Current advances in experimental and computational approaches to enhance CAR T cell manufacturing protocols and improve clinical efficacy.

Since the FDA's approval of chimeric antigen receptor (CAR) T cells in 2017, significant improvements have been made in the design of chimeric antigen receptor constructs and in the manufacturing of CAR T cell therapies resulting in increased in vivo CAR T cell persistence and improved clinical outcome in certain hematological malignancies. Despite the remarkable clinical response seen in some patients, challenges remain in achieving durable long-term tumor-free survival, reducing therapy associated malignancies and toxicities, and expanding on the types of cancers that can be treated with this therapeutic modality. Careful analysis of the biological factors demarcating efficacious from suboptimal CAR T cell responses will be of paramount importance to address these shortcomings. With the ever-expanding toolbox of experimental approaches, single-cell technologies, and computational resources, there is renowned interest in discovering new ways to streamline the development and validation of new CAR T cell products. Better and more accurate prognostic and predictive models can be developed to help guide and inform clinical decision making by incorporating these approaches into translational and clinical workflows. In this review, we provide a brief overview of recent advancements in CAR T cell manufacturing and describe the strategies used to selectively expand specific phenotypic subsets. Additionally, we review experimental approaches to assess CAR T cell functionality and summarize current in silico methods which have the potential to improve CAR T cell manufacturing and predict clinical outcomes.

Simulating the Evolution of Signaling Signatures During CART-Cell and Tumor Cell Interactions.

Immunotherapies have been proven to have significant therapeutic efficacy in the treatment of cancer. The last decade has seen adoptive cell therapies, such as chimeric antigen receptor T-cell (CART-cell) therapy, gain FDA approval against specific cancers. Additionally, there are numerous clinical trials ongoing investigating additional designs and targets. Nevertheless, despite the excitement and promising potential of CART-cell therapy, response rates to therapy vary greatly between studies, patients, and cancers. There remains an unmet need to develop computational frameworks that more accurately predict CART-cell function and clinical efficacy. Here we present a coarse-grained model simulated with logical rules that demonstrates the evolution of signaling signatures following the interaction between CART-cells and tumor cells and allows for in silico based prediction of CART-cell functionality prior to experimentation.Clinical Relevance- Analysis of CART-cell signaling signatures can inform future CAR receptor design and combination therapy approaches aimed at improving therapy response.

Neutralizing antibodies with neurotropic factor treatment maintain neurodevelopmental gene expression upon exposure to human cytomegalovirus.

IMPORTANCE: Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development.

Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection.

Innate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquitinating complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated in infection and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection.

Human Cytomegalovirus UL138 Interaction with USP1 Activates STAT1 in infection.

Innate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquintase complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection. Importance: Human cytomegalovirus (HCMV) is one of nine herpesviruses that infect humans. Following a primary infection, HCMV establishes a life-long latent infection that is marked by sporadic, and likely frequent reactivation events. While these reactivation events are asymptomatic in the immune competent host, they pose important disease risks for the immune compromised, including solid organ or stem cell transplant recipients. Its complex interactions with host biology and deep coding capacity make it an excellent model for defining mechanisms important for viral latency and reactivation. Here we define an interaction with host proteins that commandeer typically antiviral innate immune signaling for the establishment of latency.

Simulating the Evolution of Signaling Signatures during CART-Cell - Tumor Cell Interactions.

Immunotherapies have been proven to have significant therapeutic efficacy in the treatment of cancer. The last decade has seen adoptive cell therapies, such as chimeric antigen receptor T-cell (CART-cell) therapy, gain FDA approval against specific cancers. Additionally, there are numerous clinical trials ongoing investigating additional designs and targets. Nevertheless, despite the excitement and promising potential of CART-cell therapy, response rates to therapy vary greatly between studies, patients, and cancers. There remains an unmet need to develop computational frameworks that more accurately predict CART-cell function and clinical efficacy. Here we present a coarse-grained model simulated with logical rules that demonstrates the evolution of signaling signatures following the interaction between CART-cells and tumor cells and allows for in silico based prediction of CART-cell functionality prior to experimentation.

Crosstalk between Plk1, p53, cell cycle, and G2/M DNA damage checkpoint regulation in cancer: computational modeling and analysis.

