Biology subjects

Goodrum, F.

Publications and source records attributed to Goodrum, F..

15 recordsLinked to original sources

bioRxiv2026

Host DNA repair factors empower a mechanism of antiviral nucleoside analog resistance

How host functions affect resistance to antiviral drugs is poorly understood. Ganciclovir, a chain-terminating nucleoside analog, is a first-line therapy against human cytomegalovirus, a widespread herpesvirus that causes life-threatening disease in immunocompromised individuals and newborns. Ganciclovir resistance, which is caused by mutations that affect the viral kinase, UL97 and/or the viral polymerase, UL54, can cause treatment failures. Among these mutations, those reducing the exonuclease activity of the viral DNA polymerase permit ganciclovir incorporation without chain termination. However, the fate of DNA strands containing the incorporated nucleotide analog is unknown. We show here that template DNA containing ganciclovir fails to support DNA synthesis of the complementary strand by exonuclease-mutant polymerase. Moreover, while DNA synthesis and ganciclovir incorporation are limited in drug-treated fibroblasts infected by virus with wild-type polymerase, an exonuclease-resistant mutant virus can better synthesize full-length genomes and incorporate substantially more ganciclovir into DNA. Notably, ganciclovir is lost from DNA when drug is removed, suggesting that ganciclovir-containing templates are repaired. We identify the host nucleotide excision repair component, XPA, and the repair enzyme, polymerase kappa, as each being necessary for mutant virus ganciclovir resistance and polymerase kappa as being required for the mutants cidofovir resistance, demonstrating a role for host DNA repair machinery in a mechanism of antiviral resistance. We propose a model for this mechanism, which has relevance for at least one other antiviral drug and likely other nucleoside analog therapeutics, and highlights the participation of host DNA repair machinery during human cytomegalovirus DNA replication. IMPORTANCENucleoside analogues such as ganciclovir, which is a leading drug for preventing and treating human cytomegalovirus, are a critical defense against viral diseases, but antiviral resistance often results in treatment failures. This study reveals a critical role for host DNA repair in a mechanism of resistance to ganciclovir, and identifies at least one specific repair pathway that permits viral DNA synthesis in the presence of ganciclovir, defining a mechanism by which cellular DNA repair pathways conspire to enable antiviral drug resistance. This mechanism is relevant to at least one other antiviral drug and may apply to other antiviral and anticancer agents. The study also showcases the participation of host DNA repair machinery during human cytomegalovirus DNA synthesis.

microbiology↗

bioRxiv2025

Human cytomegalovirus regulates host DNA repair machinery for viral genome integrity

The DNA damage response (DDR) encompasses a multitude of interconnected pathways that serve as a cellular defense to protect genome integrity. Dysregulation or failure of these pathways results in cancers and genetic disease. DNA viruses, including the herpesvirus cytomegalovirus (CMV), activate DDR signaling during their replicative program. The mechanisms by which they commandeer these responses for replication of their genome is less well understood. Here, we define a viral protein, UL138, that modulates the activity of host DDR pathways. The loss of UL138 results in structural variants, including inversion, deletions, duplication, with signature of homology-directed repair and other DDR pathways. The actions of UL138 are due, in part, to its modulation of pathways regulated by the cellular deubiquitinating complex that targets proliferating cell nuclear antigen (PCNA) and Fanconi Anemia effectors, FANCD2 and FANCI. However, we also show that UL138 accesses pathways independent of USP1-PCNA/FANCD2/FANCI. Disruption of UL138 or these pathways impacts viral genome replication and had consequences for viral genome integrity. This work provides mechanistic insight into the long-standing questions of how DNA viruses recruit, modulate and use cellular DDR pathways. It also puts forth CMV as a model system for further defining these pathways in human cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/683233v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@121e55borg.highwire.dtl.DTLVardef@58d8b7org.highwire.dtl.DTLVardef@fc1628org.highwire.dtl.DTLVardef@12b32a_HPS_FORMAT_FIGEXP M_FIG C_FIG Major findingsO_LIUL138 protects HCMV genome integrity through its interaction with USP1-PCNA/FANCD2/FANCI and repeat sequence-mediated pathways C_LIO_LIUL138 protects HCMV genome integrity in the UL region by mechanisms independent of repeat sequences and USP1-PCNA/FANCD2/FANCI C_LIO_LICollectively, US and UL regions of the HCMV genome have fundamentally different pathways acting on them and UL138 modulates DNA damage and repair pathway choice. C_LI

microbiology↗

bioRxiv2025

Human cytomegalovirus promotes de novo PC synthesis during early virus replication

