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Viruses must negotiate cellular antiviral responses in order to replicate. Human cytomegalovirus (HCMV) is a prevalent betaherpesvirus that encodes a number of viral gene products that modulate cellular antiviral signaling. The HCMV UL26 gene has previously been found to attenuate cytokine-activated NF-κB signaling, yet the role that UL26 plays in modulating the host cell’s global transcriptional response to infection is not clear. Here, we find that infection with a UL26 deletion virus (ΔUL26) induces a proinflammatory transcriptional environment that includes substantial increases in the expression of cytokine signaling genes relative to wild-type HCMV. These increases include NF-κB-regulated genes as well as interferon-stimulated genes (ISGs), such as ISG15 and bone marrow stromal cell antigen 2 (BST2). The ΔUL26 mutant-mediated induction of ISG15 expression was found to drive increases in global protein ISGylation during ΔUL26 mutant infection. However, short hairpin RNA (shRNA) and CRISPR-mediated targeting of ISG15 indicated that its induction does not restrict HCMV infection. In contrast, shRNA-mediated targeting of BST2 demonstrated that BST2 restricts HCMV cell-to-cell spread. In addition, the increased expression of both of these ISGs and the global enhancement in protein ISGylation were found to be dependent on the activity of the canonical inhibitor of NF-κB kinase beta (IKKβ). Both CRISPR-based and pharmacologically mediated inhibition of IKKβ blocked the induction of ISG15 and BST2. These results suggest significant cross-talk between the NF-κB and interferon signaling pathways and highlight the importance of IKK signaling and the HCMV UL26 protein in shaping the antiviral response to HCMV. IMPORTANCE Modulation of cellular antiviral signaling is a key determinant of viral pathogenesis. Human cytomegalovirus (HCMV) is a significant source of morbidity in neonates and the immunosuppressed that contains many genes that modulate antiviral signaling, yet how these genes contribute to shaping the host cell’s transcriptional response to infection is largely unclear. Our results indicate that the HCMV UL26 protein is critical in preventing the establishment of a broad cellular proinflammatory transcriptional environment. Further, we find that the host gene IKKβ is an essential determinant governing the host cell’s antiviral transcriptional response. Given their importance to viral pathogenesis, continuing to elucidate the functional interactions between viruses and the cellular innate immune response could enable the development of therapeutic strategies to limit viral infection. [ABSTRACT FROM AUTHOR]

Human Cytomegalovirus (HCMV) infection induces several metabolic activities that are essential for viral replication. Despite the important role that this metabolic modulation plays during infection, the viral mechanisms involved are largely unclear. We find that the HCMV UL38 protein is responsible for many aspects of HCMV-mediated metabolic activation, with UL38 being necessary and sufficient to drive glycolytic activation and induce the catabolism of specific amino acids. UL38’s metabolic reprogramming role is dependent on its interaction with TSC2, a tumor suppressor that inhibits mTOR signaling. Further, shRNA-mediated knockdown of TSC2 recapitulates the metabolic phenotypes associated with UL38 expression. Notably, we find that in many cases the metabolic flux activation associated with UL38 expression is largely independent of mTOR activity, as broad spectrum mTOR inhibition does not impact UL38-mediated induction of glycolysis, glutamine consumption, or the secretion of proline or alanine. In contrast, the induction of metabolite concentrations observed with UL38 expression are largely dependent on active mTOR. Collectively, our results indicate that the HCMV UL38 protein induces a pro-viral metabolic environment via inhibition of TSC2. [ABSTRACT FROM AUTHOR]

In contrast to many viruses, human cytomegalovirus (HCMV) is unable to productively infect most cancer-derived cell lines. The mechanisms of this restriction are unclear. To explore this issue, we tested whether defined oncogenic alleles, including the simian virus 40 (SV40) T antigen (TAg) and oncogenic H-Ras, inhibit HCMV infection. We found that expression of SV40 TAg blocks HCMV infection in human fibroblasts, whereas the replication of a related herpesvirus, herpes simplex virus 1 (HSV-1), was not impacted. The earliest restriction of HCMV infection involves a block of viral entry, as TAg expression prevented the nuclear delivery of viral DNA and pp65. Subsequently, we found that TAg expression reduces the abundance of platelet-derived growth factor receptor α (PDGFRα), a host protein important for HCMV entry. Viral entry into TAg-immortalized fibroblasts could largely be rescued by PDGFRα overexpression. Similarly, PDGFRα overexpression in HeLa cells markedly increased the levels of HCMV gene expression and DNA replication. However, the robust production of viral progeny was not restored by PDGFRα overexpression in either HeLa cells or TAg-immortalized fibroblasts, suggesting additional restrictions associated with transformation and TAg expression. In TAg-expressing fibroblasts, expression of the immediate early 2 (IE2) protein was not rescued to the same extent as that of the immediate early 1 (IE1) protein, suggesting that TAg expression impacts the accumulation of major immediate early (MIE) transcripts. Transduction of IE2 largely rescued HCMV gene expression in TAg-expressing fibroblasts but did not rescue the production of infectious virions. Collectively, our data indicate that oncogenic alleles induce multiple restrictions to HCMV replication. [ABSTRACT FROM AUTHOR]