Different cancer cell lines can have varying responses to the same perturbations or stressful conditions. Cancer cells that have DNA damage checkpoint-related mutations are often more sensitive to gene perturbations including altered Plk1 and p53 activities than cancer cells without these mutations. The perturbations often induce a cell cycle arrest in the former cancer, whereas they only delay the cell cycle progression in the latter cancer. To study crosstalk between Plk1, p53, and G2/M DNA damage checkpoint leading to differential cell cycle regulations, we developed a computational model by extending our recently developed model of mitotic cell cycle and including these key interactions. We have used the model to analyze the cancer cell cycle progression under various gene perturbations including Plk1-depletion conditions. We also analyzed mutations and perturbations in approximately 1800 different cell lines available in the Cancer Dependency Map and grouped lines by genes that are represented in our model. Our model successfully explained phenotypes of various cancer cell lines under different gene perturbations. Several sensitivity analysis approaches were used to identify the range of key parameter values that lead to the cell cycle arrest in cancer cells. Our resulting model can be used to predict the effect of potential treatments targeting key mitotic and DNA damage checkpoint regulators on cell cycle progression of different types of cancer cells.

Nitric Oxide Circumvents Virus-Mediated Metabolic Regulation during Human Cytomegalovirus Infection.

Nitric oxide is a versatile and critical effector molecule that can modulate many cellular functions. Although recognized as a regulator of infections, the inhibitory mechanism of nitric oxide against human cytomegalovirus (HCMV) replication remains elusive. We demonstrate that nitric oxide attenuates viral replication by interfering with HCMV-mediated modulation of several cellular processes. Nitric oxide exposure reduced HCMV genome synthesis and infectious viral progeny with cell-type-dependent differences observed. Mitochondrial respiration was severely reduced in both uninfected and HCMV-infected cells during exposure with little impact on ATP levels indicating changes in cellular metabolism. Metabolomics identified significantly altered small molecules in multiple pathways during nitric oxide exposure including nucleotide biosynthesis, tricarboxylic acid (TCA) cycle, and glutamine metabolism. Glutathione metabolites were increased coinciding with a reduction in the glutathione precursor glutamine. This shift was accompanied by increased antioxidant enzymes. Glutamine deprivation mimicked defects in HCMV replication and mitochondrial respiration observed during nitric oxide exposure. These data suggest that nitric oxide limits glutaminolysis by shuttling glutamine to glutathione synthesis. In addition, lipid intermediates were severely altered, which likely contributes to the observed increase in defective viral particles. Nitric oxide disrupts multiple cellular processes, and we had limited success in rescuing replication defects by supplementing with metabolic intermediates. Our studies indicate that nitric oxide attenuation of HCMV is multifactorial with interference in viral manipulation of cellular metabolism playing a central role.IMPORTANCE Human cytomegalovirus is a prevalent pathogen that can cause serious disease in patients with compromised immune systems, including transplant patients and during congenital infection. HCMV lytic replication likely occurs in localized sites of infection with immune cells infiltrating and releasing nitric oxide with other effector molecules. This nonspecific immune response results in both uninfected and infected cells exposed to high levels of nitric oxide. The absence of nitric oxide synthase has been associated with lethal HCMV infection. We demonstrate that nitric oxide inhibition of HCMV replication is multifactorial and cell type dependent. Our results indicate that nitric oxide controls replication by interfering with viral modulation of cellular metabolism while also affecting proliferation and mitochondrial respiration of neighboring uninfected cells. These studies identify the mechanism and contribution of nitric oxide during immune control of HCMV infection and provide insight into its role in other viral infections.

Prospects for Clinical Development of Stat5 Inhibitor IST5-002: High Transcriptomic Specificity in Prostate Cancer and Low Toxicity In Vivo.

Stat5 is of significant interest in the search for new therapeutics for prostate cancer (PC) and hematopoietic disorders. We evaluated the transcriptomic specificity of the Stat5a/b inhibitor IST5-002 (IST5) in PC, defined more closely its mechanisms of action, and investigated the in vivo toxicity of IST5 for further optimization for clinical development. The transcriptomic specificity of IST5 vs. genetic Stat5 knockdown was evaluated by RNA-seq analysis, which showed high similarity with the Pearson correlation coefficient ranging from 0.98-0.99. The potency of IST5 vs. its derivative lacking the phosphate group in suppressing Stat5 was evaluated in two separate but complementary assays. The inhibitory activity of IST5 against kinases was investigated in cell-free assays followed by more focused evaluation in a cell-based assay. IST5 has no specific inhibitory activity against 54 kinases, while suppressing Stat5 phosphorylation and subsequent dimerization in PC cells. The phosphate group was not critical for the biological activity of IST5 in cells. The acute, sub-chronic and chronic toxicity studies of IST5 were carried out in mice. IST5 did not cause any significant toxic effects or changes in the blood profiles. The present work supports further optimization of IST5 for oral bioavailability for clinical development for therapies for solid tumors, hematological and myeloproliferative disorders.

