Introduction: Cytomegalovirus (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.Methods: We 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.Results: Our 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.Conclusion: The 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.

Cilj: Prikazati pacijenta s porodičnom hiperkolesterolemijom (engl. familial hypercholesterolemia; FH) i panhipopituitarizmom kod kojeg je unatoč trojnoj hipolipemijskoj terapiji ciljna vrijednost LDL kolesterola gotovo postignuta tek nakon uvođenja...

Background: During infection with the cytomegalovirus (CMV), the membrane system of the infected cell is remodelled into a megastructure called the assembly compartment (AC). These extensive changes may involve the manipulation of the host cell proteome by targeting a pleiotropic function of the cell such as ubiquitination (Ub). In this study, we investigate whether the Ub system is required for the establishment and maintenance of the AC in murine CMV (MCMV)-infected cells Methods: NIH3T3 cells were infected with wild-type and recombinant MCMVs and the Ub system was inhibited with PYR-41. The expression of viral and host cell proteins was analyzed by Western blot. AC formation was monitored by immunofluorescence with confocal imaging and long-term live imaging as the dislocation of the Golgi and expansion of Rab10-positive tubular membranes (Rab10 TMs). A cell line with inducible expression of hemagglutinin (HA)-Ub was constructed to monitor ubiquitination. siRNA was used to deplete host cell factors. Infectious virion production was monitored using the plaque assay. Results: The Ub system is required for the establishment of the infection, progression of the replication cycle, viral gene expression and production of infectious virions. The Ub system also regulates the establishment and maintenance of the AC, including the expansion of Rab10 TMs. Increased ubiquitination of WASHC1, which is recruited to the machinery that drives the growth of Rab10 TMs, is consistent with Ub-dependent rheostatic control of membrane tubulation and the continued expansion of Rab10 TMs. Conclusions: The Ub system is intensively utilized at all stages of the MCMV replication cycle, including the reorganization of the membrane system into the AC. Disruption of rheostatic control of the membrane tubulation by ubiquitination and expansion of Rab10 TREs within the AC may contribute to the development of a sufficient amount of tubular membranes for virion envelopment.