scholarly journals Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Timo W. M. De Groof ◽  
Elizabeth G. Elder ◽  
Eleanor Y. Lim ◽  
Raimond Heukers ◽  
Nick D. Bergkamp ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Early Gene ◽  
Latent Reservoir ◽  
Solid Organ ◽  
Cell Transplant ◽  
Viral Receptor ◽  
Transplant Patients ◽  
Latently Infected ◽  
Infected Cells

AbstractLatent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients.

scholarly journals Targeting the latent human cytomegalovirus reservoir with virus specific nanobodies

2020 ◽  
Author(s):  
Timo W.M. De Groof ◽  
Elizabeth G. Elder ◽  
Raimond Heukers ◽  
Eleanor Y. Lim ◽  
Mark Wills ◽  
...  
Keyword(s):  
Cytotoxic T Lymphocytes ◽  
Human Cytomegalovirus ◽  
Stem Cell Transplant ◽  
Latent Reservoir ◽  
Solid Organ ◽  
Cell Transplant ◽  
Viral Pathogens ◽  
Viral Receptor ◽  
Transplant Patients ◽  
Latently Infected

AbstractLatent reservoirs of viral pathogens are significant barriers to eradication of these viruses. During latency, herpesviruses maintain their genome, with little gene expression, making latent infections refractory to current treatments targeting viral replication. In the case of human cytomegalovirus (HCMV), sporadic reactivation events are well controlled by the immune system. However, in immunocompromised or immunosuppressed individuals, HCMV reactivation often results in morbidity in solid organ and stem cell transplant patients. Clearance of the latent reservoir could lower the incidence and severity of HCMV-associated disease. Here, we develop a virus specific nanobody (VUN100b) that partially inhibits signaling of the viral receptor US28. VUN100b treatment partially reverses latency without fully reactivating the virus. Moreover, VUN100b treatment drives recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals. This study shows the potential of VUN100b as a therapy to clear the HCMV latent reservoir of transplant patients.

scholarly journals Bromodomain proteins regulate human cytomegalovirus latency and reactivation allowing epigenetic therapeutic intervention

2021 ◽  
Vol 118 (9) ◽  
pp. e2023025118 ◽  
Author(s):  
Ian J. Groves ◽  
Sarah E. Jackson ◽  
Emma L. Poole ◽  
Aharon Nachshon ◽  
Batsheva Rozman ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Histone Deacetylase Inhibitors ◽  
Virus Genome ◽  
Early Gene ◽  
Latent Reservoir ◽  
Solid Organ ◽  
Virus Reactivation ◽  
Genes Encoding ◽  
Latently Infected ◽  
Infected Cells

Reactivation of human cytomegalovirus (HCMV) from latency is a major health consideration for recipients of stem-cell and solid organ transplantations. With over 200,000 transplants taking place globally per annum, virus reactivation can occur in more than 50% of cases leading to loss of grafts as well as serious morbidity and even mortality. Here, we present the most extensive screening to date of epigenetic inhibitors on HCMV latently infected cells and find that histone deacetylase inhibitors (HDACis) and bromodomain inhibitors are broadly effective at inducing virus immediate early gene expression. However, while HDACis, such as myeloid-selective CHR-4487, lead to production of infectious virions, inhibitors of bromodomain (BRD) and extraterminal proteins (I-BETs), including GSK726, restrict full reactivation. Mechanistically, we show that BET proteins (BRDs) are pivotally connected to regulation of HCMV latency and reactivation. Through BRD4 interaction, the transcriptional activator complex P-TEFb (CDK9/CycT1) is sequestered by repressive complexes during HCMV latency. Consequently, I-BETs allow release of P-TEFb and subsequent recruitment to promoters via the superelongation complex (SEC), inducing transcription of HCMV lytic genes encoding immunogenic antigens from otherwise latently infected cells. Surprisingly, this occurs without inducing many viral immunoevasins and, importantly, while also restricting viral DNA replication and full HCMV reactivation. Therefore, this pattern of HCMV transcriptional dysregulation allows effective cytotoxic immune targeting and killing of latently infected cells, thus reducing the latent virus genome load. This approach could be safely used to pre-emptively purge the virus latent reservoir prior to transplantation, thereby reducing HCMV reactivation-related morbidity and mortality.