Human cytomegalovirus (HCMV) infection reprograms metabolism, including lipid synthesis. While several metabolite-related pathways have been demonstrated to have altered activity in infected cells, the alteration of lipid-related pathways by HCMV has not been examined beyond fatty acid synthesis and elongation. In this study, we addressed this lack of understanding by focusing on phosphatidylcholine (PC), a class of lipids we previously showed is increased by HCMV infection in human foreskin fibroblasts. Here, we found that HCMV infection increases the abundance of PCs in several different fibroblasts and, similarly, in endothelial and epithelial cells. Additionally, HCMV elevates PC levels regardless of the level of confluency, type of growth medium, and presence of serum. Next, we investigated if HCMV alters the activity in the three PC synthesis pathways. We demonstrate that HCMV infection promotes the activity in the de novo PC synthesis pathway using a 13C-choline isotopic tracer and liquid chromatography high resolution tandem mass spectrometry (LC-MS/MS). Infection did not alter the activity in the other two pathways. Moreover, we examined the kinetics of PC remodeling by HCMV and found that the de novo synthesis pathway is promoted and the PC lipidome shifts 24 hours post infection. That led us to examining if the early stages of replication are sufficient to alter PC levels. After inhibiting late virus replication, we found that HCMV alters the PC lipidome independent of late gene expression. Overall, this work suggests that an immediate-early or early viral protein promotes the reprogramming of host lipid metabolism to ensure the synthesis of a lipidome necessary to support HCMV replication. IMPORTANCEHuman cytomegalovirus (HCMV) is a common herpesvirus that establishes a lifelong and persistent infection in its human host (1). HCMV infection in most people does not cause overt disease (1). However, in immunocompromised individuals, severe CMV-associated disease can lead to permanent disabilities and even death (1, 2). Additionally, congenital CMV is the leading infectious cause of birth defects (3, 4). Viruses have evolved to hijack host metabolic pathways to facilitate their replication cycle. In this study, we determine that HCMV promotes the activity in the de novo pathway of phosphatidylcholine (PC) synthesis. We demonstrate that the activity in the other PC synthesis pathways, the PEMT and Lands cycle, is unaltered by HCMV infection. Moreover, we found that HCMV infection alters metabolic activity to increase the PC lipidome before 48 hpi. Additionally, we demonstrate that changes in PC lipids during virus replication is independent of late gene expression. Together, our findings demonstrate that infection promotes the de novo PC pathway to increase PC lipids during the early stages of virus replication.

microbiology↗

bioRxiv2025

Complex roles for proliferating cell nuclear antigen in human cytomegalovirus replication

