Human cytomegalovirus hijacks the autophagic machinery and LC3 homologs in order to optimize cytoplasmic envelopment of mature infectious particles.

Clémence Taisne,Audrey Esclatine,Marion Lussignol,Lina Mouna,Eva Hernandez,Arnaud Moris

doi:10.1038/s41598-019-41029-z

Abstract

During its life cycle, Human cytomegalovirus (HCMV) tightly modulates autophagy, a vesicular pathway allowing degradation and recycling of cellular components. To study the interplay between autophagy and the viral life cycle, we established various autophagy-deficient human fibroblastic cell lines. By knocking down the expression or activity of five autophagy-related proteins, we confirmed the proviral function that the autophagic machinery exerts on HCMV production. Using 3D reconstruction from confocal microscopy and electron microscopy, we demonstrated that lipidated LC3-positive vesicles accumulated at the viral assembly compartment (vAC). The vAC is a juxtanuclear ring-shaped structure containing several organelles and membranes, where assembly and final envelopment of HCMV particles occur. Two LC3 homologs, GABARAPL1 and GATE16, also accumulated during HCMV infection and were associated with the vAC, in proximity with fragmented Golgi stacks. Additionally, we observed the formation of a pre-assembly compartment (PrAC) in infected cells, which consists of a juxtanuclear structure containing both fragmented Golgi and LC3-positive vesicles. Finally, we showed that highly purified extracellular viral particles were associated with various autophagy proteins. Our results thus suggest that autophagy machinery participates to the final cytoplasmic envelopment of HCMV viral particles into the vAC and that autophagy-related proteins can be spotted in the virions.

Highlights

Human cytomegalovirus (HCMV) is one of the 8 Herpesviruses that can infect humans, along with Herpes Simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV) or Varicella Zoster virus (VZV)
We first established cell lines stably expressing a trans-dominant negative form of ATG4B (ATG4B C74A) or shRNA targeting the expression of LC3B, ATG5, BECN1 and ULK1, using transduction with different lentiviral vectors as described in the Methods section[22]
The inhibitory activity of ATG4B C74A was demonstrated by accumulation of the non lipidated form of LC3 (LC3-I) because ATG4B C74A both impairs LC3 lipidation (LC3-II formation) and sequesters LC3 from cytosol[23]

Summary

Introduction

Human cytomegalovirus (HCMV) is one of the 8 Herpesviruses that can infect humans, along with Herpes Simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV) or Varicella Zoster virus (VZV). HCMV infects many cell types, such as endothelial cells, macrophages or epithelial cells but its replication cycle is primarily studied in human fibroblasts In these cells, HCMV enters the cytoplasm by fusion with the plasma membrane and its nucleocapsid (NC) is targeted to the nucleus using the microtubule network. It is important to mention that lipidated LC3 can be inserted into single membrane compartments of the endolysosomal system, including LC3-associated phagocytosis, macropinocytosis or entosis[18,19] These processes are referred to as non-canonical autophagy and in that case, LC3 lipidation is independent from the upstream autophagic regulators, such as ULK1. We and others have observed that HCMV is able to regulate autophagy throughout the viral cycle[11,20,21] It initially activates the formation of autophagosomes to secondly block the fusion of autophagosomes with lysosomes and blocks the degradation step[10]. Invalidation of several autophagy proteins makes it possible to differentiate the specific role of a particular ATG protein or of the whole autophagic process

Methods

Results

Conclusion