Human Cytomegalovirus IE2 Research Articles

Generation of glioblastoma in mice engrafted with human cytomegalovirus-infected astrocytes

Mounting evidence is identifying human cytomegalovirus (HCMV) as a potential oncogenic virus. HCMV has been detected in glioblastoma multiforme (GB). Herewith, we present the first experimental evidence for the generation of CMV-Elicited Glioblastoma Cells (CEGBCs) possessing glioblastoma-like traits that lead to the formation of glioblastoma in orthotopically xenografted mice. In addition to the already reported oncogenic HCMV-DB strain, we isolated three HCMV clinical strains from GB tissues that transformed HAs toward CEGBCs and generated spheroids from CEGBCs that resulted in the appearance of glioblastoma-like tumors in xenografted mice. These tumors were nestin-positive mostly in the invasive part surrounded by GFAP-positive reactive astrocytes. The glioblastoma immunohistochemistry phenotype was confirmed by EGFR and cMet gene amplification in the tumor parallel to the detection of HCMV IE and UL69 genes and proteins. Our results fit with an HCMV-induced glioblastoma model of oncogenesis in vivo which will open the door to new therapeutic approaches and assess the anti-HCMV treatment as well as immunotherapy in fighting GB which is characterized by poor prognosis.

Possible roles of human cytomegalovirus immunoglobulin G and its avidity to specific human cytomegalovirus antigens in the prevention of abortion among pregnant women

Abstract Background: The human cytomegalovirus (HCMV) is a widespread viral pathogen characterized by strict host specificity and is limited to humans. It has been described as an important etiological agent of intrauterine infection during the pregnancy, that causes lifelong infection and may lead to some serious results such as miscarriage, cerebellar malformation stillbirth, and fetus developmental retardation. Objectives: The study was carried out in Kirkuk governorate from the August 2021 to April 2022 to analyze the seroprevalence of HCMV-immunoglobulin G (IgG) against some HCMV antigens and its relation to the history of abortion. Materials and Methods: A total of 220 pregnant women were examined for the seroprevalence of HCMV-IgG by using electro-chemo-luminescence technique and then examined their reactivity and avidity for specific HCMV antigens using line immune assay. Results: The findings of the study showed that among 120 HCMV-IgG seropositive pregnant women, the rates of IgG antibodies were 161 (73.18%). In terms of reactivity and avidity of HCMV-IgG against different HCMV antigens, the rates were as follows: 67 (55.83%) for HCMV IE1, 52 (43.33%) for CM2, 116 (96.66%) for p150, 68 (56.66%) for p65, 117 (97.5%) for gB1, and 82 (68.33%) for gB2 antigens. Among pregnant women with no previous history of abortion, the prevalence of HCMV-IgG reactivity against gB2 was 75.60%. Furthermore, the rates of HCMV-IgG avidity for HCMV antigens were 90.24% for CM2 and 83.05% for p65 antigens. Conclusions: This finding may refer to the possible role of the specificity of HCMV-IgG and its high avidity for specific HCMV antigens in the prevention of abortion among pregnant women infected with HCMV infection.

Human cytomegalovirus infection enhances 5‑lipoxygenase and cycloxygenase‑2 expression in colorectal cancer.

Colorectal cancer (CRC) is one of the most common and fatal types of cancer. Inflammation promotes CRC development, however, the underlying etiological factors are unknown. Human cytomegalovirus (HCMV), a virus that induces inflammation and other cancer hallmarks, has been detected in several types of malignancy, including CRC. The present study investigated whether HCMV infection was associated with expression of the pro‑inflammatory enzymes 5‑lipoxygenase (5‑LO) and cyclooxygenase‑2 (COX‑2) and other molecular, genetic and clinicopathological CRC features. The present study assessed 146individual paraffin‑embedded CRC tissue microarray (TMA) cores already characterized for TP53 and KRAS mutations, microsatellite instability (MSI) status, Ki‑67 index and EGFR by immunohistochemistry (IHC). The cores were further analyzed by IHC for the expression of two HCMV proteins (Immediate Early, IE and pp65) and the inflammatory markers 5‑LO and COX‑2. The CRC cell lines Caco‑2 and LS‑174T were infected with HCMV strain VR1814, treated with antiviral drug ganciclovir (GCV) and/or anti‑inflammatory drug celecoxib (CCX) and analyzed by reverse transcription‑quantitative PCR and immunofluorescence for 5‑LO, COX‑2, IE and pp65 transcripts and proteins. HCMV IE and pp65 proteins were detected in ~90% of the CRC cases tested; this was correlated with COX‑2, 5‑LO and KI‑67 expression, but not with EGFR immunostaining, TP53 and KRAS mutations or MSI status. Invitro, HCMV infection upregulated 5‑LO and COX‑2 transcript and proteins in both Caco‑2 and LS‑174T cells and enhanced cell proliferation as determined by MTT assay. Treatment with GCV and CCX significantly decreased the transcript levels of COX‑2, 5‑LO, HCMV IE and pp65 in infected cells. HCMV was widely expressed in CRC and may promote inflammation and serve as a potential new target for CRC therapy.

