Human immunoglobulins are transported to HCMV viral envelope by viral Fc gamma receptors-dependent and independent mechanisms

Abstract

Human cytomegaloviruses (HCMVs) employ many different mechanisms to escape and subvert the host immune system, including expression of the viral IgG Fcγ receptors (vFcγRs) RL11 (gp34), RL12 (gp95), RL13 (gpRL13), and UL119 (gp68) gene products. The role of vFcγRs in HCMV pathogenesis has been reported to operate in infected cells by interfering with IgG-mediated effector functions. We found that gp34 and gp68 are envelope proteins that bind and internalize human IgGs on the surface of infected cells. Internalized IgGs are then transported on the envelope of viral particles in a vFcR-dependent mechanism. This mechanism is also responsible for the incorporation on the virions of the anti-gH neutralizing antibody MSL-109. Intriguingly, we show that gp68 is responsible for MSL-109 incorporation, but it is dispensable for other anti-HCMV antibodies that do not need this function to be transported on mature virions. HCMV-infected cells grown in presence of anti-HCMV monoclonal antibodies generate a viral progeny still infective and possible to be neutralized. This is the first example of a virus carrying neutralizing IgGs on its surface and their possible role is discussed.

Keywords: HCMV neutralizing antibodies; gp34 (RL11); gp68 (UL119); gp95 (RL12); human IgGs; human cytomegalovirus; viral Fcγ-binding proteins.

Figure 1

gp68 and gp34 localize within the viral assembly complex in HCMV-infected cells and colocalize with human Fc. (A) HFF-1 cells were infected with TR-UL119Flag and TR-RL11HA (top and bottom rows, of the two panels, respectively). 5 d.p.i. cells were fixed, permeabilized, and stained. gp68 and gp34 (green color) were detected with anti-tag antibodies. Envelope glycoprotein gL and trans-Golgi network protein TGN46 were stained with antibodies (red color). Z-stacks were collected with a confocal microscope. Representative single confocal slices of each sample are shown. Scale bars: 5 μm. (B) HFF-1 were infected with TR-UL119Flag or TR-RL11HA. 2 d.p.i. 6.6 μg/ml of 649 Dylight-conjugated human IgG Fc fragment was added to the culture media. After 48 h cells were washed, fixed and stained with anti-tag antibodies (green color). Images were collected through confocal microscopy. Single confocal slices of representative pictures are shown. Superimposition of signals from human Fc (Fc, red color), DAPI stained nuclei (blue color), and anti-tags antibodies are shown in the merge panels. Scale bars: 5 μm.

Figure 2

Endocytosed human Fc fragment is retained in the early endosomes. (A) HFF-1 were infected with HCMV TR strain. Five d.p.i. cells were incubated with 647 DyLight conjugated human IgG Fc fragment on ice for 60 min before being washed and shifted to 37°C to allow for internalization. At the indicated time points, cells were fixed and stained with antibodies against early endosomes and/or lysosomes (green color, EEA1 and Lamp1, respectively). Images were collected through confocal microscopy. Z-stack projection of representative pictures is shown. Superimposition of signals from human Fc (Fc, red color), cellular markers are shown in the merge panels. Scale bar: 5 μm. White arrows indicate signals overlapping. (B) Quantification of data from (A). Z-stacks have been deconvolved and Fc-positive objects have been segmented. For each of them, Pearson’s colocalization coefficients (PCC) between Fc signal and endosomes (EEA1) or lysosomes (Lamp1) was calculated. Each dot corresponds to an Fc-positive object. N = number of cells analyzed for each condition. Numbers in green represent the percentage of Fc-positive vesicles colocalizing with the respective cellular marker (Pearson’s coefficient ≥ 0.5).

Figure 3

HFF-1 were infected with TR-UL119_YFP and 2 d.p.i incubated with 6.6 μg/ml of human anti-gH antibody MSL-109 for additional 3 days before being fixed, permeabilized, and stained. Secondary antibody Alexa fluor 647 conjugated anti-human was used to detect human antibodies (Red). Lysosomes were stained with anti-Lamp1 antibody. YFP and lamp1 signals are shown in green (A,B panels, respectively). Signal intensity profiles along the dotted lines in the zoom panel are shown on the right. Scale bar: 5 μm.