Network mechanisms and dysfunction within an integrated computational model of progression through mitosis in the human cell cycle.

The cellular protein-protein interaction network that governs cellular proliferation (cell cycle) is highly complex. Here, we have developed a novel computational model of human mitotic cell cycle, integrating diverse cellular mechanisms, for the purpose of generating new hypotheses and predicting new experiments designed to help understand complex diseases. The pathogenic state investigated is infection by a human herpesvirus. The model starts at mitotic entry initiated by the activities of Cyclin-dependent kinase 1 (CDK1) and Polo-like kinase 1 (PLK1), transitions through Anaphase-promoting complex (APC/C) bound to Cell division cycle protein 20 (CDC20), and ends upon mitotic exit mediated by APC/C bound to CDC20 homolog 1 (CDH1). It includes syntheses and multiple mechanisms of degradations of the mitotic proteins. Prior to this work, no such comprehensive model of the human mitotic cell cycle existed. The new model is based on a hybrid framework combining Michaelis-Menten and mass action kinetics for the mitotic interacting reactions. It simulates temporal changes in 12 different mitotic proteins and associated protein complexes in multiple states using 15 interacting reactions and 26 ordinary differential equations. We have defined model parameter values using both quantitative and qualitative data and using parameter values from relevant published models, and we have tested the model to reproduce the cardinal features of human mitosis determined experimentally by numerous laboratories. Like cancer, viruses create dysfunction to support infection. By simulating infection of the human herpesvirus, cytomegalovirus, we hypothesize that virus-mediated disruption of APC/C is necessary to establish a unique mitotic collapse with sustained CDK1 activity, consistent with known mechanisms of virus egress. With the rapid discovery of cellular protein-protein interaction networks and regulatory mechanisms, we anticipate that this model will be highly valuable in helping us to understand the network dynamics and identify potential points of therapeutic interventions.

Tumor Necrosis Factor Alpha Induces Reactivation of Human Cytomegalovirus Independently of Myeloid Cell Differentiation following Posttranscriptional Establishment of Latency.

We used the Kasumi-3 model to study human cytomegalovirus (HCMV) latency and reactivation in myeloid progenitor cells. Kasumi-3 cells were infected with HCMV strain TB40/Ewt-GFP, flow sorted for green fluorescent protein-positive (GFP+) cells, and cultured for various times to monitor establishment of latency, as judged by repression of viral gene expression (RNA/DNA ratio) and loss of virus production. We found that, in the vast majority of cells, latency was established posttranscriptionally in the GFP+ infected cells: transcription was initially turned on and then turned off. We also found that some of the GFP- cells were infected, suggesting that latency might be established in these cells at the outset of infection. We were not able to test this hypothesis because some GFP- cells expressed lytic genes and thus it was not possible to separate them from GFP- quiescent cells. In addition, we found that the pattern of expression of lytic genes that have been associated with latency, including UL138, US28, and RNA2.7, was the same as that of other lytic genes, indicating that there was no preferential expression of these genes once latency was established. We confirmed previous studies showing that tumor necrosis factor alpha (TNF-α) induced reactivation of infectious virus, and by analyzing expression of the progenitor cell marker CD34 as well as myeloid cell differentiation markers in IE+ cells after treatment with TNF-α, we showed that TNF-α induced transcriptional reactivation of IE gene expression independently of differentiation. TNF-α-mediated reactivation in Kasumi-3 cells was correlated with activation of NF-κB, KAP-1, and ATM.IMPORTANCE HCMV is an important human pathogen that establishes lifelong latent infection in myeloid progenitor cells and reactivates frequently to cause significant disease in immunocompromised people. Our observation that viral gene expression is first turned on and then turned off to establish latency suggests that there is a host defense, which may be myeloid cell specific, responsible for transcriptional silencing of viral gene expression. Our observation that TNF-α induces reactivation independently of differentiation provides insight into molecular mechanisms that control reactivation.

Human Cytomegalovirus UL135 Interacts with Host Adaptor Proteins To Regulate Epidermal Growth Factor Receptor and Reactivation from Latency.

