Zika virus is transmitted in neural progenitor cells via cell-to-cell spread and infection is inhibited by the autophagy inducer trehalose

Abstract

Zika virus (ZIKV) is a mosquito-borne human pathogen that causes congenital Zika syndrome and neurological symptoms in some adults. There are currently no approved treatments or vaccines for ZIKV, and exploration of therapies targeting host processes could avoid viral development of drug resistance. The purpose of our study was to determine if the non-toxic and widely used disaccharide trehalose, which showed antiviral activity against Human Cytomegalovirus (HCMV) in our previous work, could restrict ZIKV infection in clinically relevant neural progenitor cells (NPCs). Trehalose is known to induce autophagy, the degradation and recycling of cellular components. Whether autophagy is proviral or antiviral for ZIKV is controversial and depends on cell type and specific conditions used to activate or inhibit autophagy. We show here that trehalose treatment of NPCs infected with recent ZIKV isolates from Panama and Puerto Rico significantly reduces viral replication and spread. In addition, we demonstrate that ZIKV infection in NPCs spreads primarily cell-to-cell as an expanding infectious center, and NPCs are infected via contact with infected cells far more efficiently than by cell-free virus. Importantly, ZIKV was able to spread in NPCs in the presence of neutralizing antibody.Importance Zika virus causes birth defects and can lead to neurological disease in adults. While infection rates are currently low, ZIKV remains a public health concern with no treatment or vaccine available. Targeting a cellular pathway to inhibit viral replication is a potential treatment strategy that avoids development of antiviral resistance. We demonstrate in this study that the non-toxic autophagy-inducing disaccharide trehalose reduces spread and output of ZIKV in infected neural progenitor cells (NPCs), the major cells infected in the fetus. We show that ZIKV spreads cell-to-cell in NPCs as an infectious center and that NPCs are more permissive to infection by contact with infected cells than by cell-free virus. We find that neutralizing antibody does not prevent the spread of the infection in NPCs. These results are significant in demonstrating anti-ZIKV activity of trehalose and in clarifying the primary means of Zika virus spread in clinically relevant target cells.

FIG 1

ZIKV spreads cell-to-cell in NPCs and infects NPCs less efficiently than Vero cells. NPCs (WT126) were infected 1 day postseeding on coverslips with ZIKV (PRV). Cells were washed at 2 hpi. Cells were fixed in 4% paraformaldehyde (PFA) and processed for immunofluorescence with the indicated antibodies. Nuclei were counterstained with Hoechst 33342. Images are representative of 2 independent experiments. (A) NPCs (WT126) were infected at an MOI of 5. At 72 hpi, cells were fixed for immunofluorescence with antibodies against Pax6 and dsRNA. (B) NPCs (WT126) were infected at an MOI of 0.5 with ZIKV (PRV). Cells were washed at 2 hpi, and medium was added and refreshed every day. Slips were fixed at the indicated times and stained with antibody against capsid protein. (C) Vero cells (top) or NPCs (WT126, bottom) were infected at an MOI of 0.5 on coverslips with ZIKV PRV. At 24 hpi, slips were fixed and stained with antibody against capsid protein. (D) Quantification of panel C. Infected cells were counted for 6 random fields per coverslip. Mean and SD of infected cells per field (left) and percentage infected (right) are displayed.

FIG 2

ZIKV infection via cell-cell spread is more effective than infection via cell-free virus. NPCs (WT126) were infected with ZIKV PRV. Infected cells or cell-free supernatants were subsequently transferred to uninfected NPCs on coverslips to visualize foci by IFA or quantified by plaque assay on Vero cells (diagrammed in panel A). At 48 hpi, NPCs on coverslips were fixed and stained with antibody against capsid protein and counterstained with Hoechst 33342 to visualize focal spread of infection (foci containing greater than 4 infected cells). (B) Representative fields. (C) The number of infectious centers (plaques on Vero cells and foci on NPCs) from infected NPCs for 4 cultures from 2 independent experiments. Means were compared by Mann-Whitney test. *, P < 0.05.

FIG 3

ZIKV spreads in the presence of neutralizing antibody. (A) A total of 10,000 PFU of ZIKV (PRV) were incubated with increasing concentrations of ZV-54 ZIKV neutralizing antibody or IgG2A control for 1 h. Virus-antibody mixtures were serially diluted, and infectious titers were quantified by plaque assay on Vero cells. The graph shows the means of two experiments. (B to D) In 2 independent experiments, NPCs (WT126) on coverslips were infected with 25,000 PFU of ZIKV PRV. At 2 hpi, cells were washed and incubated with medium. At 9 hpi, cells were switched to medium with 25 μg/ml ZV-54 or control IgG2A. Control cells received virus that had been preincubated with 25 μg/ml ZV-54 or IgG2A control Ab (Preinc) or received virus with no antibody (No Ab). At 48 hpi, coverslips were fixed and stained for capsid protein. Images were acquired of entire coverslips. (B) The infectious centers per coverslip were counted and shown as the mean and SD of the 2 experiments. (C) The cells per infectious center were counted for 100 randomly chosen infectious centers and are displayed as individual values with the median (red bars) for two independent experiments. Kruskal-Wallis test for significance and Dunn’s multiple-comparison test were applied. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Representative images of infectious centers are displayed.