Human Cytomegalovirus IE2 Disrupts Neural Progenitor Development and Induces Microcephaly in Transgenic Mouse

Human cytomegalovirus (HCMV) is a significant contributor to congenital birth defects. Limited by the lack of animal models, the pathogenesis of neurological damage in vivo caused by HCMV infection and the role of individual viral genes remain to be elucidated. Immediate early (IE2) protein may play a function in neurodevelopmental problems caused by HCMV infection. Here, this study intended to investigate IE2’s long-term effects on development of the brain in IE2-expressing transgenic mice (Rosa26-LSL-IE2+/−, Camk2α-Cre) aimed to observe the phenotype of postnatal mice. The expression of IE2 in transgenic mice was confirmed by PCR and Western blot technology. We collected mouse brain tissue at 2, 4, 6, 8, and 10 days postpartum to analyze the developmental process of neural stem cells by immunofluorescence. We discovered that transgenic mice (Rosa26-LSL-IE2+/−, Camk2α-Cre) can reliably produce IE2 in the brain at various postpartum phases. Furthermore, we also observed the symptoms of microcephaly in postnatal transgenic mice, and IE2 can damage the amount of neural stem cells, prevent them from proliferating and differentiating, and activate microglia and astrocytes, creating an unbalanced environment in the brain’s neurons. In conclusion, we demonstrate that long-term expression of HCMV-IE2 can cause microcephaly through molecular mechanisms affecting the differentiation and development of neural stem cells in vivo. This work establishes a theoretical and experimental foundation for elucidating the molecular mechanism of fetal microcephaly brought by HCMV infection in throughout the period of neural development of pregnancy.Graphical

Rationally designed synthetic vectors for therapeutic nucleic acid delivery against human cytomegalovirus infection.

RNA therapy represents a great way to precisely regulate cellular processes by modulating the gene expression. Despite this potential, a profound gap exists in our knowledge of how to subsequently deliver these RNAs into the specific target cells and turn therapeutically active RNAs into practical medicines. An advanced series of interlocked, thermodynamically self-regulated processes that enable the precise assembly of functional synthetic carriers of siRNA to the target cells in vivo was developed. To demonstrate the efficacy of this delivery system, we used it to treat human cytomegalovirus (HCMV) infection in a humanized mouse model. In this study, we use small interfering RNA (siRNA) and small complementary RNA (scRNA) to inhibit the expressions of two HCMV genes, IE1 and IE2. The auto-regulated nanocarrier polywraplex with core-shell structure was designed to condense and package these RNAs for delivering. To allow these particles recognize the HCMV-infected cells, a ligand was coupled on the surface of nanoparticle, which would specifically target the HCMV-encoded CX3 CL1 chemokine receptor presented in the HCMV-infected cells. The results demonstrated that the polywraplex conjugated with the target molecule CX3 CL1 effectively and specifically delivered the siRNA/scRNA to HCMV infected cells and inhibited virus growth in vitro and in vivo.