scholarly journals Dysregulation of Cyclin E Gene Expression in Human Cytomegalovirus-Infected Cells Requires Viral Early Gene Expression and Is Associated with Changes in the Rb-Related Protein p130

2000 ◽  
Vol 74 (9) ◽  
pp. 4192-4206 ◽  
Author(s):  
Anita K. McElroy ◽  
Roopashree S. Dwarakanath ◽  
Deborah H. Spector
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Human Cytomegalovirus ◽  
Cyclin E ◽  
Early Gene ◽  
Immediate Early ◽  
Mrna Levels ◽  
Early Gene Expression ◽  
Cell Extracts ◽  
Infected Cells

ABSTRACT We have previously shown that many cell cycle regulatory gene products are markedly affected by infection of primary fibroblasts with human cytomegalovirus (HCMV) (F. M. Jault, J. M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697–6704, 1995). One of these proteins, cyclin E, is a key determinant of cell cycle progression during G1, and its mRNA levels are significantly increased in HCMV-infected fibroblasts (B. S. Salvant, E. A. Fortunato, and D. H. Spector, J. Virol. 72:3729–3741, 1998). To determine the molecular basis of this effect, we have examined the events that occur at the endogenous cyclin E promoter during the course of infection. In vivo dimethyl sulfate footprinting of the cyclin E promoter revealed several regions of protection and hypersensitivity that were unique to infected cells. In accord with this observation, we find that the virus-induced cyclin E transcripts initiate downstream of the start site identified in mock-infected cells, in regions where these newly appearing protected and hypersensitive sites occur. Viral gene expression is required for this induction. However, the viral immediate-early proteins IE1-72 and IE2-86, either alone or in combination, cannot induce expression of the endogenous cyclin E. The virus must progress past the immediate-early phase and express an early gene product(s) for activation of cyclin E expression. Moreover, IE1-72 does not appear to be required, as infection of cells with an HCMV mutant containing a deletion in the IE1-72 gene leads to full upregulation of cyclin E expression. Using electrophoretic mobility shift assays with infected cell extracts and a region of the cyclin E promoter that includes two previously defined E2F sites as the probe, we detected the appearance of an infection-specific banding pattern. One of the infection-specific bands contained the proteins E2F-4, DP-1, and p130, which were maintained in the infected cells as uniquely phosphorylated species. These results suggest that an altered E2F-4–DP-1–p130 complex along with viral early gene expression may play a role in the transcriptional regulation of cyclin E mRNA during HCMV infection.

scholarly journals Human Cytomegalovirus Gene Expression Is Silenced by Daxx-Mediated Intrinsic Immune Defense in Model Latent Infections Established In Vitro

2007 ◽  
Vol 81 (17) ◽  
pp. 9109-9120 ◽  
Author(s):  
Ryan T. Saffert ◽  
Robert F. Kalejta
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Chromatin Structure ◽  
Lytic Replication ◽  
Immune Defense ◽  
Latent Infections ◽  
Abortive Infection ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT In addition to productive lytic infections, herpesviruses such as human cytomegalovirus (HCMV) establish a reservoir of latently infected cells that permit lifelong colonization of the host. When latency is established, the viral immediate-early (IE) genes that initiate the lytic replication cycle are not expressed. HCMV IE gene expression at the start of a lytic infection is facilitated by the viral pp71 protein, which is delivered to cells by infectious viral particles. pp71 neutralizes the Daxx-mediated cellular intrinsic immune defense that silences IE gene expression by generating a repressive chromatin structure on the viral major IE promoter (MIEP). In naturally latently infected cells and in cells latently infected in vitro, the MIEP also adopts a similar silenced chromatin structure. Here we analyze the role of Daxx in quiescent HCMV infections in vitro that mimic some, but not all, of the characteristics of natural latency. We show that in these “latent-like” infections, the Daxx-mediated defense that represses viral gene expression is not disabled because pp71 and Daxx localize to different cellular compartments. We demonstrate that Daxx is required to establish quiescent HCMV infections in vitro because in cells that would normally foster the establishment of these latent-like infections, the loss of Daxx causes the lytic replication cycle to be initiated. Importantly, the lytic cycle is inefficiently completed, which results in an abortive infection. Our work demonstrates that, in certain cell types, HCMV must silence its own gene expression to establish quiescence and prevent abortive infection and that the virus usurps a Daxx-mediated cellular intrinsic immune defense mechanism to do so. This identifies Daxx as one of the likely multiple viral and cellular determinants in the pathway of HCMV quiescence in vitro, and perhaps in natural latent infections as well.