Human cytomegalovirus, HCMV, is a betaherpesvirus that establishes a lifelong latent infection in its host that is marked by recurrent episodes of reactivation. The molecular mechanisms by which the virus and host regulate entry into and exit from latency remain poorly understood. We have previously reported that UL135 is critical for reactivation, functioning in part by overcoming suppressive effects of the latency determinant UL138 We have demonstrated a role for UL135 in diminishing cell surface levels and targeting epidermal growth factor receptor (EGFR) for turnover. The attenuation of EGFR signaling promotes HCMV reactivation in combination with cellular differentiation. In this study, we sought to define the mechanisms by which UL135 functions in regulating EGFR turnover and viral reactivation. Screens to identify proteins interacting with pUL135 identified two host adaptor proteins, CIN85 and Abi-1, with overlapping activities in regulating EGFR levels in the cell. We mapped the amino acids in pUL135 necessary for interaction with Abi-1 and CIN85 and generated recombinant viruses expressing variants of pUL135 that do not interact with CIN85 or Abi-1. These recombinant viruses replicate in fibroblasts but are defective for reactivation in an experimental model for latency using primary CD34+ hematopoietic progenitor cells (HPCs). These UL135 variants have altered trafficking of EGFR and are defective in targeting EGFR for turnover. These studies demonstrate a requirement for pUL135 interactions with Abi-1 and CIN85 for regulation of EGFR and mechanistically link the regulation of EGFR to reactivation.IMPORTANCE Human cytomegalovirus (HCMV) establishes a lifelong latent infection in the human host. While the infection is typically asymptomatic in healthy individuals, HCMV infection poses life-threatening disease risk in immunocompromised individuals and is the leading cause of birth defects. Understanding how HCMV controls the lifelong latent infection and reactivation of replication from latency is critical to developing strategies to control HCMV disease. Here, we identify the host factors targeted by a viral protein that is required for reactivation. We define the importance of this virus-host interaction in reactivation from latency, providing new insights into the molecular underpinnings of HCMV latency and reactivation.

Cytomegalovirus Late Protein pUL31 Alters Pre-rRNA Expression and Nuclear Organization during Infection.

The replication cycle of human cytomegalovirus (CMV) leads to drastic reorganization of domains in the host cell nucleus. However, the mechanisms involved and how these domains contribute to infection are not well understood. Our recent studies defining the CMV-induced nuclear proteome identified several viral proteins of unknown functions, including a protein encoded by the UL31 gene. We set out to define the role of UL31 in CMV replication. UL31 is predicted to encode a 74-kDa protein, referred to as pUL31, containing a bipartite nuclear localization signal, an intrinsically disordered region overlapping arginine-rich motifs, and a C-terminal dUTPase-like structure. We observed that pUL31 is expressed with true late kinetics and is localized to nucleolin-containing nuclear domains. However, pUL31 is excluded from the viral nuclear replication center. Nucleolin is a marker of nucleoli, which are membrane-less regions involved in regulating ribosome biosynthesis and cellular stress responses. Other CMV proteins associate with nucleoli, and we demonstrate that pUL31 specifically interacts with the viral protein, pUL76. Coexpression of both proteins altered pUL31 localization and nucleolar organization. During infection, pUL31 colocalizes with nucleolin but not the transcriptional activator, UBF. In the absence of pUL31, CMV fails to reorganize nucleolin and UBF and exhibits a replication defect at a low multiplicity of infection. Finally, we observed that pUL31 is necessary and sufficient to reduce pre-rRNA levels, and this was dependent on the dUTPase-like motif in pUL31. Our studies demonstrate that CMV pUL31 functions in regulating nucleolar biology and contributes to the reorganization of nucleoli during infection.IMPORTANCE Nucleolar biology is important during CMV infection with the nucleolar protein, with nucleolin playing a role in maintaining the architecture of the viral nuclear replication center. However, the extent of CMV-mediated regulation of nucleolar biology is not well established. Proteins within nucleoli regulate ribosome biosynthesis and p53-dependent cellular stress responses that are capable of inducing cell cycle arrest and/or apoptosis, and they are proposed targets for cancer therapies. This study establishes that CMV protein pUL31 is necessary and sufficient to regulate nucleolar biology involving the reorganization of nucleolar proteins. Understanding these processes will help define approaches to stimulate cellular intrinsic stress responses that are capable of inhibiting CMV infection.

Opposing Regulation of the EGF Receptor: A Molecular Switch Controlling Cytomegalovirus Latency and Replication.

Herpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous ß-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling.

Targeted analysis of recombinant NF kappa B (RelA/p65) by denaturing and native top down mass spectrometry.

