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.

The human cytomegalovirus decathlon: Ten critical replication events provide opportunities for restriction

“…Recent work has suggested that there are multiple populations of heterogeneous vesicles clustering within the vAC, potentially containing distinct microdomains (Zeltzer et al, 2018), consistent with the extensive re-modelling of host organelles throughout infection (Jean Beltran et al, 2016). Organelle remodelling and vAC formation is also observed in murine CMV (MCMV) infected cells, with all the same features as HCMV (Lučin et al, 2020;Marcelić et al, 2021;Pavišić et al, 2021;Štimac et al, 2021), showing important conservation. Observations of HCMV infection by live cell imaging has provided unprecedented insights into the dynamic nature of this structure (Procter et al, 2018).…”

“…Examples include the Golgi residents syntaxin 5 (STX5) (Cruz et al, 2017) and Golgi reassembly stacking protein 65 kD (GRASP65) (Rebmann et al, 2016), ER chaperone BiP (Buchkovich et al, 2008;Buchkovich et al, 2009), and nuclear WDR5 that translocates to the vAC during HCMV infection (Yang et al, 2021;Yang et al, 2022). Proper functioning of the host endocytic system is required for vAC formation (Archer et al, 2017), including dynamin (Hasan et al, 2018;Štimac et al, 2021). Endocytic involvement is further supported by the localisation and requirement for ADP ribosylation factor 1 (ARF1), ARF3,…”

Host Cell Signatures of the Envelopment Site within Beta-Herpes Virions

“…Secondary envelopment occurs within the AC, a typical large cytoplasmic membranous organelle cluster established in beta-herpesvirus-infected cells ( Figure 1 A), as demonstrated in HCMV [ 19 , 20 , 21 , 22 , 23 ] and MCMV [ 24 , 25 , 26 , 27 ] studies and presented in several reviews [ 1 , 2 , 3 ]. The inner area (inner AC) contains many vesicular and tubular elements derived from early endosomes (EEs), the endosomal recycling compartment (ERC), and the trans-Golgi network (TGN).…”

“…These organelles are surrounded by several layers of membranous elements (outer AC), which contain the modified Golgi stacks and trans-Golgi elements that project toward the inner area. The inner/outer AC configuration is established early in infection [ 21 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ] and is initiated by the extensive displacement of the Golgi from the cell center and its realignment in such a way that the trans side accumulates and expands around the cell center along with the EE-ERC-derived elements [ 21 , 24 , 26 ]. The endoplasmic reticulum (ER), late endosomes (LEs), and lysosomes appear to be relocated to the cell periphery [ 1 , 2 , 19 , 20 , 26 ], but it is unclear whether ER- and LE-derived elements contribute to the inner AC [ 19 , 20 ].…”

“…Similarly, clathrin heavy chains and dynamin 2 were highly enriched in most of the virion preparations [ 10 , 11 , 12 ] but also in l EVs, s EVs, and NVEPs [ 16 ] (data not shown). Nevertheless, the AP complexes, clathrin, and dynamin may play an important role in CMV assembly as they are enriched at the membranes of the inner AC of MCMV- [ 24 , 26 ] and HCMV- [ 29 , 163 , 177 ] infected cells.…”