Stabilization of the human cytomegalovirus UL136p33 reactivation determinant overcomes the requirement for UL135 for replication in hematopoietic cells.

Human cytomegalovirus (HCMV) is a beta herpesvirus that persists indefinitely in the human host through a 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 and is required for reactivation. By contrast, UL136 is expressed with later kinetics and encodes multiple proteins with differential roles in latency and reactivation. Like UL135, the largest UL136 isoform, UL136p33, is required for reactivation from latency in HPCs; viruses failing to express either protein are unresponsive to reactivation stimuli. 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 replication. We generated recombinant viruses lacking UL135 that expressed a stabilized variant of UL136p33. Stabilizing UL136p33 did not impact the replication of the UL135 mutant virus in fibroblasts. However, in the context of infection in HPCs, stabilization of UL136p33 strikingly compensated for the loss of UL135, resulting in increased replication in CD34+ HPCs and in humanized NOD-scid IL2Rγcnull (huNSG) mice. This finding suggests that while UL135 is essential for replication in HPCs, it functions largely at steps preceding the accumulation of UL136p33, and that stabilized expression of UL136p33 largely overcomes the requirement for UL135. Taken together, our genetic evidence indicates an epistatic relationship between UL136p33 and UL135, whereby UL135 may initiate events early in reactivation that drive the accumulation of UL136p33 to a threshold required for productive reactivation. IMPORTANCE Human cytomegalovirus (HCMV) is one of nine human herpesviruses and a significant human pathogen. While HCMV establishes a lifelong latent infection that is typically asymptomatic in healthy individuals, its reactivation from latency can have devastating consequences in the immunocompromised. Defining viral genes important in the establishment of or reactivation from latency is important 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 cells and humanized mouse models.

Liver X Receptor-Inducible Host E3 Ligase IDOL Targets a Human Cytomegalovirus Reactivation Determinant.

Liver X receptor (LXR) signaling broadly restricts virus replication; however, the mechanisms of restriction are poorly defined. Here, we demonstrate that the cellular E3 ligase LXR-inducible degrader of low-density lipoprotein receptor (IDOL) 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 hypothesis, knockdown of IDOL impacts viral gene expression in wild-type (WT) HCMV infection but not in infection where UL136p33 has been stabilized. Furthermore, the induction of LXR signaling restricts WT HCMV reactivation from latency but does not affect the 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. IMPORTANCE Herpesviruses establish lifelong latent infections, which pose an important risk for disease particularly in the immunocompromised. Our work is focused on the betaherpesvirus 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 for 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 to replicate.

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.

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 IL2Rγ c null (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. SIGNIFICANCE: Human 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.

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.

Host translesion polymerases are required for viral genome integrity.

Human cells encode up to 15 DNA polymerases with specialized functions in chromosomal DNA synthesis and damage repair. In contrast, complex DNA viruses, such as those of the herpesviridae family, encode a single B-family DNA polymerase. This disparity raises the possibility that DNA viruses may rely on host polymerases for synthesis through complex DNA geometries. We tested the importance of error-prone Y-family polymerases involved in translesion synthesis (TLS) to human cytomegalovirus (HCMV) infection. We find most Y-family polymerases involved in the nucleotide insertion and bypass of lesions restrict HCMV genome synthesis and replication. In contrast, other TLS polymerases, such as the polymerase ζ complex, which extends past lesions, was required for optimal genome synthesis and replication. Depletion of either the polζ complex or the suite of insertion polymerases demonstrate that TLS polymerases suppress the frequency of viral genome rearrangements, particularly at GC-rich sites and repeat sequences. Moreover, while distinct from HCMV, replication of the related herpes simplex virus type 1 is impacted by host TLS polymerases, suggesting a broader requirement for host polymerases for DNA virus replication. These findings reveal an unexpected role for host DNA polymerases in ensuring viral genome stability.

Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency.

