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Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection.

ABSTRACT: Cytomegalovirus (CMV) infection initiates massive rearrangement of cytoplasmic organelles to generate assembly compartment (AC). The earliest events, the establishment of the preAC, are initiated in the early phase as an extensive reorganization of early endosomes (EEs), endosomal recycling compartment (ERC), trans-Golgi network (TGN), and the Golgi. Here, we demonstrate that dynamin inhibitors (Dynasore, Dyngo-4a, MiTMAB, and Dynole-34-2) block the establishment of the preAC in murine CMV (MCMV) infected cells. In this study, we extensively analyzed the effect of Dynasore on the Golgi reorganization sequence into the outer preAC. We also monitored the development of the inner preAC using a set of markers that define EEs (Rab5, Vps34, EEA1, and Hrs), the EE-ERC interface (Rab10), the ERC (Rab11, Arf6), three layers of the Golgi (GRASP65, GM130, Golgin97), and late endosomes (Lamp1). Dynasore inhibited the pericentriolar accumulation of all markers that display EE-ERC-TGN interface in the inner preAC and prevented Golgi unlinking and dislocation to the outer preAC. Furthermore, in pulse-chase experiments, we demonstrated that the presence of dynasore only during the early phase of MCMV infection (4-14 hpi) is sufficient to prevent not only AC formation but also the synthesis of late-phase proteins and virion production. Therefore, our results indicate that dynamin-2 acts as a part of the machinery required for AC generation and rearrangement of EE/ERC/Golgi membranes in the early phase of CMV infection.

SUBMITTER: Stimac I

PROVIDER: S-EPMC8469281 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection.

Štimac Igor I Jug Vučko Natalia N Blagojević Zagorac Gordana G Marcelić Marina M Mahmutefendić Lučin Hana H Lučin Pero P

Life (Basel, Switzerland) 20210825 9

Cytomegalovirus (CMV) infection initiates massive rearrangement of cytoplasmic organelles to generate assembly compartment (AC). The earliest events, the establishment of the preAC, are initiated in the early phase as an extensive reorganization of early endosomes (EEs), endosomal recycling compartment (ERC), trans-Golgi network (TGN), and the Golgi. Here, we demonstrate that dynamin inhibitors (Dynasore, Dyngo-4a, MiTMAB, and Dynole-34-2) block the establishment of the preAC in murine CMV (MCMV ...[more]

PMID: 34575026

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Project description:IntroductionCytomegalovirus (CMV) infection reorganizes early endosomes (EE), recycling endosome (RE), and trans-Golgi network (TGN) and expands their intermediates into a large perinuclear structure that forms the inner part of the cytoplasmic assembly complex (AC). The reorganization begins and results with the basic configuration (known as pre-AC) in the early (E) phase of infection, but the sequence of developmental steps is not yet well understood. One of the first signs of the establishment of the inner pre-AC, which can be observed by immunofluorescence, is the accumulation of Rab10. This study aims to investigate whether Rab10-positive domain (Rab10-PD) is expanded during the E phase of infection.MethodsWe performed long-term live imaging of EGFP-Rab10 with epifluorescence imaging-enhanced digital holotomographic microscopy (DHTM), confocal imaging of known Rab10 interactors and identification of important Rab10 interactors with the proximity-dependent biotin identification assay (BioID). The accumulation of Rab10-PD was analyzed after knock-down of EHBP1 and Rabin8, two proteins that facilitate Rab10 recruitment to membranes, and after blocking of PI(4,5)P2 by PI(4,5)P2-binding protein domains.ResultsOur study shows the gradual expansion of Rab10-PD in the inner pre-AC, the association of Rab10 with EHBP1 and MICAL-L1, and the dependence of Rab10-PD expansion on EHBP1 and PI(4,5)P2 but not Rabin8, indicating the expansion of EE-derived tubular recycling endosome-like membranes in the pre-AC. Silencing of Rab10 and EHBP1 suggests that Rab10-PD expansion is not required for the establishment of the inner pre-AC nor for the expansion of downstream tubular domains.ConclusionThe present work characterizes one of the earliest sequences in the establishment of pre-AC and suggests that subsets of EE-derived tubular membranes may serve as the earliest biomarkers in pre-AC biogenesis. Our study also indicates that the pre-AC biogenesis is complex and likely involves multiple parallel processes, of which Rab10-PD expansion is one. Our experiments, particularly our silencing experiments, show that Rab10 and EHBP-1 do not play a significant role in the later stages of inner pre-AC biogenesis or in the expansion of downstream tubular domains. A more comprehensive understanding of the tubular domain expansion remains to be established.