scholarly journals Human Cytomegalovirus IE72 Protein Interacts with the Transcriptional Repressor hDaxx To Regulate LUNA Gene Expression during Lytic Infection

2010 ◽  
Vol 84 (14) ◽  
pp. 7185-7194 ◽  
Author(s):  
Matthew Reeves ◽  
David Woodhall ◽  
Teresa Compton ◽  
John Sinclair
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Direct Interaction ◽  
Lytic Infection ◽  
Early Gene ◽  
Productive Infection ◽  
Viral Promoters ◽  
Infected Cells ◽  
Sirna Knockdown ◽  
Interfering Rna

ABSTRACT A putative latency-associated transcript (LUNA) complementary to the human cytomegalovirus (HCMV) UL81-82 region previously identified in seropositive donors' monocytes is also expressed during lytic infection. Thus, the LUNA promoter is active during both lytic and latent infection. Consequently, the mechanisms regulating this promoter may provide further insight into factors that determine whether the outcome of HCMV infection is latent or lytic. By transfection, the LUNA promoter exhibited low but reproducible activity. Substantial activation by virus infection suggested that a viral factor was important for LUNA expression during lytic infection. IE72, a known transactivator of viral promoters, activated the LUNA promoter in cotransfection assays. Furthermore, coinfection with wild-type HCMV but not an IE72 deletion virus (CR208) also activated the LUNA promoter. Finally, diminished LUNA gene expression in CR208 virus-infected cells supported a role for IE72 in LUNA gene expression. The initial regulation of herpesvirus immediate-early gene expression is associated with proteins found at cellular nuclear domain 10 (ND10) bodies, such as PML, hDaxx, and ATRX. hDaxx transfection repressed LUNA promoter activity. Furthermore, we observed binding of hDaxx to the LUNA promoter, which was abrogated by IE72 gene expression via direct interaction. Finally, we show that small interfering RNA (siRNA) knockdown of the hDaxx interaction partner ATRX rescued LUNA gene expression in CR208-infected cells. Overall, these data show that hDaxx/ATRX-mediated repression of LUNA during lytic infection absolutely requires IE72 gene expression. It also suggests that the targeting of cellular factors by IE72 is important throughout the different phases of HCMV gene expression during productive infection.

scholarly journals Characterization of phenyl pyrimidine derivatives that inhibit cytomegalovirus immediate-early gene expression

2018 ◽  
Vol 26 ◽  
pp. 204020661876319 ◽  
Author(s):  
Koh-Hei Yamada ◽  
Ryuichi Majima ◽  
Toyofumi Yamaguchi ◽  
Naoki Inoue
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Early Gene ◽  
Immediate Early ◽  
Murine Cytomegalovirus ◽  
Viral Attachment ◽  
Varicella Zoster ◽  
Infected Cells

Background Previously, we established a reporter cell line for human cytomegalovirus and screened anti-human cytomegalovirus compounds using the cell line. In this study, we characterized one of the identified compounds, 2,4-diamino-6–(4-methoxyphenyl)pyrimidine (coded as 35C10). Methods 50% Effective concentrations (EC50s) and 50% cytotoxic concentrations (CC50s) of 35C10 and its derivatives in human fibroblasts were determined by X-gal staining of the cells infected with human cytomegalovirus Towne strain expressing β-galactosidase. Results EC50 and CC50 of 35C10 were 4.3 µM and >200 µM, respectively. Among several 35C10 derivatives, only one lacking 4-amino group of pyrimidine showed a similar EC50. 35C10 weakly inhibited murine cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. A “time of addition” experiment suggested that 35C10 inhibited an early phase of the infection. Although 35C10 did not inhibit viral attachment to the cells nor the delivery of viral DNA to the nuclei, it decreased the number of infected cells expressing immediate-early 1 and 2 (IE1/IE2) proteins. 35C10 also inhibited the activation of a promoter for TRL4 in the reporter cells upon human cytomegalovirus infection, but not in the same reporter cells transfected with a plasmid expressing IE2. Conclusion Our findings suggest that 35C10 is a novel compound that inhibits IE gene expression in human cytomegalovirus-infected cells.