Human Cytomegalovirus IE2 Both Activates and Represses Initiation and Modulates Elongation in a Context-Dependent Manner

ABSTRACTHuman cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional transcription factor that is essential for lytic HCMV infection. IE2 functions as an activator of viral early genes, negatively regulates its own promoter, and is required for viral replication. The mechanisms by which IE2 executes these distinct functions are incompletely understood. Using PRO-Seq, which profiles nascent transcripts, and a recently developed DFF-chromatin immunoprecipitation (DFF-ChIP; employs chromatin digestion by the endonuclease DNA fragmentation factor prior to IP) approach that resolves occupancy and local chromatin environment, we show that IE2 controls viral gene transcription in three distinct capacities during late HCMV infection and reveal mechanisms that involve direct binding of IE2 to viral DNA. IE2 represses a subset of viral promoters by binding within their core promoter regions and blocking the assembly of preinitiation complexes (PICs). Remarkably, IE2 forms a repressive complex at the major immediate-early promoter region involving direct association of IE2 with nucleosomes and TBP. IE2 stimulates transcription by binding nearby, but not within, core promoter regions. In addition, IE2 functions as a direct roadblock to transcription elongation. At one locus, this function of IE2 appears to be important for the synthesis of a spliced viral RNA. Consistent with the minimal observed effects of IE2 depletion on host gene transcription, IE2 does not functionally engage the host genome. Our results reveal mechanisms of transcriptional control by IE2, uncover a previously unknown function of IE2 as a Pol II elongation modulator, and demonstrate that DFF-ChIP is a useful tool for probing transcription factor occupancy and interactions between transcription factors and nucleosomes at high resolution.

Development and Characterization of an HCMV Multi-Antigen Therapeutic Vaccine for Glioblastoma Using the UNITE Platform.

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with a median survival of 15 months that has remained unchanged despite advances in the standard of care. GBM cells express human cytomegalovirus (HCMV) proteins, providing a unique opportunity for targeted therapy. We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins pp65, gB, and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated membrane protein 1 (LAMP1) with target ntigens. We demonstrate evidence of increased antigen presentation by both MHC-I and -II, delivering a robust antigen-specific CD4 and CD8 T-cell response in addition to a strong humoral response. Using a syngeneic orthotopic GBM mouse model, therapeutic treatment with the ITI-1001 vaccine resulted in ~56% survival of tumor-bearing mice. Investigation of the tumor microenvironment showed significant CD4 infiltration as well as enhanced Th1 and cytotoxic CD8 T activation. Regulatory T cells were also upregulated after ITI-1001 vaccination. In addition, tumor burden negatively correlated with activated interferon (IFN)γ+ CD4 T cells, reiterating the importance of CD4 activation in ITI-1001 efficacy and in identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+, and CD8+ T cells in responders compared to non-responders. Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant antitumor activity, leading to enhanced survival in a mouse model of GBM.

Human Cytomegalovirus-Encoded microRNAs Can Be Found in Saliva Samples from Renal Transplant Recipients.

Human cytomegalovirus (HCMV) infections are common following renal transplantation and may have long-lasting effects. HCMV can be measured directly by viral DNA or indirectly via host immune responses. HCMV-encoded microRNA (miRNA) may alter the pathobiology of HCMV infections and contribute to the progression of HCMV disease. HCMV-encoded miRNAs can be detected in blood but have not been sought in saliva. We investigated saliva samples from 32 renal transplant recipients (RTR) and 12 seropositive healthy controls for whom immunological data was available. Five HCMV-encoded miRNAs (miR-UL112-5p, miR-US5-2-3p, miR-UL36, miR-US25-2-3p and miR-UL22A) were sought using primer probe assays. HCMV miRNA species were detected in saliva from 15 RTR and 3 healthy controls, with miR-US5-2-3p most commonly detected. The presence of HCMV miRNAs associated with increased T-cell responses to HCMV IE-1 in RTR, suggesting a link with frequent reactivations of HCMV.

