Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to infect fibroblastic, epithelial, endothelial and hematopoietic cells. Over the past ten years, several groups have provided direct evidence that dendritic cells (DCs) fully support the HCMV lytic cycle. We previously demonstrated that the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) has a prominent role in the docking of HCMV on monocyte-derived DCs (MDDCs). The DC-SIGN/HCMV interaction was demonstrated to be a crucial and early event that substantially enhanced infection in trans, i.e., from one CMV-bearing cell to another non-infected cell (or trans-infection), and rendered susceptible cells fully permissive to HCMV infection. Nevertheless, nothing is yet known about how HCMV enters MDDCs. In this study, we demonstrated that VHL/E HCMV virions (an endothelio/dendrotropic strain) are first internalized into MDDCs by a macropinocytosis-like process in an actin- and cholesterol-dependent, but pH-independent, manner. We observed the accumulation of virions in large uncoated vesicles with endosomal features, and the virions remained as intact particles that retained infectious potential for several hours. This trans-infection property was specific to MDDCs because monocyte-derived macrophages or monocytes from the same donor were unable to allow the accumulation of and the subsequent transmission of the virus. Together, these data allowed us to delineate the early mechanisms of the internalization and entry of an endothelio/dendrotropic HCMV strain into human MDDCs and to propose that DCs can serve as a "Trojan horse" to convey CMV from entry sites to other locations that may favor the occurrence of either latency or acute infection

Endosomal pH neutralization does not inhibit HCMV internalization or MDDC infection.

<p>A) Cells were pre-incubated with NH₄Cl-containing buffer (50, 5, 0.5 mM) or bafilomycin A1 (320, 32, 3.2 nM) and compared to the vehicle (DMSO; 1/100). The cells were processed as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034795#pone-0034795-g001" target="_blank">Figure 1D</a>. For NH₄Cl, n= 3 independent experiments with 3 different donors in total, for bafilomycin A1 n= 6 independent experiments with 8 different donors in total. ns: not significant (p=0,0939). B) TEM picture of (50 nM) bafilomycin A1 treated MDDCs incubated with HCMV (VHL/E; MOI=10). Black arrows indicate HCMV virions. C) Quantification of infectious HCMV particles by TEM immobilized at the plasma membrane (OUT, white bars) or internalized into vacuoles (IN, black bars) of (50 nM) BafA1-treated MDDCs incubated for two hours with VHL/E (MOI=10) (n=5-6 cells per condition). The results are displayed as the median values of the percentage (±SD) of plasma membrane-associated and internalized HCMV particles. These results are representative of two distinct experiments.</p

Cholesterol depletion is detrimental to the HCMV entry into MDDCs.

<p>A) Cells were pre-incubated with filipin (7.66, 1.5, 0.3 µM), nystatin (21.2, 4.3, 0.85 µM) or methyl-β-cyclodextrin (MβCD; 5, 1, 0.2 mM) or with vehicle (DMSO, 1/100) and were processed as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034795#pone-0034795-g001" target="_blank">Figure 1D</a>. For nystatin, n= 2 independent experiments with 2 different donors in total; for Filipin and MβCD n=4 independent experiments with 6 different donors in total. <i>ns</i>: not significant (p=0,0535).</p

HCMV internalization into MDDCs is impaired by macropinocytosis inhibitors.

<p>A) MDDCs were pre-incubated with drugs blocking macropinocytosis (Gö6983∶ 13, 7.3, 3.75 nM and amiloride, Ami: 100, 20 µM) or clathrin-mediated endocytosis (chlorpromazine, CPZ: 30, 6, 1.2 µM) or with vehicle (DMSO, 1/100) prior to be infected with HCMV (MOI=2) for two hours. Cells were extensively washed then subcultured for 48 hours. The cells were then prepared and analyzed as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034795#pone-0034795-g001" target="_blank">Figure 1D</a>. For Gö6983, n=2 independent experiments with 4 different donors in total, for Ami and CPZ n=5 independent experiments with 7 different donors in total. ns: not significant (p=0,0733) B) TEM picture of (500µM) amiloride-treated MDDCs incubated with HCMV (VHL/E; MOI=10). Black arrows indicate HCMV virions. C) Quantification of infectious HCMV particles by TEM immobilized at the plasma membrane (OUT, white bars) or internalized into vacuoles (IN, black bars) of (500 µM) amiloride-treated MDDCs incubated for two hours with VHL/E (MOI=10) (n=6 cells per conditions). The results are displayed as the median values of the percentage (±SD) of plasma membrane-associated and internalized HCMV particles.</p

MDDCs can mediate HCMV <i>trans</i>-infection through both plasma membrane-associated virions and the release of internalized virions.

<p>MDDCs, MDMs or monocytes from the same blood donor were obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034795#s2" target="_blank">Materials and Methods</a> section. Cells were pretreated with 40 nM BafA1 (black bars) or the vehicle (DMSO) (white bars) prior to incubation with the VHL/E HCMV strain for two hours (MOI=2). Cells were then extensively washed with a low-pH buffer (glycine 0.2M, pH=2.8) or with PBS alone as indicated above each panel of the figure and were subcultured in close contact with HFFs. After 48 hours, fibroblasts were processed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034795#pone.0034795-Halary1" target="_blank">[14]</a> to evaluate the infection rate due to <i>trans</i>-infection by HCMV-loaded cells (absolute number of IE/E antigen-positive fibroblasts among 10⁵ total cells). n= 4 independent experiments with four different donors in total.</p

CMV internalization into large uncoated vesicles in MDDCs is an active process that requires actin cytoskeleton polymerization.