Viral infection frequently triggers activation of host innate immune pathways that attempt to limit viral spread. The NF-κB pathway is a critical component that governs this response. We have found that the human cytomegalovirus (HCMV) UL26 protein antagonizes NF-κB activation. Upon infection, an HCMV strain lacking the UL26 gene (ΔUL26) induced the nuclear translocation of the NF-κB RelB subunit and activated expression and secretion of interleukin-6 (IL-6), an NF-κB target gene. The ΔUL26 mutant was also more sensitive to challenge with tumor necrosis factor alpha (TNF-), a canonical NF-κB inducer. Further, expression of UL26 in the absence of other viral proteins blocked NF-κB activation induced by either TNF- treatment or infection with Sendai virus (SeV). Our results indicate that UL26 expression is sufficient to block TNF-α-induced NF-κB nuclear translocation and IκB degradation. Last, UL26 blocks TNF--induced IκB-kinase (IKK) phosphorylation, a key step in NF-κB activation. Combined, our results indicate that UL26 is part of a viral program to antagonize innate immunity through modulation of NF-κB signaling. [ABSTRACT FROM AUTHOR]

Interactions between acute myeloid leukemia (AML) and the bone marrow microenvironment (BMME) are critical to leukemia progression and chemoresistance. Altered metabolite levels in the tumor microenvironment contribute to immunosuppression in solid tumors, while this has not been studied yet in the leukemic BMME. Metabolomics of AML patient bone marrow serum detected elevated metabolites, including lactate, compared to age- and sex-matched controls. Excess lactate has been implicated in solid tumors for inducing suppressive tumor-associated macrophages (TAMs) and correlates with poor prognosis. We describe the role of lactate in the polarization of leukemia-associated macrophages (LAMs) using a murine model of blast crisis chronic myelogenous leukemia (bcCML) and mice genetically lacking the lactate receptor GPR81. LAMs were CD206 hi and suppressive in transcriptomics and cytokine profiling. Yet, LAMs had a largely unique expression profile from other types of TAMs. We demonstrate GPR81 signaling as a mechanism of both LAM polarization and the direct support of leukemia cell growth and self-repopulation. Furthermore, LAMs and elevated lactate diminished the function of hematopoietic progenitors and stromal support, while knockout of GPR81 had modest protective effects on the hematopoietic system. We report microenvironmental lactate as a critical driver of AML-induced immunosuppression and leukemic progression, thus identifying GPR81 signaling as an exciting and novel therapeutic target for treating this devastating disease., Competing Interests: Competing Interests: Authors have no competing interest to declare.

Importance: Viruses modulate host cell metabolism to support the mass production of viral progeny. For human cytomegalovirus, we find that the viral U L 38 protein is critical for driving these pro-viral metabolic changes. However, our results indicate that these changes come at a cost, as U L 38 induces an anabolic rigidity that leads to a metabolic vulnerability. We find that U L 38 decouples the link between glucose availability and fatty acid biosynthetic activity. Normal cells respond to glucose limitation by down-regulating fatty acid biosynthesis. Expression of U L 38 results in the inability to modulate fatty acid biosynthesis in response to glucose limitation, which results in cell death. We find this vulnerability in the context of viral infection, but this linkage between fatty acid biosynthesis, glucose availability, and cell death could have broader implications in other contexts or pathologies that rely on glycolytic remodeling, for example, oncogenesis., Competing Interests: The authors declare no conflict of interest.

Human cytomegalovirus (HCMV) modulates cellular metabolism to support productive infection, and the HCMV U L 38 protein drives many aspects of this HCMV-induced metabolic program. However, it remains to be determined whether virally-induced metabolic alterations might induce novel therapeutic vulnerabilities in virally infected cells. Here, we explore how HCMV infection and the U L 38 protein modulate cellular metabolism and how these changes alter the response to nutrient limitation. We find that expression of U L 38, either in the context of HCMV infection or in isolation, sensitizes cells to glucose limitation resulting in cell death. This sensitivity is mediated through U L 38's inactivation of the TSC complex subunit 2 (TSC2) protein, a central metabolic regulator that possesses tumor-suppressive properties. Further, expression of U L 38 or the inactivation of TSC2 results in anabolic rigidity in that the resulting increased levels of fatty acid biosynthesis are insensitive to glucose limitation. This failure to regulate fatty acid biosynthesis in response to glucose availability sensitizes cells to glucose limitation, resulting in cell death unless fatty acid biosynthesis is inhibited. These experiments identify a regulatory circuit between glycolysis and fatty acid biosynthesis that is critical for cell survival upon glucose limitation and highlight a metabolic vulnerability associated with viral infection and the inactivation of normal metabolic regulatory controls., Competing Interests: Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo . GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production., Competing Interests: DECLARATION OF INTERESTS All other authors declare no competing interests.