HCMV infection induces AD pathology in astrocytes in vitro

AbstractBackgroundInfectious agents have long been suspected to be risk factors for Alzheimer’s disease (AD). Some studies have identified a link between neurotropic viruses and AD. While these studies demonstrated an association between AD and neuroviruses, most do not establish a causal link. However, many AD pathological hallmarks, including beta amyloid plaques (Aβ), astrogliosis, neuroinflammation, and neuronal damage, overlap with pathologies caused by neuroviruses. Approximately 80% of adults are seropositive for human cytomegalovirus (HCMV), a virus that is neuroinvasive, productively infects brain cells, and is immune‐dominant in aging populations. Using post‐mortem tissues from the Rush Alzheimer’s Disease Center we previously reported that HCMV infection is associated with AD clinical markers. In the present study, we assessed the ability of HCMV infection to induce two AD pathological hallmarks in astrocytes: Aβ production and astrocyte reactivity.MethodTo determine the impact of HCMV infection on astrocytes, we generated low‐passage clinical HCMV isolates for infection of human fetal astrocytes (HFA) and astroglioma (U251) cells. To assess the ability of HCMV to produce Aβ, we infected HFAs and U251s with HCMV for 3‐9 days then immunostained for Aβ and HCMV proteins. To test whether HCMV infection results in astrocyte reactivity, we infected HFAs with HCMV for 5 days then quantified levels of reactivity‐associated transcripts via quantitative PCR.ResultHFA and U251 infected cells showed HCMV cytopathic effects characteristic of permissive cells, including enlargement and plaque formation. HCMV infection of HFA and U251 cells induced the expression of Aβ as observed by co‐staining of Aβ and HCMV proteins. Aβ production was observed within 3 days and persisted until at least 9 days post‐infection. To assess the amyloidogenicity of HCMV, we employed the Waltz algorithm, which predicted two amyloidogenic sequences within immunodominant HCMV proteins pp65 and IE1 (average score: 98.3). Further, HCMV infection of HFAs mediated a 2‐ to 4‐ fold induction in CXCL10, CD44, and IRGM, reactivity‐associated genes also involved in immune and inflammatory pathways.ConclusionOur data demonstrate that HCMV infection can contribute to AD pathology in cultured astrocytes. These findings could ultimately inform the use of antiviral therapy to prevent and/or reduce AD severity.

The HSV-1 ICP4 Transcriptional Auto-Repression Circuit Functions as a Transcriptional "Accelerator" Circuit.

Herpes simplex virus-1 (HSV-1) is a significant human pathogen. Upon infection, HSV-1 expresses its immediate early (IE) genes, and the IE transcription factor ICP4 (infectious cell protein-4) plays a pivotal role in initiating the downstream gene-expression cascade. Using live-cell time-lapse fluorescence microscopy, flow cytometry, qPCR, and chromatin immunoprecipitation, we quantitatively monitored the expression of ICP4 in individual cells after infection. We find that extrinsic stimuli can accelerate ICP4 kinetics without increasing ICP4 protein or mRNA levels. The accelerated ICP4 kinetics—despite unchanged steady-state ICP4 protein or mRNA level—correlate with increased HSV-1 replicative fitness. Hence, the kinetics of ICP4 functionally mirror the kinetics of the human herpesvirus cytomegalovirus IE2 “accelerator” circuit, indicating that IE accelerator circuitry is shared among the alpha and beta herpesviruses. We speculate that this circuit motif is a common evolutionary countermeasure to throttle IE expression and thereby minimize the inherent cytotoxicity of these obligate viral transactivators.

Human cytomegalovirus IE2 drives transcription initiation from a select subset of late infection viral promoters by host RNA polymerase II.

Herpesvirus late promoters activate gene expression after viral DNA synthesis has begun. Alphaherpesviruses utilize a viral immediate-early protein to do this, whereas beta- and gammaherpesviruses primarily use a 6-member set of viral late-acting transcription factors (LTF) that are drawn to a TATT sequence in the late promoter. The betaherpesvirus, human cytomegalovirus (HCMV), produces three immediate-early 2 protein isoforms, IE2-86, IE2-60, IE2-40, late in infection, but whether they activate late viral promoters is unknown. Here, we quickly degrade the IE2 proteins in late infection using dTag methodology and analyze effects on transcription using customized PRO-Seq and computational methods combined with multiple validation methods. We discover that the IE2 proteins selectively drive RNA Pol II transcription initiation at a subset of viral early-late and late promoters common to different HCMV strains, but do not substantially affect Pol II transcription of the 9,942 expressed host genes. Most of the IE2-activated viral late infection promoters lack the TATT sequence bound by the HCMV UL87-encoded LTF. The HCMV TATT-binding protein is not mechanistically involved in late RNA expression from the IE2-activated TATT-less UL83 (pp65) promoter, as it is for the TATT-containing UL82 (pp71) promoter. While antecedent viral DNA synthesis is necessary for transcription from the late infection viral promoters, continued viral DNA synthesis is unnecessary. We conclude that in late infection the IE2 proteins target a distinct subset of HCMV early-late and late promoters for transcription initiation by RNA Pol II. Commencement of viral DNA replication renders the HCMV genome late promoters susceptible to late-acting viral transcription factors.