DNA viruses at once elicit and commandeer host pathways, including DNA repair pathways for virus replication. Despite encoding its own DNA polymerase and processivity factor, human cytomegalovirus (HCMV) recruits the cellular processivity factor, proliferating cell nuclear antigen (PCNA) and specialized host DNA polymerases involved in translesion synthesis (TLS) to replication compartments (RCs) where viral DNA (vDNA) is synthesized. While the recruitment of TLS polymerases is important for viral genome stability, the role of PCNA is poorly understood. PCNA function in DNA repair is regulated by monoubiquitination (mUb) or SUMOylation of PCNA at lysine 164 (K164). We find that mUb-PCNA increases over the course of infection, and modification of K164 is required for PCNA-mediated restriction of virus replication. mUb-PCNA plays important known roles in recruiting TLS polymerases to DNA, which we have shown are important for viral genome integrity and diversity, represented by novel junctions and single nucleotide variants (SNVs), respectively. We find that PCNA drives SNVs on vDNA similar to Y-family TLS polymerases, but that this did not require modification at K164. Unlike TLS polymerases, PCNA was dispensable for preventing large scale rearrangements on vDNA. These striking results suggest separable PCNA-dependent and - independent functions of TLS polymerases on vDNA. By extension, these results imply roles for TLS polymerase beyond their canonical function in TLS in host biology. These findings highlight PCNA as a complex restriction factor for HCMV infection, likely with multiple distinct roles, and provides new insights into the PCNA-mediated regulation of DNA synthesis and repair in viral infection. IMPORTANCEGenome synthesis is a critical step of virus life cycles and a major target of antiviral drugs. Human cytomegalovirus, like other herpesviruses, encodes machinery sufficient for viral DNA synthesis and relies on host factors for efficient replication. We have shown that host DNA repair factors play important roles in HCMV replication, but our understanding of this is incomplete. Building on previous findings that specialized host DNA polymerases contribute to HCMV genome integrity and diversity, we sought to determine the importance of PCNA, the central polymerase regulator. PCNA associates with nascent viral DNA and restricts HCMV replication. While PCNA is dispensable for genome integrity, it contributes to genome diversity. Our findings suggest that host polymerases function on viral genomes by separable PCNA-dependent and - independent mechanisms. Through revealing complex roles for PCNA in HCMV replication, this study expands the repertoire of host DNA synthesis and repair proteins hijacked by this ubiquitous herpesvirus.

microbiology↗

bioRxiv2024

Viral and host network analysis of the human cytomegalovirus transcriptome in latency.

HCMV genes UL135 and UL138 play opposing roles regulating latency and reactivation in CD34+ human progenitor cells (HPCs). Using the THP-1 cell line model for latency and reactivation, we designed an RNA sequencing study to compare the transcriptional profile of HCMV infection in the presence and absence of these genes. The loss of UL138 results in elevated levels of viral gene expression and increased differentiation of cell populations that support HCMV gene expression and genome synthesis. The loss of UL135 results in diminished viral gene expression during an initial burst that occurs as latency is established and no expression of eleven viral genes from the ULb region even following stimulation for differentiation and reactivation. Transcriptional network analysis revealed host transcription factors with potential to regulate the ULb genes in coordination with pUL135. These results reveal roles for UL135 and UL138 in regulation of viral gene expression and potentially hematopoietic differentiation.

microbiology↗

bioRxiv2023

Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

The phosphoinositide 3-kinase (PI3K)/AKT pathway plays crucial roles in cell viability and protein synthesis and is frequently co-opted by viruses to support their replication. Although many viruses maintain high levels of AKT activity during infection, other viruses, such as vesicular stomatitis virus and human cytomegalovirus (HCMV), cause AKT to accumulate in an inactive state. To efficiently replicate, HCMV requires FoxO transcription factors to localize to the infected cell nucleus (Zhang et. al. mBio 2022), a process directly antagonized by AKT. Therefore, we sought to investigate how HCMV inactivates AKT to achieve this. Subcellular fractionation and live cell imaging studies indicated that AKT failed to recruit to membranes upon serum-stimulation of infected cells. However, UV-inactivated virions were unable to render AKT non-responsive to serum, indicating a requirement for de novo viral gene expression. Interestingly, we were able to identify that UL38 (pUL38), a viral activator of mTORC1, is required to diminish AKT responsiveness to serum. mTORC1 contributes to insulin resistance by causing proteasomal degradation of insulin receptor substrate (IRS) proteins, such as IRS1, which are necessary for the recruitment of PI3K to growth factor receptors. In cells infected with a recombinant HCMV disrupted for UL38, AKT responsiveness to serum is retained and IRS1 is not degraded. Furthermore, ectopic expression of UL38 in uninfected cells induces IRS1 degradation, inactivating AKT. These effects of UL38 were reversed by the mTORC1 inhibitor, rapamycin. Collectively, our results demonstrate that HCMV relies upon a cell-intrinsic negative feedback loop to render AKT inactive during productive infection.

microbiology↗

bioRxiv2023

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. ImportanceHuman 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.