Sustained phosphotinositide3-kinase (PI3K) signaling is critical to the maintenance of alpha and beta 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. How signaling downstream of EGFR is regulated and how this impacts CMV infection and latency is not fully understood. We demonstrate that CMV downregulates EGFR early in the productive infection, which blunts the activation of EGFR and its downstream pathways in response to stimuli. However, CMV infection sustains basal levels of EGFR and downstream pathway activity in the context of latency in CD34+ hematopoietic progenitor cells (HPCs). Inhibition of MEK/ERK, STAT or PI3K/AKT pathways downstream of EGFR increases viral reactivation from latently infected CD34+ HPCs, defining a role for these pathways in latency. We hypothesized that CMV modulation of EGFR signaling might impact viral transcription important to latency. 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 the MEK/ERK pathway and is important for the maintenance of hematopoietic stemness. We demonstrate that EGR1 binds the viral genome upstream of UL138 and is sufficient to promote UL138 expression. Further, disruption of EGR1 binding upstream of UL138 prevents the establishment of latency in CD34+ HPCs. Our results indicate a model whereby UL138 modulation of EGFR signaling feeds back to promote UL138 gene expression and suppression of replication for latency. By this mechanism, the virus has hardwired itself into host cell biology to sense and respond to changes in homeostatic host cell signaling.

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.

Virus Control of Trafficking from Sorting Endosomes.

The maintenance of cell surface proteins is critical to the ability of a cell to sense and respond to information in its environment. As such, modulation of cell surface composition and receptor trafficking is a potentially important target of control in virus infection. Sorting endosomes (SEs) are control stations regulating the recycling or degradation of internalized plasma membrane proteins. Here we report that human cytomegalovirus (HCMV), a ubiquitous betaherpesvirus, alters the fate of internalized clathrin-independent endocytosis (CIE) cargo proteins, retaining them in virally reprogrammed SEs. We show that the small G protein ARF6 (ADP ribosylation factor 6), a regulator of CIE trafficking, is highly associated with SE membranes relative to uninfected cells. Combined with the observation of accumulated CIE cargo at the SE, these results suggest that infection diminishes the egress of ARF6 and its cargo from the SE. Expression of ubiquitin-specific protease 6 (USP6), also known as TRE17, was sufficient to restore ARF6 and some ARF6 cargo trafficking to the cell surface in infected cells. The USP activity of TRE17 was required to rescue both ARF6 and associated cargo from SE retention in infection. The finding that TRE17 expression does not rescue the trafficking of all CIE cargos retained at SEs in infection suggests that HCMV hijacks the normal sorting machinery and selectively sorts specific cargos into endocytic microdomains that are subject to alternative sorting fates.IMPORTANCE Cells maintain their surface composition, take up nutrients, and respond to their environment through the internalization and recycling of cargo at the cell surface through endocytic trafficking pathways. During infection with human cytomegalovirus (HCMV), host endocytic membranes are reorganized into a juxtanuclear structure associated with viral assembly and egress. Less appreciated is the effect of this reorganization on the trafficking of host proteins through the endocytic pathway. We show that HCMV retains internalized cargo and the effector of clathrin-independent endocytosis at sorting endosomes. The retention of some cargo, but not all, was reversed by overexpression of a ubiquitin-specific protease, TRE17. Our results demonstrate that HCMV induces profound reprogramming of endocytic trafficking and influences cargo sorting decisions. Further, our work suggests the presence of a novel ubiquitin-regulated checkpoint for the recycling of cargo from sorting endosome. These findings have important implications for host signaling and immune pathways in the context of HCMV 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.

Gastrulation EMT Is Independent of P-Cadherin Downregulation.

Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved process during which cells lose epithelial characteristics and gain a migratory phenotype. Although downregulation of epithelial cadherins by Snail and other transcriptional repressors is generally considered a prerequisite for EMT, recent studies have challenged this view. Here we investigate the relationship between E-cadherin and P-cadherin expression and localization, Snail function and EMT during gastrulation in chicken embryos. Expression analyses show that while E-cadherin transcripts are detected in the epiblast but not in the primitive streak or mesoderm, P-cadherin mRNA and protein are present in the epiblast, primitive and mesoderm. Antibodies that specifically recognize E-cadherin are not presently available. During EMT, P-cadherin relocalizes from the lateral surfaces of epithelial epiblast cells to a circumferential distribution in emerging mesodermal cells. Cells electroporated with an E-cadherin expression construct undergo EMT and migrate into the mesoderm. An examination of Snail function showed that reduction of Slug (SNAI2) protein levels using a morpholino fails to inhibit EMT, and expression of human or chicken Snail in epiblast cells fails to induce EMT. In contrast, cells expressing the Rho inhibitor peptide C3 rapidly exit the epiblast without activating Slug or the mesoderm marker N-cadherin. Together, these experiments show that epiblast cells undergo EMT while retaining P-cadherin, and raise questions about the mechanisms of EMT regulation during avian gastrulation.