Human Cytomegalovirus (HCMV)-infected Astrocytoma Cells Impair the Function of HCMV-specific Cytotoxic T Cells.

BackgroundHuman cytomegalovirus (HCMV) infection in glioblastoma multiforme (GBM) is associated with a poor prognosis and may affect the pathogenesis of GBM. In this study, we investigated the role of HCMV-infected astrocytoma cells in impairing the activity of cytotoxic T lymphocytes (CTLs) specific to the HCMV protein.MethodsCTLs specific to HCMV immediate early (IE)-1 were expanded from peripheral blood mononuclear cells of healthy donors by stimulating CD8+ T lymphocytes with U373MG cells (ATCC HTB-17: male) expressing HCMV IE-1. The death rate of the target and the effector cells was determined by the total count of the remaining respective cells after the interaction of them.ResultsThe death rate of the target cells by CTLs increased depending on HLA restriction and the effector:target (E:T) ratio. The death rate of effector cells in the HCMV-infected U373MG cell culture was 37.1% on day 4 post-infection. The removal of the culture supernatant from HCMV-infected U373MG cells prior to adding the effector cells increased target cell death from 8.4% to 40.8% at E:T = 1:1, but not at E:T = 3:1. The transfer of cells from a 24-hour co-culture of the HCMV-infected U373MG cells and CTLs to HCMV IE-1-expressing target cells resulted in decreasing the cell death rate of the target cells from 31.1% to 13.0% at E:T = 1:1, but not at E:T = 3:1. HCMV infection of U373MG cells decreases the activity of CTLs specific to HCMV when the number of CTLs is low.ConclusionThese results suggest that HCMV could impair CTL activity and facilitate glioblastoma growth unchecked by CTLs.

Human cytomegalovirus IE86 protein aa 136-289 mediates STING degradation and blocks the cGAS-STING pathway.

We previously reported that human cytomegalovirus (HCMV) 86 kDa immediate-early 2 gene product (IE86) promotes proteasome-dependent degradation of STING. In the present study, we determined the specific residues of IE86 responsible for STING degradation using a STING-firefly luciferase fusion protein expression system for quantitative meas-urement of STING protein levels. IE86 amino acids (aa) 136-289 were sufficient to promote STING degradation and further induced down-regulation of 2'3'-cyclic GMP-AMP (cGAMP)-mediated IFN-β promoter activation. Interestingly, transactivation domains (TAD) of the IE86 protein located at the N- and C-termini were required for down-regulation of Toll/interleukin-1 receptor (TIR) domain-containing adaptor-inducing interferon β (IFN-β) (TRIF)-mediated IFN-β-and p65/RelA-induced NF-κB-dependent promoter activation while amino acids (aa) 136-289 had no significant effects. Our collective data suggest that the IE86 protein utilizes the aa 136-289 region to promote STING degradation and inhibit the cGAS-STING pathway.

Human cytomegalovirus IE2 protein regulates macrophage-mediated immune escape by upregulating GRB2 expression in UL122 genetically modified mice.

Although cytomegalovirus (HCMV) infection is asymptomatic in healthy individuals, the virus can remain latent for many years due to its ability to evade host immune surveillance. However, reactivation of HCMV can lead to life-threatening disease. Recent studies have shown that HCMV infection mediates immune escape by regulating macrophage activity, although the role of the HCMV-encoded IE2 protein is unclear. A ul122 transgenic mouse model was created to stably expresses the IE2 protein, and the proportion of M1 and M2 macrophage populations in their spleen and bone marrow was compared to that in wild-type controls. In addition, the phagocytic function of the macrophages was evaluated in terms of neutral red dye uptake. Spleen and bone marrow macrophages in IE2-expressing mice were mainly of the M2 phenotype and displayed enhanced phagocytic function compared to that in control mice. The relative levels of expression of macrophage-related GRB2 and of IL-4, IFN-γ, IL-13, and TNF-α were also analyzed in the spleen and bone marrow of the two groups. The IE2-expressing mice had increased expression of GRB2 and increased expression of the M2-related cytokines IL-4 and IL-13. Taken together, the current results suggest that HCMV IE2 polarizes the host macrophages to the M2 type via a GRB2/IL-4-related pathway, which enables long-term survival of the virus in the host.