FIG 4

Low infectivity of NPCs compared to Vero is not due to changes in tropism of cell-free virus during replication in NPCs. (A) ZIKV (PRV) was propagated on NPCs (WT126) or on Vero cells, and the titers were quantified via plaque assay on Vero cells. Vero (top) or NPCs (WT126, bottom) were infected with virus derived from each cell type at an MOI of 0.5. Infected supernatants were collected every 24 h, and the titers were quantified on Vero cells. Graphs show the mean and SD from 3 cultures. (B) NPCs (WT126) were infected at an MOI of 0.5 with ZIKV PRV derived from NPCs (top) or Vero cells (bottom) on coverslips. At 48 hpi, coverslips were fixed and stained for capsid protein.

FIG 5

Trehalose reduces ZIKV infection in neural lineage cells. (A) NPCs (WT126) were infected with ZIKV PRV at an MOI of 5 in the presence or absence of 100 mM trehalose. Cell pellets were collected at 72 hpi and processed for Western blot analysis using antibodies against LC3B and tubulin as the loading control. (B) Uninfected WT126 NPCs were treated with 100 mM trehalose beginning 1 day postplating. At the indicated times posttrehalose addition, cells were dissociated with Accutase and counted in Trypan blue to exclude dead cells. The graph shows the mean and SD of two cultures. A t test was performed at each time point. (C to G) NPC lines WT126 (C and E) and NSC11 (D and F) and early cortical neurons (G) were pretreated with 0 or 100 mM trehalose for 2 h. Cells were then infected with the indicated isolates of ZIKV at an MOI of 0.5 in the presence or absence of 100 mM trehalose. Medium with or without trehalose was refreshed, and supernatants were collected every 24 h, and the titers were quantified on Vero cells. The graphs show means and SD from 4 to 8 wells from 2 to 4 independent experiments (panels C to F) and of 2 to 3 cultures from 2 experiments (panel G). A t test was performed at each time point. (H) NPCs (WT126) were infected with ZIKV (PRV) at an MOI of 0.05. Trehalose was added 2 h before (t = −2), at the time of (t = 0), or 2 h after (t = +2) infection. Medium with or without trehalose was refreshed every 24 h. The graph shows the mean and SD of 3 cultures. Results were confirmed with a second independent experiment. At each time point, ordinary unpaired one-way analysis of variance (ANOVA) comparing all means was performed on log-transformed data (black bars). Additional ordinary unpaired one-way ANOVA compared means excluding 0 tre (absence of trehalose; gray bars). ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

Trehalose reduces ZIKV production and spread in NPCs at various MOIs. NPCs (WT126) were infected with ZIKV (PRV) at the indicated MOIs. After 2 h of adsorption, cells were washed and medium with or without 100 mM trehalose was added. Medium was refreshed every 24 h. (A) Infected supernatants were collected every 24 h, and the titer was quantified by plaque assay on Vero cells. Graphs show the means and SD of 3 to 4 cultures from 1 to 2 experiments. Significance was determined by t test at each time point for log-transformed data. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) NPCs (WT126) on coverslips were infected with ZIKV (PRV) at an MOI of 0.5. Coverslips were fixed at 48 hpi and stained for capsid protein. Nuclei were counterstained with Hoechst 33342.

FIG 7

Trehalose reduces ZIKV protein and RNA levels. WT126 NPCs were infected with ZIKV strain PRV at an MOI of 1 to follow the accumulation of viral protein (A) or 0.1 to follow the accumulation of viral RNA (B). At various times postinfection (p.i.), cell pellets were collected. (A) Pellets were processed for Western blotting using antibodies to detect viral capsid, envelope, NS3, and PrM and cellular GAPDH as the loading control. A 2× serial dilution of lysate indicates the range of each antibody. A representative blot of 2 independent experiments is shown. (B) Total RNA was extracted. The ZIKV genome and GAPDH cDNA were reverse-transcribed using sequence-specific primers. Copies of the ZIKV genome were quantified by qPCR via comparison to the standard curve of known copies of in vitro-synthesized ZIKV RNA subjected to the same reverse-transcription conditions as the samples. Copy numbers were normalized to GAPDH. The dotted line indicates the limit of detection. The graph shows the mean and SD of 2 experiments. A t test was performed at each time point. *, P < 0.05.