of DNA damage was due to actively replicating cells with uncondensed chromosomes entering directly into mitosis when the DDR was inhibited in BKPyV-infected cells. This study clarifies the previously enigmatic role of the DDR during BKPyV infection by demonstrating that the virus activates the DDR to maintain the cells in S phase in order to promote viral replication and that disruption of this cell cycle arrest can lead to catastrophic DNA damage for the host.

Phylogenetic utility, and variability in structure and content, of complete mitochondrial genomes among genetic lineages of the Hawaiian anchialine shrimp Halocaridina rubra Holthuis 1963 (Atyidae:Decapoda).

The Atyidae are caridean shrimp possessing hair-like setae on their claws and are important contributors to ecological services in tropical and temperate fresh and brackish water ecosystems. Complete mitochondrial genomes have only been reported from five of the 449 species in the family, thus limiting understanding of mitochondrial genome evolution and the phylogenetic utility of complete mitochondrial sequences in the Atyidae. Here, comparative analyses of complete mitochondrial genomes from eight genetic lineages of Halocaridina rubra, an atyid endemic to the anchialine ecosystem of the Hawaiian Archipelago, are presented. Although gene number, order, and orientation were syntenic among genomes, three regions were identified and further quantified where conservation was substantially lower: (1) high length and sequence variability in the tRNA-Lys and tRNA-Asp intergenic region; (2) a 317-bp insertion between the NAD6 and CytB genes confined to a single lineage and representing a partial duplication of CytB; and (3) the putative control region. Phylogenetic analyses utilizing complete mitochondrial sequences provided new insights into relationships among the H. rubra genetic lineages, with the topology of one clade correlating to the geologic sequence of the islands. However, deeper nodes in the phylogeny lacked bootstrap support. Overall, our results from H. rubra suggest intra-specific mitochondrial genomic diversity could be underestimated across the Metazoa since the vast majority of complete genomes are from just a single individual of a species.

Quantitative Proteomic Analysis of Enriched Nuclear Fractions from BK Polyomavirus-Infected Primary Renal Proximal Tubule Epithelial Cells.

Polyomaviruses are a family of small DNA viruses that are associated with a number of severe human diseases, particularly in immunocompromised individuals. The detailed virus-host interactions during lytic polyomavirus infection are not fully understood. Here, we report the first nuclear proteomic study with BK polyomavirus (BKPyV) in a primary renal proximal tubule epithelial cell culture system using stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling coupled with liquid chromatography-tandem mass spectrometry. We demonstrated the feasibility of SILAC labeling in these primary cells and subsequently performed reciprocal labeling-infection experiments to identify proteins that are altered by BKPyV infection. Our analyses revealed specific proteins that are significantly up- or down-regulated in the infected nuclear proteome. The genes encoding many of these proteins were not identified in a previous microarray study, suggesting that differential regulation of these proteins may be independent of transcriptional control. Western blotting experiments verified the SILAC proteomic findings. Finally, pathway and network analyses indicated that the host cell DNA damage response signaling and DNA repair pathways are among the cellular processes most affected at the protein level during polyomavirus infection. Our study provides a comprehensive view of the host nuclear proteomic changes during polyomavirus lytic infection and suggests potential novel host factors required for a productive polyomavirus infection.

Polyomavirus interaction with the DNA damage response.

Viruses are obligate intracellular parasites that subvert cellular metabolism and pathways to mediate their own replication-normally at the expense of the host cell. Polyomaviruses are a group of small DNA viruses, which have long been studied as a model for eukaryotic DNA replication. Polyomaviruses manipulate host replication proteins, as well as proteins involved in DNA maintenance and repair, to serve as essential cofactors for productive infection. Moreover, evidence suggests that polyomavirus infection poses a unique genotoxic threat to the host cell. In response to any source of DNA damage, cells must initiate an effective DNA damage response (DDR) to maintain genomic integrity, wherein two protein kinases, ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), are major regulators of DNA damage recognition and repair. Recent investigation suggests that these essential DDR proteins are required for productive polyomavirus infection. This review will focus on polyomaviruses and their interaction with ATM- and ATR-mediated DNA damage responses and the effect of this interaction on host genomic stability.

Viral DNA replication-dependent DNA damage response activation during BK polyomavirus infection.

UNLABELLED: BK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that its activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine what viral components are responsible for activating the two major phosphatidylinositol 3-kinase-like kinases (PI3KKs) involved in the DDR: ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3-related (ATR) kinase. Using a combination of UV treatment, lentivirus transduction, and mutant virus infection experiments, our results demonstrate that neither the input virus nor the expression of large T antigen (TAg) alone is sufficient to trigger the activation of ATM or ATR in our primary culture model. Instead, our data suggest that the activation of both the ATM- and ATR-mediated DDR pathways is linked to viral DNA replication. Intriguingly, a TAg mutant virus that is unable to activate the DDR causes substantial host DNA damage. Our study provides insight into how DDRs are activated by polyomaviruses in primary cells with intact cell cycle checkpoints and how the activation might be linked to the maintenance of host genome stability. IMPORTANCE: Polyomaviruses are opportunistic pathogens that are associated with several human diseases under immunosuppressed conditions. BK polyomavirus (BKPyV) affects mostly kidney and bone marrow transplant patients. The detailed replication mechanism of these viruses remains to be determined. We have previously reported that BKPyV activates the host DNA damage response (DDR), a response normally used by the host cell to combat genotoxic stress, to aid its own replication. In this study, we identified that the trigger for DDR activation is viral replication. Furthermore, we show that the virus is able to cause host DNA damage in the absence of viral replication and DDR activation. These results suggest an intricate relationship between viral replication, DDR activation, and host genome instability.