P2Y2 purinergic receptor modulates virus yield, calcium homeostasis, and cell motility in human cytomegalovirus-infected cells

Human cytomegalovirus (HCMV) manipulates many aspects of host cell biology to create an intracellular milieu optimally supportive of its replication and spread. Our study reveals that levels of several components of the purinergic signaling system, including the P2Y2 and P2X5 receptors, are elevated in HCMV-infected fibroblasts. Knockdown and drug treatment experiments demonstrated that P2Y2 enhances the yield of virus, whereas P2X5 reduces HCMV production. The HCMV IE1 protein induces P2Y2 expression; and P2Y2-mediated signaling is important for efficient HCMV gene expression, DNA synthesis, and the production of infectious HCMV progeny. P2Y2 cooperates with the viral UL37x1 protein to regulate cystolic Ca2+ levels. P2Y2 also regulates PI3K/Akt signaling and infected cell motility. Thus, P2Y2 functions at multiple points within the viral replication cycle to support the efficient production of HCMV progeny, and it may facilitate in vivo viral spread through its role in cell migration.

Human cytomegalovirus ie2 affects the migration of glioblastoma by mediating the different splicing patterns of RON through hnRNP A2B1.

Glioblastoma is the most aggressive intracranial tumor and diffuse migration is the leading cause of death. Recent evidence has indicated that heterogeneous nuclear ribonucleoprotein A2B1 (hnRNP A2B1) is overexpressed in human glioblastoma tissue and enhances glioblastoma invasion in vitro. We found by mass spectrometry that hnRNP A2B1 interacts with human cytomegalovirus (HCMV) immediate early 86 protein (IE86, ie2 gene-encoded) in malignant glioma cells (U87MG) infected with HCMV. However, the role of hnRNP A2 B1 in glioblastoma development remains poorly understood. Here, we report that hnRNP A2B1 is highly expressed in the HCMV·ie2 transgenic mice model. This phenomenon was confirmed in U87MG cell lines transfected with pEGFP-N3-ie2 plasmid. In addition, hnRNP A2B1 knockdown in U87MG cells inhibited tumor migration, and this effect might be mediated by hnRNP A2B1 through effects on splicing patterns of RON. Our data suggested that HCMV· ie2 promotes glioblastoma migration by regulating hnRNP A2B1 expression.

Human Cytomegalovirus Compromises Development of Cerebral Organoids.

Congenital human cytomegalovirus (HCMV) infection causes a broad spectrum of central and peripheral nervous system disorders, ranging from microcephaly to hearing loss. These ramifications mandate the study of virus-host interactions in neural cells. Neural progenitor cells are permissive for lytic infection. We infected two induced pluripotent stem cell (iPSC) lines and found these more primitive cells to be susceptible to infection but not permissive. Differentiation of infected iPSCs induced de novo expression of viral antigens. iPSCs can be cultured in three dimensions to generate cerebral organoids, closely mimicking in vivo development. Mock- or HCMV-infected iPSCs were subjected to a cerebral organoid generation protocol. HCMV IE1 protein was detected in virus-infected organoids at 52 days postinfection. Absent a significant effect on organoid size, infection induced regions of necrosis and the presence of large vacuoles and cysts. Perhaps more in parallel with the subtler manifestations of HCMV-induced birth defects, infection dramatically altered neurological development of organoids, decreasing the number of developing and fully formed cortical structure sites, with associated changes in the architectural organization and depth of lamination within these structures, and manifesting aberrant expression of the neural marker β-tubulin III. Our observations parallel published descriptions of infected clinical samples, which often contain only sparse antigen-positive foci yet display areas of focal necrosis and cellular loss, delayed maturation, and abnormal cortical lamination. The parallels between pathologies present in clinical specimens and the highly tractable three-dimensional (3D) organoid system demonstrate the utility of this system in modeling host-virus interactions and HCMV-induced birth defects.IMPORTANCE Human cytomegalovirus (HCMV) is a leading cause of central nervous system birth defects, ranging from microcephaly to hearing impairment. Recent literature has provided descriptions of delayed and abnormal maturation of developing cortical tissue in infected clinical specimens. We have found that infected induced pluripotent stem cells can be differentiated into three-dimensional, viral protein-expressing cerebral organoids. Virus-infected organoids displayed dramatic alterations in development compared to those of mock-infected controls. Development in these organoids closely paralleled observations in HCMV-infected clinical samples. Infection induced regions of necrosis, the presence of larger vacuoles and cysts, changes in the architectural organization of cortical structures, aberrant expression of the neural marker β-tubulin III, and an overall reduction in numbers of cortical structure sites. We found clear parallels between the pathologies of clinical specimens and virus-infected organoids, demonstrating the utility of this highly tractable system for future investigations of HCMV-induced birth defects.

