Characterization of human cytomegalovirus infection dynamics in human microglia

Abstract

Human cytomegalovirus (HCMV) is a β-herpesvirus that establishes asymptomatic infections in immunocompetent individuals but can cause severe or even life-threatening symptoms in immunocompromised patients. HCMV can replicate in a wide variety of cells through the engagement of diverse cell factors with the viral envelope protein gH/gL/gO (trimer) or gH/gL/UL128/UL130/UL131a (pentamer), allowing for systemic spread within the human host. This study explores HCMV infection tropism and dynamics in human microglia, demonstrating the susceptibility of microglia to both clinical and laboratory HCMV strains, albeit with lower efficacy for the laboratory strain, implying that both the gH/gL-trimer and -pentamer can mediate virus entry in microglia. The importance of the gH/gL pentamer for virus entry was demonstrated by the inhibition of virus infection upon pre-incubation with a soluble neuropilin-2 (NRP-2) entry factor. Further, we demonstrated that HCMV infection can be effectively inhibited by monoclonal antibodies specific for the gH/gL complexes and HCMV hyperimmunoglobulin. Lastly, we report that microglia infection can be prevented by newly characterized chemical entry inhibitors. Altogether, these findings underscore the potential of microglia as valuable models for studying HCMV neurotropism and strategies to block virus infection in cells that can impact neurological disorders.

Keywords: tropism; antibodies; entry inhibitors; human cytomegalovirus; microglia; neuropilin-2 (NRP-2).

Fig. 1.. Human C20 microglia cells are permissive for HCMV infection. (a) Representative whole well of HCMV-infected C20 cells. Cells were infected with HCMV strains AD169^R, AD169^IE2/YFP or TB40/E and incubated at 37 °C, 5% CO₂. After incubation, cells were fixed with 4% paraformaldehyde, permeabilised with 0.3% Triton X-100 and stained with anti-IE1 antibody, followed by Alexa647-conjugated secondary antibody and Hoechst 33 342. Imaging was performed using the Celigo imaging cytometer. (b) The percentage of infection was calculated using non-infected cells as the 0% infection baseline. All conditions were performed in technical triplicate. Error bars represent standard deviation from the mean. Statistical significance is denoted as not significant (ns), **P<0.01; ***P<0.001; ****P<0.0001. (c) Total cell lysates from C20 cells infected with AD169^R (m.o.i. 0.1) were subjected to Western blot analysis using a rabbit anti-IE1 antibody and an anti-GAPDH antibody as a loading control. The molecular weight markers and immunoreactive proteins are indicated.

Fig. 2.. NRP-2 protein efficiently inhibits the entry of AD169^R into microglia. C20 microglia and ARPE-19 epithelial cells were infected with AD169^R (a), TB40/E (b) and AD169^IE2/YFP (c)(m.o.i. 0.5) pre-incubated with soluble NRP-2 (SinoBiological, 10695-H02H, 0.05–0.15 µg ml⁻¹). A non-neutralizing antibody, 1H2 (20 µg ml⁻¹), served as a negative inhibition control. Infection levels were assessed 24 h.p.i. Relative percent infection at 24 h.p.i. was determined following fixation and indirect immunofluorescence (IF) staining for IE1. All conditions were performed in technical triplicate. Error bars represent standard deviation from the mean. Statistical significance is denoted as **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant.

Fig. 3.. Anti-gH mAbs are broadly neutralizing HCMV in C20 cells. The mAbs and Cytogam (0.04–10 µg ml⁻¹) were incubated with AD169^R (m.o.i. 0.2) to assess neutralizing capacity in C20 cells. Antibodies were diluted from 10 µg ml⁻¹ using threefold dilution. Relative infection (%) at 24 h.p.i. was determined following fixation and indirect immunofluorescence (IF) staining for IE1. All conditions were performed in technical triplicate. Error bars represent standard deviation from the mean. Statistical significance is denoted as no significance (ns); **P<0.01; ****P<0.0001 based on the untreated sample, respectively.

Fig. 4.. NAPA compounds MBX-4336 and MBX-4992 limit HCMV infection. C20 cells (10,000 cells per well) were incubated overnight with heparin (50 µg ml⁻¹) and NAPA compounds (0–20 µM) along with AD169^R (m.o.i. 0.2). Relative infection (%) at 24 h.p.i. was determined following fixation and indirect immunofluorescence (IF) staining for IE1. All conditions were performed in technical triplicate. Error bars represent standard deviation from the mean. Statistical significance is denoted as non-significant (ns); ****P<0.0001 based on the untreated sample, respectively.