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. 2019 Mar 14;9(1):4560.
doi: 10.1038/s41598-019-41029-z.

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

Clémence Taisne  1 , Marion Lussignol  1 , Eva Hernandez  1 , Arnaud Moris  1   2 , Lina Mouna  1   3 , Audrey Esclatine  4
Affiliations

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

Clémence Taisne et al. Sci Rep. .

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Several autophagic proteins are beneficial for HCMV production. Stable HFF cell lines either expressing a dominant-negative mutant of ATG4B or knockdown for the indicated autophagic proteins (LC3B, ATG5, BECN1 or ULK1) and their corresponding control cell lines (WT: wild type HFF, shSCR: scrambled shRNA, shNT: non-target shRNA) have been used. (A) Immunoblot analysis of p62, LC3B, ATG5, BECN1 and ULK1 expression confirms the inhibition of autophagy in the indicated cell lines. Actin was used as a loading control. Full-length blots are presented in Fig. S7 (B) Viral titers were determined 10 days post-infection with HCMV AD169 strain at MOI 0.01 in the different cell lines. Error bars indicate SEM from at least three independent experiments. *p < 0.05 (Student’s t test).
Figure 2
Figure 2
Vesicles containing lipidated form of LC3 accumulate in the vAC. Confocal images of HFF mock-infected (Mock) or infected with HCMV AD169 strain at MOI 0.5 for 4 days and immunostained for LC3 and (A) pp28 (viral tegument protein), (B) GM130 (Golgi marker), (C) EEA1 (early endosome marker), or (D) LAMP1 (late endosome/lysosome marker). Representative images of 3 independent experiments. See also supplementary videos S2-4. (E) Confocal images of cells stably expressing p41-LC3 or p41-LC3 G120A infected with HCMV at MOI 0.5 for 4 days and immunostained for exogenous LC3 with pp28 or GM130. (F) Representative images of WT HFF infected with HCMV at MOI 0.5 for 4 days, and permeabilized either after fixation (“normal”) or before fixation (“depleted”) to eliminate soluble cytosolic proteins. Cells were immunostained for LC3 and GM130. Nuclei were subsequently stained with DAPI. Scale bar = 10 µm.
Figure 3
Figure 3
Formation of a pre-assembly compartment (PrAC) containing Golgi stacks and LC3 starts early in the viral life cycle. (A,B) Representative images of HFF infected with HCMV AD169 strain at MOI 1 during 10 to 48 hours and immunostained for IEA (immediate-early viral antigens) and for GM130 (A) or LC3 (B). Nuclei were subsequently stained with DAPI. Representative images of 2 independent experiments. Scale bars = 10 µm.
Figure 4
Figure 4
Roles of the autophagy machinery in different steps of the viral cycle. (A) Quantification of HCMV entry by immunofluorescence detection of the viral tegument protein pp65 2 h after contact with the virus (MOI 1). DAPI was used to stain nuclei. Percentages of pp65-positive cells correspond to viral entry into the indicated cell lines compared to control cells (deficient: Autophagy-deficient). (B) Quantification of HCMV genome replication 4 dpi at MOI 0.5 by real time PCR in the indicated cell lines, compared to control cells. (C) Expression of several viral proteins after HCMV infection of control and autophagy-deficient cell lines at MOI 0.5 for 1, 2 or 5 days post infection (dpi). Actin was used as a loading control. Full-length blots are presented in Fig. S7. (D) Indicated control and deficient cells were infected at MOI 0.5 with HCMV AD169 strain. Cell-associated viruses were collected 4 dpi and the amount of infectious virus was measured. Results represent the mean values of 3 independent experiments. ns: non-significant (Student’s t test).
Figure 5
Figure 5
ATG8 homologs accumulate in the vAC in their lipidated form. (A,B) Representative images of HFF cells infected with HCMV AD169 strain at MOI 0.5 for 4 days and immunostained for (A) GABARAPL1, and pp28 (viral tegument protein), GM130 or LC3; (B) GATE16 and pp28, GM130 or LC3 (NI: non-infected cells) (C) Immunoblot analysis of GABARAPL1 and GATE16 expression in HCMV-infected cells at the indicated times after infection. Autophagic flux was measured by addition of chloroquine (CQ) 4 h before lysis. Actin was used as a loading control. Full-length blots are presented in Fig. S7 (D) Confocal images of autophagy-deficient cells stably expressing ATG4B C74A infected with HCMV at MOI 0.5 for 4 days and immunostained for GM130 (Golgi) with LC3 or GABARAPL1 or GATE16. (*) non-transduced cells. Nuclei were subsequently stained with DAPI. Scale bars = 10 µm.
Figure 6
Figure 6
Autophagic proteins accumulate during infection and are present in extracellular viral particles. (A) Immunoblot analysis of cellular proteins contained in purified extracellular viral particles. Ultra (ultracentrifuged supernatant); Purif (purified virus by tartrate/glycerol gradient centrifugation). Supernatant of mock-infected cells was ultracentrifuged and used as a negative control and cell lysate are used as a positive control. (B) Immunoblot analysis of ATG5 and BECN1 expression in HCMV-infected cells at the indicated times after infection. Actin was used as a loading control. Full-length blots are presented in Fig. S7. (C) Representative images of HFF cells infected with HCMV AD169 strain at MOI 0.5 for 4 days and immunostained for p62 and IRS1/TRS1 (viral proteins) or LC3. Nuclei were subsequently stained with DAPI. Scale bars = 10 µm.
Figure 7
Figure 7
HCMV uses autophagic membranes for its assembly. (A) Transmission electron micrographs of HFF infected by HCMV at MOI 0.5 during four days. Inserts showed a) autophagosome, b) tegumented nucleocapsid envelopment c) enveloped virus in a vesicle. Insert scale bars = 0.1 µm. (B,C) Model based on our data and prior confocal and ultrastructural analyses. (B) Schematic representation of vAC structure and localization of LC3/GABARAP-positive vesicles in vAC with respect to cellular and viral components. HCMV could use membranes from different compartments and in particular autophagic vesicles for its final envelopment. (C) Schematic representation of HCMV envelopment. (1) The viral budding process involves a small double membrane crescent-shape structure which contains viral glycoproteins; (2) this double membrane elongates and fuses around the tegumented nucleocapsid to give rise to (3) an enveloped viral particle within a single-membrane vesicle. This process shares common points with phagophore and autophagosome formation.
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