scholarly journals Inactivating a Cellular Intrinsic Immune Defense Mediated by Daxx Is the Mechanism through Which the Human Cytomegalovirus pp71 Protein Stimulates Viral Immediate-Early Gene Expression

2006 ◽  
Vol 80 (8) ◽  
pp. 3863-3871 ◽  
Author(s):  
Ryan T. Saffert ◽  
Robert F. Kalejta
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Immediate Early Gene ◽  
Nuclear Body ◽  
Immune Defense ◽  
Early Gene ◽  
Immediate Early ◽  
Innate Immune Responses ◽  
Early Gene Expression ◽  
Infected Cells

ABSTRACT Human cytomegalovirus (HCMV) masterfully evades adaptive and innate immune responses, allowing infection to be maintained and periodically reactivated for the life of the host. Here we show that cells also possess an intrinsic immune defense against HCMV that is disarmed by the virus. In HCMV-infected cells, the promyelocytic leukemia nuclear body (PML-NB) protein Daxx silences viral immediate-early gene expression through the action of a histone deacetylase. However, this antiviral tactic is efficiently neutralized by the viral pp71 protein, which is incorporated into virions, delivered to cells upon infection, and mediates the proteasomal degradation of Daxx. This work demonstrates the mechanism through which pp71 activates viral immediate-early gene expression in HCMV-infected cells. Furthermore, it provides insight into how a PML-NB protein institutes an intrinsic immune defense against a DNA virus and how HCMV pp71 inactivates this defense.

scholarly journals Transcriptional Regulation of the Human Cytomegalovirus US11 Early Gene

1999 ◽  
Vol 73 (2) ◽  
pp. 863-870 ◽  
Author(s):  
Nha H. Chau ◽  
Cynthia D. Vanson ◽  
Julie A. Kerry
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Human Cytomegalovirus ◽  
Promoter Activity ◽  
Early Gene ◽  
Mrna Levels ◽  
Wild Type ◽  
Infected Cells ◽  
Wild Type Promoter

ABSTRACT The human cytomegalovirus (HCMV) US11 early gene encodes a protein involved in the down-regulation of major histocompatibility complex class I cell surface expression in HCMV-infected cells. Consequently, this gene is thought to play an important role in HCMV evasion of immune recognition. In this study, we examined the transcriptional regulation of US11 gene expression. Analysis of deletions within the US11 promoter suggests that two sequence elements are important for activation by the viral immediate-early (IE) proteins. Deletion of a CREB site located at −83 relative to the cap site resulted in a reduction in promoter activity to 50% of the wild-type level. Deletion of an additional ATF site immediately upstream of the TATA box resulted in abrogation of responsiveness to the IE proteins. To confirm the role of the CREB and ATF sites within the US11 promoter, mutagenesis of these two sites, both individually and in combination, was carried out. Results indicate that both the CREB element and the ATF site were required for full promoter activity, with the ATF site critical for US11 promoter activation. The loss of transcriptional activation correlated with a loss of cellular proteins binding to the mutated US11 promoter elements. In combination with the viral IE proteins, the HCMV tegument protein pp71 (UL82) was found to up-regulate the US11 promoter by six- to sevenfold in transient assays. These results suggest that pp71 may contribute to the activation of the US11 promoter at early times after infection. Up-regulation by pp71 required the presence of the CREB and ATF sites within the US11 promoter for full activation. The role of the ATF and CREB elements in regulating US11 gene expression during viral infection was then assessed. The US11 gene is not required for replication of HCMV in tissue culture. This property was exploited to generate US11 promoter mutants regulating expression of the endogenous US11 gene in the natural genomic context. We generated recombinant HCMV that contained the US11 promoter with mutations in either the CREB or ATF element or both regulating the expression of the endogenous US11 gene. Northern blot analysis of infected cell mRNA revealed that mutation of the CREB element reduced US11 mRNA expression to approximately 25% of that of the wild-type promoter, with identical kinetics of expression. Mutation of the ATF site alone reduced US11 mRNA levels to 6% of that of the wild-type promoter, with mRNA detectable only at 8 h after infection. Mutation of both the CREB and ATF elements in the US11 promoter reduced US11 gene expression to undetectable levels. These results demonstrate that the CREB and ATF sites cooperate to regulate the US11 promoter in HCMV-infected cells.

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