microbiology↗

bioRxiv2023

UL135 and UL136 Epistasis Controls Reactivation of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is beta herpesvirus that persists indefinitely in the human host through a protracted, latent infection. The polycistronic UL133-UL138 gene locus of HCMV encodes genes regulating latency and reactivation. While UL138 is pro-latency, restricting virus replication in CD34+ hematopoietic progenitor cells (HPCs), UL135 overcomes this restriction for reactivation. By contrast, UL136 is expressed with later kinetics and encodes multiple protein isoforms with differential roles in latency and reactivation. Like UL135, the largest UL136 isoform, UL136p33, is required for reactivation from latency in hematopoietic cells. Furthermore, UL136p33 is unstable, and its instability is important for the establishment of latency and sufficient accumulation of UL136p33 is a checkpoint for reactivation. We hypothesized that stabilizing UL136p33 might overcome the requirement of UL135 for reactivation. To test this, we generated recombinant viruses lacking UL135 that expressed a stabilized variant of UL136p33. Stabilizing UL136p33 did not impact replication of the UL135-mutant virus in fibroblasts. However, in the context of infection in hematopoietic cells, stabilization of UL136p33 strikingly compensated for the loss of UL135, resulting in increased replication in CD34+ HPCs and in humanized NOD-scid IL2R{gamma}cnull (NSG) mice. This finding suggests that while UL135 is essential for reactivation, it functions at steps preceding the accumulation of UL136p33 and that stabilized expression of UL136p33 largely overcomes the requirement for UL135 in reactivation. Taken together, our genetic evidence indicates an epistatic relationship between UL136p33 and UL135 whereby UL135 may initiate events early in reactivation that will result in the accumulation of UL136p33 to a threshold required for productive reactivation. SIGNIFICANCEHuman cytomegalovirus (HCMV) is one of nine human herpesviruses and a significant human pathogen. While HCMV establishes a life-long latent infection that is typically asymptomatic in healthy individuals, its reactivation from latency can have devastating consequences in the immune compromised. Defining virus-host and virus-virus interactions important for HCMV latency, reactivation and replication is critical to defining the molecular basis of latent and replicative states and in controlling infection and CMV disease. Here we define a genetic relationship between two viral genes in controlling virus reactivation from latency using primary human hematopoietic progenitor cell and humanized mouse models.

microbiology↗

bioRxiv2022

LXR-inducible host E3 ligase IDOL targets a human cytomegalovirus reactivation determinant to promote latency

Liver X receptor (LXR) signaling broadly restricts virus replication; however, the mechanisms of restriction are poorly defined. Here, we demonstrate that the LXR-inducible cellular E3 ligase IDOL (inducible degrader of low-density lipoprotein receptor, LDLR) targets the human cytomegalovirus (HMCV) UL136p33 protein for turnover. UL136 encodes multiple proteins that differentially impact latency and reactivation. UL136p33 is a determinant of reactivation. UL136p33 is targeted for rapid turnover by the proteasome and its stabilization by mutation of lysine residues to arginine results in a failure to quiet replication for latency. We show that IDOL targets UL136p33 for turnover, but not the stabilized variant. IDOL is highly expressed in undifferentiated hematopoietic cells where HCMV establishes latency, but is sharply downregulated upon differentiation, a stimulus for reactivation. We hypothesize that IDOL maintains low levels of UL136p33 for the establishment of latency. Consistent with this, knockdown of IDOL impacts viral gene expression in WT HCMV infection, but not in infection where UL136p33 has been stabilized. Further, induction of LXR signaling restricts WT HCMV reactivation from latency, but does not affect replication of a recombinant virus expressing a stabilized variant of UL136p33. This work establishes the UL136p33-IDOL interaction as a key regulator of the bistable switch between latency and reactivation. It further suggests a model whereby a key viral determinant of HCMV reactivation is regulated by a host E3 ligase and acts as a sensor at the tipping point between the decision to maintain the latent state or exit latency for reactivation. ImportanceHerpesviruses establish life-long latent infections, which pose an important risk for disease particularly in the immunocompromised. Our work is focused on the beta-herpesvirus, human cytomegalovirus (HCMV) that latently infects the majority of the population worldwide. Defining the mechanisms by which HCMV establishes latency or reactivates from latency is important to controlling viral disease. Here, we demonstrate that the cellular inducible degrader of low-density lipoprotein receptor, IDOL, targets a HCMV determinant of reactivation for degradation. The instability of this determinant is important for the establishment of latency. This work defines a pivotal virus-host interaction that allows HCMV to sense changes in host biology to navigate decisions to establish latency or replicate.