<p>A) Transmission electron microscopy (TEM) picture is shown of day 6 immature MDDCs incubated with HCMV (VHL/E; MOI=10) for two hours then extensively washed with PBS. A scale bar is indicated (magnification x7,500). N=nucleus. B) Close-ups of different pictures are represented. Black arrows indicate HCMV virions. A scale bar is indicated for each picture. Cells were prepared as described in 1A. C) Kinetic analysis of HCMV internalization in MDDCs. The data represent the quantification of the mean number of particles per cell from the TEM pictures (n≥20 independent cells) of viral particles (VHL/E; MOI=10) immobilized at the plasma membrane (OUT, white bars) or internalized into vacuoles (IN, black bars) as function of time (30 minutes, two, six and 24 hours incubations). Cells were extensively washed with PBS after incubation with HCMV. These results are representative of two distinct experiments. D) MDDCs were pre-incubated with drugs (cytochalasin, cytD: 50-5-0.5 µM) or vehicle (DMSO: 1/100) prior to be infected with HCMV (MOI=2) for two hours. Cells were extensively washed then subcultured for 48 hours. The cells were coated onto poly-L-lysine-coated slides, fixed and permeabilized with acetone and were stained with mAbs against anti-IE and –E antigens (Argene Biosoft, France). Four distinct fields were digitalized and analyzed with ImageJ software to determine the percentage of I.E.A./E.A.+ MDDCs. n=6 independent experiments with eight different donors in total.</p

Internalized HCMV virions partially co localize with EEA1 and not with LAMP2.

<p>Confocal imaging was performed on immobilized immature MDDCs incubated for two hours on ice with the VHL/E strain (MOI=2) and subsequently cultured at 37°C for three hours (A) and 24 hours (B). Cells were then fixed/permeabilized and immunostained as indicated above each column with anti-EEA1 or -LAMP2 antibodies (green) and with an anti-HCMV gB (red). Images were obtained on a 510 LSM Meta (Zeiss, Germany). Single confocal planes are presented. In merged panels, green and red staining intensities were directly analyzed along a virtual section indicated by a thin white line with the ImageJ RGB profiler plugin. The results are displayed in graphs on the right (X-axis=distance in pixels; Y-axis=fluorescence intensity). White arrowheads indicate small CMV aggregates or isolated particles. C) Between 7 and 10 x 10⁷ HCMV-infected MDDCs (VHL/E; MOI=10) were subjected to three consecutive subcellular fractionation steps on sucrose and Percoll gradients to harvest early and late endosome-enriched and lysosome-enriched fractions. Here early endosomes (EE) and late endosomes (LE) enriched fractions were first pooled (EE+LE) and the EE+LE and the lysosomes (Ly) enriched fractions were concentrated before being used in a western blot analysis using anti-EEA1, LAMP-2, HCMV gB and MCP antibodies. Recombinant HCMV gB (Biomérieux, France) was used as a positive control. The molecular weight of the gB is 160 kDa and 153 kDa for the MCP. PN means post nuclear fraction.</p

Can shellfish be used to monitor SARS-CoV-2 in the coastal environment?

The emergence and worldwide spread of SARS-CoV-2 raises new concerns and challenges regarding possible environmental contamination by this virus through spillover of human sewage, where it has been detected. The coastal environment, under increasing anthropogenic pressure, is subjected to contamination by a large number of human viruses from sewage, most of them being non-enveloped viruses like norovirus. When reaching coastal waters, they can be bio-accumulated by filter-feeding shellfish species such as oysters. Methods to detect this viral contamination were set up for the detection of non-enveloped enteric viruses, and may need optimization to accommodate enveloped viruses like coronaviruses (CoV). Here, we aimed at assessing methods for the detection of CoV, including SARS-CoV-2, in the coastal environment and testing the possibility that SARS-CoV-2 can contaminate oysters, to monitor the contamination of French shores by SARS-CoV-2 using both seawater and shellfish. Using the porcine epidemic diarrhea virus (PEDV), a CoV, as surrogate for SARS-CoV-2, and Tulane virus, as surrogate for non-enveloped viruses such as norovirus, we assessed and selected methods to detect CoV in seawater and shellfish. Seawater-based methods showed variable and low yields for PEDV. In shellfish, the current norm for norovirus detection was applicable to CoV detection. Both PEDV and heat-inactivated SARS-CoV-2 could contaminate oysters in laboratory settings, with a lower efficiency than a calicivirus used as control. Finally, we applied our methods to seawater and shellfish samples collected from April to August 2020 in France, where we could detect the presence of human norovirus, a marker of human fecal contamination, but not SARS-CoV-2. Together, our results validate methods for the detection of CoV in the coastal environment, including the use of shellfish as sentinels of the microbial quality of their environment, and suggest that SARS-CoV-2 did not contaminate the French shores during the summer season