Human Cytomegalovirus Immediate Early 86-kDa Protein Blocks Transcription and Induces Degradation of the Immature Interleukin-1β Protein during Virion-Mediated Activation of the AIM2 Inflammasome.

Secretion of interleukin-1β (IL-1β) represents a fundamental innate immune response to microbial infection that, at the molecular level, occurs following activation of proteolytic caspases that cleave the immature protein into a secretable form. Human cytomegalovirus (HCMV) is the archetypal betaherpesvirus that is invariably capable of lifelong infection through the activity of numerous virally encoded immune evasion phenotypes. Innate immune pathways responsive to cytoplasmic double-stranded DNA (dsDNA) are known to be activated in response to contact between HCMV and host cells. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) genome editing to demonstrate that the dsDNA receptor absent in melanoma 2 (AIM2) is required for secretion of IL-1β following HCMV infection. Furthermore, dsDNA-responsive innate signaling induced by HCMV infection that leads to activation of the type I interferon response is also shown, unexpectedly, to play a contributory role in IL-1β secretion. Importantly, we also show that rendering virus particles inactive by UV exposure leads to substantially increased IL-1β processing and secretion and that live HCMV can inhibit this, suggesting the virus encodes factors that confer an inhibitory effect on this response. Further examination revealed that ectopic expression of the immediate early (IE) 86-kDa protein (IE86) is actually associated with a block in transcription of the pro-IL-1β gene and, independently, diminishment of the immature protein. Overall, these results reveal two new and distinct phenotypes conferred by the HCMV IE86 protein, as well as an unusual circumstance in which a single herpesviral protein exhibits inhibitory effects on multiple molecular processes within the same innate immune response.IMPORTANCE Persistent infection with HCMV is associated with the operation of diverse evasion phenotypes directed at antiviral immunity. Obstruction of intrinsic and innate immune responses is typically conferred by viral proteins either associated with the viral particle or expressed immediately after entry. In line with this, numerous phenotypes are attributed to the HCMV IE86 protein that involve interference with innate immune processes via transcriptional and protein-directed mechanisms. We describe novel IE86-mediated phenotypes aimed at virus-induced secretion of IL-1β. Intriguingly, while many viruses target the function of the molecular scaffold required for IL-1β maturation to prevent this response, we find that HCMV and IE86 target the IL-1β protein specifically. Moreover, we show that IE86 impairs both the synthesis of the IL-1β transcript and the stability of the immature protein. This indicates an unusual phenomenon in which a single viral protein exhibits two molecularly separate evasion phenotypes directed at a single innate cytokine.

A tumor-specific endogenous repetitive element is induced by herpesviruses

Tandem satellite repeats account for 3% of the human genome. One of them, Human Satellite II (HSATII), is highly expressed in several epithelial cancers and cancer cell lines. Here we report an acute induction of HSATII RNA in human cells infected with two herpes viruses. We show that human cytomegalovirus (HCMV) IE1 and IE2 proteins cooperate to induce HSATII RNA affecting several aspects of the HCMV replication cycle, viral titers and infected-cell processes. HSATII RNA expression in tissue from two chronic HCMV colitis patients correlates with the strength of CMV antigen staining. Thus, endogenous HSATII RNA synthesis after herpesvirus infections appears to have functionally important consequences for viral replication and may provide a novel insight into viral pathogenesis. The HSATII induction seen in both infected and cancer cells suggests possible convergence upon common HSATII-based regulatory mechanisms in these seemingly disparate diseases.

Effect of ATF5 in Mice Transfected with Human Cytomegalovirus IE2 Gene

Effect of ATF5 in Mice Transfected with Human Cytomegalovirus IE2 Gene