microbiology↗

bioRxiv2022

The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication

The serine/threonine protein kinase AKT is a critical mediator of growth factor signaling that broadly impacts mRNA translation, metabolism, programmed cell death and stress responses. Human cytomegalovirus (HCMV), a large dsDNA virus in the herpesvirus family, dramatically remodels host cell signaling pathways during lytic infection. Here, we show that AKT accumulates in an inactive form during HCMV infection, as indicated by hypo-phosphorylation at Thr308 and Ser473, as well as nuclear localization of FOXO3a. Moreover, we find that expression of constitutively active myristoylated AKT (myr-AKT) causes a 1-log defect in viral replication, accompanied by defects in viral DNA synthesis and viral late gene expression. These findings suggest that HCMV shutoff of AKT is not merely incidental to viral factors such as pUL38 that maintain high levels of mTORC1 activity independently of extracellular growth factor signals. Short interfering RNA knockdown of FOXO3a, but not FOXO1, phenocopied the replication defects seen during expression of myr-AKT, corroborating a role for FOXO3a during HCMV infection. Accordingly, a chimeric FOXO3a-estrogen receptor fusion protein, whose nuclear localization is regulated by the small molecule ligand 4-hydroxytamoxifen instead of AKT activity, rescues virtually all the replication defects induced by myr-AKT expression. Taken together, our results argue that HCMV dampens AKT signaling to promote nuclear localization of FOXO transcription factors, which are required for efficient viral replication. IMPORTANCEEvidence from a diverse herpesvirus infection models suggests that the PI3K / AKT signaling pathway suppresses reactivation from latency, and that inactivation of the pathway stimulates viral lytic replication. Here, we show that AKT accumulates in an inactive state during HCMV infection of lytically permissive cells while the presence of constitutive AKT activity causes substantial viral replication defects. Although AKT phosphorylates a diverse array of cellular substrates, we identify an important role for the Forkhead box class O transcription factors. Our findings show that when FoxO3a nuclear localization is decoupled from its negative regulation by AKT, the viral replication defects observed in the presence of constitutively active AKT are reversed. Collectively, our results reveal that HCMV inactivates AKT to promote the nuclear localization of FOXO transcription factors, which strongly implies that FOXOs play critical roles in transactivating cellular and/or viral genes during infection.

microbiology↗

bioRxiv2020

Cell type-specific biogenesis of novel vesicles containing viral products in human 1cytomegalovirus infection

Human cytomegalovirus (HCMV), while highly restricted for the human species, infects an unlimited array of cell types in the host. Patterns of infection are dictated by the cell type infected, but cell type-specific factors and how they impact tropism for specific cell types is poorly understood. Previous studies in primary endothelial cells showed that HCMV infection induces large multivesicular-like bodies that incorporate viral products including dense bodies and virions. Here we define the nature of these large vesicles using a recombinant virus where UL32, encoding the pp150 tegument protein, is fused in frame with green fluorescent protein (GFP, TB40/E-UL32-GFP). Cells were fixed and labeled with antibodies against subcellular compartment markers and imaged using confocal and super-resolution microscopy. In fibroblasts, UL32-GFP-positive vesicles were marked with classical markers of MVBs, including CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as the clathrin and LAMP1. Unexpectedly, UL32-GFP-positive vesicles in endothelial cells were not labeled by CD63, and LBPA was completely lost from infected cells. We defined these UL32-positive vesicles in endothelial cells using markers for the cis-Golgi (GM130), lysosome (LAMP1), and autophagy (LC3B). These findings suggest that virus-containing MVBs in fibroblasts are derived from the canonical endocytic pathway and takeover classical exosomal release pathway. Virus containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less characterized early Golgi-LAMP1-associated non-canonical secretory autophagy pathway. These results reveal striking cell-type specific membrane trafficking differences in host pathways that are exploited by HCMV. ImportanceHuman cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, that establishes a life long infection. HCMV remains a significant cause of morbidity and mortality in the immunocompromised and HCMV seropositivity is associated with increased risk vascular disease. HCMV infects many cells in the human and the biology underlying the different patterns of infection in different cell types is poorly understood. Endothelial cells are important target of infection that contribute to hematogenous spread of the virus to tissues. Here we define striking differences in the biogenesis of large vesicles that incorporate virions in fibroblasts and endothelial cells. In fibroblasts, HCMV is incorporated into canonical MVBs derived from an endocytic pathway, whereas HCMV matures through vesicles derived from the biosynthetic pathway in endothelial cells. This work defines basic biological differences between these cell types that may impact the outcome of infection.

