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.
Highlights
At 6 h postinfection, the EE system of infected cells is compacted around the cell center and accessible to the incoming endosomal flow, as demonstrated by 45 min internalization of transferrin (Figure 1A)
∆m138-murine CMV (MCMV) infected (MOI of 10, 6 hpi) Balb/3T3 cells were incubated for 45 min with 50 μg/mL of Tf-AF555, fixed, and stained with mAb against immediate-early 1 (IE1) protein of MCMV, which was visualized with AF680 -conjugated anti-mouse IgG1 . (B) Balb/3T3 cells were transfected with Murine Stem Cell retroviral vectors (MSCV) containing YFP-PXP40phox PI(3)P-binding module and
Data represent mean ± standard error of the mean (SEM) per cell (n = 10–15). (C) Western blot analysis of IE1, Vps34, and β-actin in the course of MCMV infection. (D) Triple immunofluorescence images of 6 h infected cells stained with anti-GM130, anti-Vps34, and anti-IE1, visualized with the appropriate fluorochrome-conjugated non-crossreactive secondary reagents
Summary
Cytomegaloviruses (CMV) are large DNA viruses that extensively reorganize infected cells. They arrange the membranous system of the cell into a large factory for the final packaging of nascent virions, known as the cytoplasmic assembly compartment (AC). This structure occupies an area as large as the nucleus and contains reorganized membranous organelles, which accumulate viral structural proteins required for the final packaging of nascent capsids into membrane-enveloped virions [1,2]. The establishment of the AC occurs sequentially throughout the CMV replication cycle [8,9] and involves the interaction of a large number of viral proteins with normal host-cell functions [3,5]
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