Figure 4

gp68 and gp34, but no gp95, are envelope proteins. (A) HFF-1 cells were infected at MOI 1 with either HCMV TR-UL119Flag or RL11HA in duplicate. Human Fcγ fragments were added to one sample for each virus while the other was grown in normal medium. At day 6 p.i., supernatants were harvested, virions purified through a glycerol/tartrate density gradient and tegument/capsid fraction (Pellet) was separated from envelope proteins (Soluble) through triton X-114 procedure. Lysates of infected cells and fractions of purified virions were analyzed through western blot using antibodies against gL, pp65, human IgG, anti-Flag, or anti-HA. (B) HFF-1 cells were infected at MOI 1 with HCMV TR-RL12Flag. Infected cells were incubated with human Fcs as described above. At day 6 p.i., supernatants were harvested, and virions were purified through a glycerol/tartrate density gradient. Tegument/capsid fraction (Pellet) was separated from envelope proteins (soluble) through detergent extraction. Lysates of infected cells and fractions of purified virions were analyzed through western blot using indicated antibodies.

Figure 5

gp68 is the main protein responsible for transporting human Fc and IgGs on virions. HFF-1 cells were infected with HCMV TR-wt, TR-UL119-null, TR-ΔRL11-12-13, and TR-vFcγRs-null mutant. 48 hpi, 6.6 μg/ml of human Fcγ (A) or 13.2 μg/ml of recombinant human IgGs (B) were added to the culture media. At day 4 pi medium was replaced with fresh DMEM 15% FBS and supernatant was collected at 6 dpi. Viral particles were purified through a sucrose cushion. Pelleted viruses were treated for SDS-PAGE and western blot analysis. Anti-human HRP-conjugated IgG antibody was used to reveal Fcs (A) and IgGs (B). Anti-pp65 was used as markers of viral tegument. Anti-GAPDH was used to exclude cellular contaminations.

Figure 6

Monoclonal anti HCMV glycoproteins are transported on virions by vFcγRs dependent and independent mechanisms. HFF-1 cells were infected with HCMV TR-wt or TR-vFcRnull. At day 2 p.i., human monoclonal antibodies (indicated at the bottom of the figure) were added to the culture media. After incubation at 37°C for 48 h, cells were washed and incubated with fresh medium for additional 48 h. Pelleted viruses were treated with SDS-PAGE and western blot analysis. Human IgG heavy chains (middle panel) were revealed with HRP-conjugated anti-human IgG antibody. Anti-gB (upper panel) and anti-GAPDH (bottom panel) were used as viral protein marker and to exclude cellular contaminations, respectively.

Figure 7

Neutralization assays on untreated and mAbs-coated TRvFcγRs-null and parental strain. HFF-1 were infected with either TR-wt or TR-vFcγRs-null at an MOI of 1 and infection carried on for 48 h before adding 13.2 μg/ml of the mAb as indicated in the legend. Medium was changed at day 4 p.i. and replaced with fresh medium. At day 6 p.i., culture media were collected, cleared from cellular debris, and viral genomes quantified by Digital PCR. Fresh HFF-1 were infected at MOI 1 with viruses untreated or pre-incubated with the mAb indicated on the x-axis. Antibodies used for neutralization are specified on each bar. Percentage of infected cells was calculated 24 hpi by cytofluorimetry using an anti-CMV 488-conjugated antibody.

Figure 8

Neutralization assay on mAbs-coated viruses with anti-human antibodies. TR-wt and TR-vFcγRsnull coated with the mAb indicated on each bar and untreated were prepared as described in legend of Figure 7. Viral genomes were quantified by Digital PCR and fresh HFF-1 infected at MOI 1 with viruses pre-incubated with anti-human antibodies. Percentage of infected cells was calculated by cytofluorimetry using an anti-CMV 488-conjugated antibody.

Figure 9

(1) HCMV-infected cells in absence of antibodies, produce viruses that can be neutralized by anti-HCMV specific IgGs, but not by anti-human IgG. (2) In presence of human IgGs, antibodies are internalized via gp68 and incorporated on the virion envelope with their Fc portion bound to the vFcR. Virions harboring nonspecific IgGs on their surface can be neutralized by anti-HCMV antibodies but not by anti-human IgGs. (3) The anti-gH neutralizing antibody MSL-109 can also be transported via gp68 on the virion surface. In this scenario, the virions are resistant to further neutralization by MSL-109, but sensitive to neutralization with anti-human and other neutralizing anti-HCMV antibodies. (4) Antibodies targeting HCMV envelope glycoproteins can also be incorporated on the viral envelope likely through their Fab moiety bound to the specific glycoprotein. Such virions are susceptible to neutralization by anti-HCMV as well as anti-human antibodies. Image made with BioRender.