microbiology↗

bioRxiv2020

A Molecular Mechanism for Probabilistic Bet-hedging and its Role in Viral Latency

Probabilistic bet hedging, a strategy to maximize fitness in unpredictable environments by matching phenotypic variability to environmental variability, is theorized to account for the evolution of various fate-specification decisions, including viral latency. However, the molecular mechanisms underlying bet-hedging remain unclear. Here, we report that large variability in protein abundance within individual herpesvirus virion particles enables probabilistic bet hedging between viral replication and latency. Super-resolution imaging of individual virions of the human herpesvirus cytomegalovirus (CMV) showed that virion-to-virion levels of pp71 tegument protein--the major viral transactivator protein--exhibit extreme variability. This super-Poissonian tegument variability promoted alternate replicative strategies: high virion pp71 levels enhance viral replicative fitness but, strikingly, impede silencing, whereas low virion pp71 levels reduce fitness but promote silencing. Overall, the results indicate that stochastic tegument packaging provides a mechanism enabling probabilistic bet hedging between viral replication and latency. SIGNIFICANCEProbabilistic bet hedging is a generalized diversification strategy to maximize fitness in unpredictable environments, and has been proposed as an evolutionary basis for herpesvirus latency. However, the molecular mechanisms enabling probabilistic bet hedging have remained elusive. Here, we find that the human herpesvirus cytomegalovirus--a major cause of birth defects and transplant failures--utilizes stochastic variability in the abundance of a protein packaged into individual viral particles to enable probabilistic bet hedging between alternate viral states.

molecular biology↗

bioRxiv2020

FOXO Transcription Factors Activate Alternative Major Immediate Early Promoters to Induce Human Cytomegalovirus Reactivation

A key step during viral reactivation from latency is the re-expression of viral genes. Hematopoietic progenitor cells (HPCs) support human cytomegalovirus (HCMV) latency, and their differentiation triggers cellular cues that drive reactivation. A key step during HCMV reactivation in latently infected HPCs is re-expression of viral genes. We recently determined that the major immediate early promoter (MIEP), which is primarily responsible for MIE gene expression during lytic replication, remains silent during reactivation. Instead, alternative promoters in the MIE locus are induced by reactivation stimuli. Here, we find that forkhead family (FOXO) transcription factors are critical for activation of alternative MIE promoters during HCMV reactivation, as mutating FOXO binding sites in alternative MIE promoters decreased HCMV IE gene expression upon reactivation and significantly decreased the production of infectious virus from latently infected primary CD34+ HPCs. These findings establish a mechanistic link by which infected cells sense environmental cues to regulate latency and reactivation, and emphasize the role of contextual activation of alternative MIE promoters as the primary drivers of reactivation. SignificanceHuman cytomegalovirus infection is lifelong and persistent. Periodic reactivation of cytomegalovirus poses serious disease risk for immune-compromised patients. A critical driver of reactivation is expression of viral genes from the major immediate early locus. Recent paradigm-shifting evidence shows that reactivation is driven from promoters distinct from those that drive replication in permissive cells. Understanding the contextual control of these promoters and how they specifically respond to cellular cues that drive reactivation is critical for establishing future therapies that prevent reactivation. Our findings mechanistically define a previously enigmatic relationship between differentiation and reactivation, and provide potential targets for therapeutic intervention to prevent HCMV reactivation and disease.