Measuring post-translational modifications on transcription factors by targeted mass spectrometry is hampered by low protein abundance and inefficient isolation. Here, we utilized HaloTag technology to overcome these limitations and evaluate various top down mass spectrometry approaches for measuring NF-κB p65 proteoforms isolated from human cells. We show isotopic resolution of N-terminally acetylated p65 and determined it is the most abundant proteoform expressed following transfection in 293T cells. We also show MS(1) evidence for monophosphorylation of p65 under similar culture conditions and describe a high propensity for p65 proteoforms to fragment internally during beam-style MS(2) fragmentation; up to 71% of the fragment ions could be matched as internals in some fragmentation spectra. Finally, we used native spray mass spectrometry to measure proteins copurifying with p65 and present evidence for the native detection of p65, 71kDa heat shock protein, and p65 homodimer. Collectively, our work demonstrates the efficient isolation and top down mass spectrometry analysis of p65 from human cells, and we discuss the perturbations of overexpressing tagged proteins to study their biochemistry. This article is part of a Special Issue entitled: Protein Species. BIOLOGICAL SIGNIFICANCE: Characterizing transcription factor proteoforms in human cells is of high value to the field of molecular biology; many agree that post-translational modifications and combinations thereof play a critical role in modulating transcription factor activity. Thus, measuring these modifications promises increased understanding of molecular mechanisms governing the regulation of complex gene expression outcomes. To date, comprehensive characterization of transcription factor proteoforms within human cells has eluded measurement, owing primarily-with regard to top down mass spectrometry-to large protein size and low relative abundance. Here, we utilized HaloTag technology and recombinant protein expression to overcome these limitations and show top down mass spectrometry characterization of proteoforms of the 60kDa NF-kB protein, p65. By optimizing the analytical procedure (i.e. purification, MS(1), and MS(2)), our results make important progress toward the ultimate goal of targeted transcription factor characterization from endogenous loci.

Antagonistic Relationship between Human Cytomegalovirus pUL27 and pUL97 Activities during Infection.

UNLABELLED: Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously shown that expression of HCMV pUL27 results in increased levels of the cyclin-dependent kinase (CDK) inhibitor p21(Cip1). In addition, pUL27 is necessary for the full antiviral activity of the pUL97 kinase inhibitor maribavir (MBV). The purpose of this study was to define the relationship between pUL27 and pUL97 and its role in MBV antiviral activity. We observed that expression of wild-type but not kinase-inactive pUL97 disrupted pUL27-dependent induction of p21(Cip1). Furthermore, pUL97 associated with and promoted the phosphorylation of pUL27. During infection, inhibition of the kinase resulted in elevated levels of p21(Cip1) in wild-type virus but not a pUL27-deficient virus. We manipulated the p21(Cip1) levels to evaluate the functional consequence to MBV. Overexpression of p21(Cip1) restored MBV activity against a pUL27-deficient virus, while disruption reduced activity against wild-type virus. We provide evidence that the functional target of p21(Cip1) in the context of MBV activity is CDK1. One CDK-like activity of pUL97 is to phosphorylate nuclear lamin A/C, resulting in altered nuclear morphology and increased viral egress. In the presence of MBV, we observed that infection using a pUL27-deficient virus still altered the nuclear morphology. This was prevented by the addition of a CDK inhibitor. Overall, our results demonstrate an antagonistic relationship between pUL27 and pUL97 activities centering on p21(Cip1) and support the idea that CDKs can complement some activities of pUL97. IMPORTANCE: HCMV infection results in severe disease upon immunosuppression and is a leading cause of congenital birth defects. Effective antiviral compounds exist, yet they exhibit high levels of toxicity, are not approved for use during pregnancy, and can result in antiviral resistance. Our studies have uncovered new information regarding the antiviral efficacy of the HCMV pUL97 kinase inhibitor MBV as it relates to the complex interplay between pUL97 and a second HCMV protein, pUL27. We demonstrate that pUL97 functions antagonistically against pUL27 by phosphorylation-dependent inactivation of pUL27-mediated induction of p21(Cip1). In contrast, we provide evidence that p21(Cip1) functions to antagonize overlapping activities between pUL97 and cellular CDKs. In addition, these studies further support the notion that CDK inhibitors or p21(Cip1) activators might be useful in combination with MBV to effectively inhibit HCMV infections.

Human cytomegalovirus IE1 protein disrupts interleukin-6 signaling by sequestering STAT3 in the nucleus.

