Endosomal Phosphatidylinositol-3-Phosphate-Associated Functions Are Dispensable for Establishment of the Cytomegalovirus Pre-Assembly Compartment but Essential for the Virus Growth

Abstract

Murine cytomegalovirus (MCMV) initiates the stepwise establishment of the pre-assembly compartment (pre-AC) in the early phase of infection by the expansion of the early endosome (EE)/endosomal recycling compartment (ERC) interface and relocation of the Golgi complex. We depleted Vps34-derived phosphatidylinositol-3-phosphate (PI(3)P) at EEs by VPS34-IN1 and inhibited PI(3)P-associated functions by overexpression of 2xFYVE- and p40PX PI(3)P-binding modules to assess the role of PI(3)P-dependent EE domains in the pre-AC biogenesis. We monitored the accumulation of Rab10 and Evectin-2 in the inner pre-AC and the relocation of GM130-positive cis-Golgi organelles to the outer pre-AC by confocal microscopy. Although PI(3)P- and Vps34-positive endosomes build a substantial part of pre-AC, the PI(3)P depletion and the inhibition of PI(3)P-associated functions did not prevent the establishment of infection and progression through the early phase. The PI(3)P depletion in uninfected and MCMV-infected cells rapidly dispersed PI(3)P-bond proteins and reorganized EEs, including ablation of EE-to-ERC transport and relocation of Rab11 endosomes. The PI(3)P depletion one hour before pre-AC initiation and overexpression of 2xFYVE and p40PX domains neither prevented Rab10- and Evectin-2 accumulation, nor Golgi unlinking and relocation. These data demonstrate that PI(3)P-dependent functions, including the Rab11-dependent EE-to-ERC route, are dispensable for pre-AC initiation. Nevertheless, the virus growth was drastically reduced in PI(3)P-depleted cells, indicating that PI(3)P-associated functions are essential for the late phase of infection.

Early Endosomal Vps34-Derived Phosphatidylinositol-3-Phosphate Is Indispensable for the Biogenesis of the Endosomal Recycling Compartment

“…Endosomal recycling processes are crucial for many pathophysiological states, such as the biological behavior of tumor cells [95] or the assembly and egress of viruses [96,97]. Despite the growing knowledge of the physiology of endosomal recycling and ERC, many aspects of these processes remain to be elucidated.…”

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

“…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 ].…”

“…SNX3 remained the most important signature that can provide information about the envelopment organelle. Membranes containing SNX3 are highly enriched in the inner AC area of MCMV-infected cells [ 33 ]. This SNX has only the PX domain and binds to membranes via an interaction with phosphatidylinositol-3-phosphate [PI(3)P] [ 187 ], which is essential for its recruitment, as acute depletion of PI(3)P leads to complete dissociation of SNX3 from membranes [ 26 , 33 ].…”

“…Membranes containing SNX3 are highly enriched in the inner AC area of MCMV-infected cells [ 33 ]. This SNX has only the PX domain and binds to membranes via an interaction with phosphatidylinositol-3-phosphate [PI(3)P] [ 187 ], which is essential for its recruitment, as acute depletion of PI(3)P leads to complete dissociation of SNX3 from membranes [ 26 , 33 ]. Although PI(3)P membranes are dispensable for the biogenesis of AC, PI(3)P itself is essential for the release of infectious MCMV virions [ 33 ].…”

“…The distribution of PIs in the AC was studied in MCMV-infected cells using immunofluorescent PI-binding probes [ 33 ]. The inner AC membranes were enriched in PI(3)P [ 33 ], whereas the outer AC membranes were enriched in PI(4)P ( Figure 8 A), although PI(4)P membranes could also be found in the inner AC area. This distribution is consistent with the distribution of other membrane organelle markers [ 26 ].…”