microbiology↗

bioRxiv2019

EGR1 transcriptional control of human cytomegalovirus latency

Sustained phosphotinositide3-kinase (PI3K) signaling is critical to the maintenance of herpesvirus latency. We have previously shown that the beta-herpesvirus, human cytomegalovirus (CMV), regulates epidermal growth factor receptor (EGFR), upstream of PI3K, to control states of latency and reactivation. Inhibition of EGFR signaling enhances CMV reactivation from latency and increases viral replication, but the mechanisms by which EGFR impacts replication and latency is not known. We demonstrate that HCMV downregulates MEK/ERK and AKT phosphorylation, but not STAT3 or PLC{gamma} for productive replication. Similarly, inhibition of either MEK/ERK or PI3K/AKT, but not STAT or PLC{gamma}, pathways increases viral reactivation from latently infected CD34+ hematopoietic progenitor cells (HPCs), defining a role for these pathways in latency. We hypothesized that CMV modulation of EGFR signaling might impact viral transcription. Indeed, EGF-stimulation increased expression of the UL138 latency gene, but not immediate early or early viral genes, suggesting that EGFR signaling promotes latent gene expression. The early growth response-1 (EGR1) transcription factor is induced downstream of EGFR signaling through both PI3K/AKT and MEK/ERK pathways. EGR1 expression is important for the maintenance of HPC stemness and its downregulation drives HPC differentiation and mobilization. We demonstrate that EGR1 binds upstream of UL138 and is sufficient to promote UL138 expression. Further, disruption of EGR1 binding upstream of UL138 prevented CMV from establishing a latent infection in CD34+ HPCs. Our results indicate a model whereby UL138 modulation of EGFR signaling feeds back to promote UL138 expression and suppression of replication to establish or maintain viral quiescence.\n\nAUTHOR SUMMARYCMV regulates EGFR signaling to balance states of viral latency and replication. CMV blocks downstream PI3K/AKT and MEK/ERK signaling pathways through down-regulation of EGFR at the plasma membrane. PI3K/AKT and MEK/ERK signaling increases expression of the EGR1 transcription factor that is necessary for the maintenance of stem cell stemness. A decrease in EGR1 expression promotes HPC mobilization to the periphery and differentiation, a known stimulus for CMV reactivation. We identified functional EGR1 binding sites upstream of the UL138 gene and EGR-1 binding stimulates UL138 expression. Additionally, down-regulation of EGR1 by CMV miR-US22 decreases UL138 expression emphasizing the importance of this transcription factor in expression of this latency gene. Lastly, we demonstrate that a CMV mutant virus lacking an upstream EGR1 binding site is unable to establish latency in CD34+ HPCs. This study defines one mechanism by which EGFR signaling impacts viral gene expression to promote CMV latency.

molecular biology↗

bioRxiv2019

HCMV miR-US22 down-regulation of EGR-1 regulates CD34+ hematopoietic progenitor cell proliferation and viral reactivation

Reactivation of latent Human Cytomegalovirus (HCMV) in CD34+ hematopoietic progenitor cells (HPCs) is closely linked to hematopoiesis. Viral latency requires maintenance of the progenitor cell quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14+ macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34+ HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34+ HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation during latency. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus.\n\nAuthor summaryHuman cytomegalovirus (HCMV) is a widespread herpesvirus that persists in the host and remains a significant cause of morbidity and mortality in solid organ and stem cell transplant patients. HCMV latency is complex, and the molecular mechanisms for establishment, maintenance, and reactivation from latency are poorly understood.\n\nQuiescent stem cells in the bone marrow represent a critical reservoir of latent HCMV, and the mobilization and differentiation of these cells is closely linked to viral reactivation from latency. HCMV encodes small regulatory RNAs, called miRNAs that play important roles in the regulation of viral and cellular gene expression. In this study, we show that HCMV miR-US22 targets Early growth response gene 1 (EGR-1) a host transcription factor that is necessary for stem cell quiescence and self-renewal in the bone marrow. Expression of this miR-US22 down-regulates expression of EGR-1 that reduces CD34+ HPCs proliferation and total hematopoietic colony formation. An HCMV miR-US22 mutant is unable to reactivate from latency suggesting that the ability of the miRNA to disrupt CD34+ HPC renewal in the bone marrow niche to initiate a differentiation pathway is critical for viral reactivation.

microbiology↗