In the canonical STAT3 signaling pathway, binding of agonist to receptors activates Janus kinases that phosphorylate cytoplasmic STAT3 at tyrosine 705 (Y705). Phosphorylated STAT3 dimers accumulate in the nucleus and drive the expression of genes involved in inflammation, angiogenesis, invasion, and proliferation. Here, we demonstrate that human cytomegalovirus (HCMV) infection rapidly promotes nuclear localization of STAT3 in the absence of robust phosphorylation at Y705. Furthermore, infection disrupts interleukin-6 (IL-6)-induced phosphorylation of STAT3 and expression of a subset of IL-6-induced STAT3-regulated genes, including SOCS3. We show that the HCMV 72-kDa immediate-early 1 (IE1) protein associates with STAT3 and is necessary to localize STAT3 to the nucleus during infection. Furthermore, expression of IE1 is sufficient to disrupt IL-6-induced phosphorylation of STAT3, binding of STAT3 to the SOCS3 promoter, and SOCS3 gene expression. Finally, inhibition of STAT3 nuclear localization or STAT3 expression during infection is linked to diminished HCMV genome replication. Viral gene expression is also disrupted, with the greatest impact seen following viral DNA synthesis. Our study identifies IE1 as a new regulator of STAT3 intracellular localization and IL-6 signaling and points to an unanticipated role of STAT3 in HCMV infection.

A conserved gammaherpesvirus protein kinase targets histone deacetylases 1 and 2 to facilitate viral replication in primary macrophages.

Gammaherpesviruses are ubiquitious pathogens that establish lifelong infection and are associated with several malignancies. All gammaherpesviruses encode a conserved protein kinase that facilitates viral replication and chronic infection and thus represents an attractive therapeutic target. In this study, we identify a novel function of gammaherpesvirus protein kinase as a regulator of class I histone deacetylases (HDAC). Mouse gammaherpesvirus 68 (MHV68)-encoded protein kinase orf36 interacted with HDAC1 and 2 and prevented association of these HDACs with the viral promoter driving expression of RTA, a critical immediate early transcriptional activator. Furthermore, the ability to interact with HDAC1 and 2 was not limited to the MHV68 orf36, as BGLF4, a related viral protein kinase encoded by Epstein-Barr virus, interacted with HDAC1 in vitro. Importantly, targeting of HDAC1 and 2 by orf36 was independent of the kinase's enzymatic activity. Additionally, orf36 expression, but not its enzymatic activity, induced changes in the global deacetylase activity observed in infected primary macrophages. Combined deficiency of HDAC1 and 2 rescued attenuated replication and viral DNA synthesis of the orf36 null MHV68 mutant, indicating that the regulation of HDAC1 and 2 by orf36 was relevant for viral replication. Understanding the mechanism by which orf36 facilitates viral replication, including through HDAC targeting, will facilitate the development of improved therapeutics against gammaherpesvirus kinases.

Human cytomegalovirus pUL29/28 and pUL38 repression of p53-regulated p21CIP1 and caspase 1 promoters during infection.

During infection by human cytomegalovirus (HCMV), the tumor suppressor protein p53, which promotes efficient viral gene expression, is stabilized. However, the expression of numerous p53-responsive cellular genes is not upregulated. The molecular mechanism used to manipulate the transcriptional activity of p53 during infection remains unclear. The HCMV proteins IE1, IE2, pUL44, and pUL84 likely contribute to the regulation of p53. In this study, we used a discovery-based approach to identify the protein targets of the HCMV protein pUL29/28 during infection. Previous studies have demonstrated that pUL29/28 regulates viral gene expression by interacting with the chromatin remodeling complex NuRD. Here, we observed that pUL29/28 also associates with p53, an additional deacetylase complex, and several HCMV proteins, including pUL38. We confirmed the interaction between p53 and pUL29/28 in both the presence and absence of infection. HCMV pUL29/28 with pUL38 altered the activity of the 53-regulatable p21CIP1 promoter. During infection, pUL29/28 and pUL38 contributed to the inhibition of p21CIP1 as well as caspase 1 expression. The expression of several other p53-regulating genes was not altered. Infection using a UL29-deficient virus resulted in increased p53 binding and histone H3 acetylation at the responsive promoters. Furthermore, expression of pUL29/28 and its interacting partner pUL38 contributed to an increase in the steady-state protein levels of p53. This study identified two additional HCMV proteins, pUL29/28 and pUL38, which participate in the complex regulation of p53 transcriptional activity during infection.

Proteasome-dependent disruption of the E3 ubiquitin ligase anaphase-promoting complex by HCMV protein pUL21a.

The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109-110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity.