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. 2018 Nov:524:97-105.
doi: 10.1016/j.virol.2018.08.008. Epub 2018 Aug 27.

Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins

Sonia Zicari  1 , Anush Arakelyan  1 , Rogers Alberto Ñahui Palomino  1 , Wendy Fitzgerald  1 , Christophe Vanpouille  1 , Anna Lebedeva  2 , Alain Schmitt  3 , Morgane Bomsel  4 , William Britt  5 , Leonid Margolis  6
Affiliations

Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins

Sonia Zicari et al. Virology. 2018 Nov.

Abstract

Extracellular vesicles (EVs) released by virus-infected cells typically incorporate host and viral components inside the vesicles (cargo molecules). Here, we investigated if human cytomegalovirus (HCMV) proteins are incorporated in EV outer membrane released by HCMV-infected cells. We separated EVs from HCMV using an iodixanol step-gradient and found that the separated vesicles carried EV markers such as the tetraspanin CD63 and Rab27A. Flow analysis of individual EVs demonstrated that on average, 15 ± 3.7% of EVs were positive for gB, 5.3 ± 2.3% were positive for gH and 3.74 ± 1.5% were positive for both gB and gH. In light of previous findings demonstrating HIV envelope proteins in EV membranes, the presence of viral protein at the surface of EVs released by HCMV-infected cells indicated that viral membrane proteins incorporated in EVs released by virus-infected cells may be a general phenomenon.

Keywords: Extracellular vesicles; HCMV; gB; gH.

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Conflict of interest statement

Conflict of interests

The authors declare that they have no competing financial interests.

Figures

Fig. 1.
Fig. 1.. Characterization of EVs in the EV fraction.
(A) HCMV DNA quantification in iodixanol fractions. After separation on a discontinuous iodixanol gradient using 1 ml of UL32-EGFP-HCMV as input (insert), the amount of virus present in each fraction was evaluated with qPCR and presented as a percentage of the viral input. (B) NanoSight analysis of HCMV preparation (left panel) and of isolated EV fraction (right panel). (C) Flow analysis of single EVs. DiI-stained EVs were serially diluted two-fold from 1:2–1:1024. The events were acquired in an LSRII flow cytometer, set to be triggered by DiI fluorescence. Presented are the numbers of events as a function of the dilution factor (left panel) and the median fluorescence intensity (MFI) of each dilution (right panel).
Fig. 2.
Fig. 2.. EVs carry HCMV surface proteins.
DiI-labeled EVs isolated from UL32-EGFP-HCMV were stained with anti-gB AF647 and anti-gH PB antibodies or with their isotype controls. (A) Visualization of EVs as DiI-positive/GFP-negative events. (B) EVs stained with anti-gB AF647 antibodies or (C) with anti-gH PB antibodies. (D) Distribution of viral antigens gB and gH on EVs isolated from UL32-EGFP-HCMV viral preparation. Presented are means ( ± SEM) of EVs evaluated in three experiments. (E) Isotype control to B. (F) Isotype control to C. (G–J) DiI-labeled EVs, isolated from supernatant of control uninfected cells, were stained with anti-gB AF647 antibodies and anti-gH PB antibodies or with their isotype controls. (G) EV fraction stained with anti-gB AF647 antibodies. (H) EV fraction stained with anti-gH PB antibodies. (I) Isotype control to G. (J) Isotype control to H. (K-N) Detection of HCMV minor capsid protein UL-85 in EVs and HCMV by flow cytometry. EV fraction (left panel) or HCMV fraction (right panel) were stained with (upper row) or with its corresponding isotype control (lower row). (K) DiI+GVP- particles (EVs) stained with anti UL-85-AF647 antibody (L) DiI+GFP+ particles (HCMV) stained with anti UL-85-AF647 antibody. (M) Isotype control to K. (N) Isotype control to L.
Fig. 2.
Fig. 2.. EVs carry HCMV surface proteins.
DiI-labeled EVs isolated from UL32-EGFP-HCMV were stained with anti-gB AF647 and anti-gH PB antibodies or with their isotype controls. (A) Visualization of EVs as DiI-positive/GFP-negative events. (B) EVs stained with anti-gB AF647 antibodies or (C) with anti-gH PB antibodies. (D) Distribution of viral antigens gB and gH on EVs isolated from UL32-EGFP-HCMV viral preparation. Presented are means ( ± SEM) of EVs evaluated in three experiments. (E) Isotype control to B. (F) Isotype control to C. (G–J) DiI-labeled EVs, isolated from supernatant of control uninfected cells, were stained with anti-gB AF647 antibodies and anti-gH PB antibodies or with their isotype controls. (G) EV fraction stained with anti-gB AF647 antibodies. (H) EV fraction stained with anti-gH PB antibodies. (I) Isotype control to G. (J) Isotype control to H. (K-N) Detection of HCMV minor capsid protein UL-85 in EVs and HCMV by flow cytometry. EV fraction (left panel) or HCMV fraction (right panel) were stained with (upper row) or with its corresponding isotype control (lower row). (K) DiI+GVP- particles (EVs) stained with anti UL-85-AF647 antibody (L) DiI+GFP+ particles (HCMV) stained with anti UL-85-AF647 antibody. (M) Isotype control to K. (N) Isotype control to L.
Fig. 3.
Fig. 3.. EVs captured by anti-gB-magnetic nano-particles.
DiI labeled EVs were captured by AF488-labeled anti-gB-magnetic nanoparticles (MNPs). A typical experiment out of three is shown. (A) DiI-labeled EVs released by HCMV-infected cells and captured by AF488labeled anti-gB-MNPs; thresholding on DiI fluorescence. (B) DiI-labeled EVs released by HCMV-infected cells captured by AF488-la-beled anti-gB-MNPs; thresholding on AF488 fluorescence. (C) DiI-labeled EVs released by control uninfected cells and captured by AF488-labeled anti-gB-MNPs; thresholding on AF488 fluorescence. (D) Visualization in transmission electron microscopy of EVs from HCMV-infected cells captured by anti-gB-MNPs. Note the excess of MNPs that according to the protocol (Arakelyan et al., 2013) should be applied to capture virions and exclude their aggregation.
Fig. 4.
Fig. 4.. Detection of HCMV and EV markers in HCMV and EV fractions by western blot.
(A) stain-free blot representing the spectrum of proteins obtained for HCMV and EV fractions after separation of AD169 HCMV pre-paration using a iodixanol step-gradient centrifugation. (B) Detection of EV-associated proteins CD63 (~60 kDa), Rab27A (~30 kDa), and calnexin (~25 kDa) proteins in both fractions. (C) Detection of HCMV capsid protein MCP (~150 kDa) and HCMV tegument protein, pp150 (~160 kDa) in HCMV and EV fractions after separation of AD169 viral preparation using a iodixanol step-gradient centrifugation.
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