Project description:BackgroundPrimary cytomegalovirus (CMV) infection during pregnancy carries a risk of congenital infection and possible severe sequelae. There is no established intervention for preventing congenital CMV infection.MethodsIn this multicenter, double-blind trial, pregnant women with primary CMV infection diagnosed before 24 weeks' gestation were randomly assigned to receive a monthly infusion of CMV hyperimmune globulin (at a dose of 100 mg per kilogram of body weight) or matching placebo until delivery. The primary outcome was a composite of congenital CMV infection or fetal or neonatal death if CMV testing of the fetus or neonate was not performed.ResultsFrom 2012 to 2018, a total of 206,082 pregnant women were screened for primary CMV infection before 23 weeks of gestation; of the 712 participants (0.35%) who tested positive, 399 (56%) underwent randomization. The trial was stopped early for futility. Data on the primary outcome were available for 394 participants; a primary outcome event occurred in the fetus or neonate of 46 of 203 women (22.7%) in the group that received hyperimmune globulin and of 37 of 191 women (19.4%) in the placebo group (relative risk, 1.17; 95% confidence interval [CI] 0.80 to 1.72; P = 0.42). Death occurred in 4.9% of fetuses or neonates in the hyperimmune globulin group and in 2.6% in the placebo group (relative risk, 1.88; 95% CI, 0.66 to 5.41), preterm birth occurred in 12.2% and 8.3%, respectively (relative risk, 1.47; 95% CI, 0.81 to 2.67), and birth weight below the 5th percentile occurred in 10.3% and 5.4% (relative risk, 1.92; 95% CI, 0.92 to 3.99). One participant in the hyperimmune globulin group had a severe allergic reaction to the first infusion. Participants who received hyperimmune globulin had a higher incidence of headaches and shaking chills while receiving infusions than participants who received placebo.ConclusionsAmong pregnant women, administration of CMV hyperimmune globulin starting before 24 weeks' gestation did not result in a lower incidence of a composite of congenital CMV infection or perinatal death than placebo. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Center for Advancing Translational Sciences; ClinicalTrials.gov number, NCT01376778.).

Project description:BackgroundTriplex vaccine was developed to enhance cytomegalovirus (CMV)-specific T cells and prevent CMV reactivation early after hematopoietic stem cell transplant (HCT).ObjectiveTo determine the safety and efficacy of Triplex.DesignFirst-in-patient, phase 2 trial. (ClinicalTrials.gov: NCT02506933).Setting3 U.S. HCT centers.Participants102 CMV-seropositive HCT recipients at high risk for CMV reactivation.InterventionIntramuscular injections of Triplex or placebo were given on days 28 and 56 after HCT. Triplex is a recombinant attenuated poxvirus (modified vaccinia Ankara) expressing immunodominant CMV antigens.MeasurementsThe primary outcomes were CMV events (CMV DNA level ≥1250 IU/mL, CMV viremia requiring antiviral treatment, or end-organ disease), nonrelapse mortality, and severe (grade 3 or 4) graft-versus-host disease (GVHD), all evaluated through 100 days after HCT, and grade 3 or 4 adverse events (AEs) within 2 weeks after vaccination that were probably or definitely attributable to injection.ResultsA total of 102 patients (51 per group) received the first vaccination, and 91 (89.2%) received both vaccinations (46 Triplex and 45 placebo). Reactivation of CMV occurred in 5 Triplex (9.8%) and 10 placebo (19.6%) recipients (hazard ratio, 0.46 [95% CI, 0.16 to 1.4]; P = 0.075). No Triplex recipient died of nonrelapse causes during the first 100 days or had serious AEs, and no grade 3 or 4 AEs related to vaccination were observed within 2 weeks after vaccination. Incidence of severe acute GVHD after injection was similar between groups (hazard ratio, 1.1 [CI, 0.53 to 2.4]; P = 0.23). Levels of long-lasting, pp65-specific T cells with effector memory phenotype were significantly higher in Triplex than placebo recipients.LimitationThe lower-than-expected incidence of CMV events in the placebo group reduced the power of the trial.ConclusionNo vaccine-associated safety concerns were identified. Triplex elicited and amplified CMV-specific immune responses, and fewer Triplex-vaccinated patients had CMV viremia.Primary funding sourceNational Cancer Institute and Helocyte.

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Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection.

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Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection.

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