Immature dengue virus: a veiled pathogen?

Abstract

Cells infected with dengue virus release a high proportion of immature prM-containing virions. In accordance, substantial levels of prM antibodies are found in sera of infected humans. Furthermore, it has been recently described that the rates of prM antibody responses are significantly higher in patients with secondary infection compared to those with primary infection. This suggests that immature dengue virus may play a role in disease pathogenesis. Interestingly, however, numerous functional studies have revealed that immature particles lack the ability to infect cells. In this report, we show that fully immature dengue particles become highly infectious upon interaction with prM antibodies. We demonstrate that prM antibodies facilitate efficient binding and cell entry of immature particles into Fc-receptor-expressing cells. In addition, enzymatic activity of furin is critical to render the internalized immature virus infectious. Together, these data suggest that during a secondary infection or primary infection of infants born to dengue-immune mothers, immature particles have the potential to be highly infectious and hence may contribute to the development of severe disease.

Figure 1. Immature DENV particles become highly infectious in the presence of anti-prM antibodies.

K562 cells were infected with immature (prM) or wild-type (wt) DENV-2 strain 16681 at MOG 100 in the presence or absence of anti-prM 70–21. (A) Representative read-out of the percentage of infected cells. Prior to infection, immature DENV particles were incubated with 40 ng/ml 70–21 antibody for 1 h at room temperature. At 43 hpi, the cells were fixed, stained intracellularly with Alexa-647-coupled anti-E antibody 3H5.1, and subjected to flow-cytometric analysis. Mock-infected cells and an IgG isotype control (murine IgG2a) antibody were used as controls. Titrations of the virus particles released at 43 hpi from infected K562 cells (B), U937 cells (C), and primary human PBMCs (D) were performed by plaque assay on BHK-15 cells. Data are expressed as means of at least three independent experiments. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”.

Figure 2. Anti-prM antibody stimulates binding of immature DENV particles to cells through interaction with FcγIIR.

(A) Binding of immature and wild-type virions with and without prior opsonization to antibodies (40 ng/ml) to K562 cells. Virus-cell binding was measured after 1 h incubation at 4°C by q-PCR analysis. (B) Effect of anti-CD32 mAb and 70–21 F(ab')₂ fragments on virus particle production. Virus particle production was determined as described in the legend to Figure 1B. Data are expressed as means and SD of three independent experiments. Two-tailed Student's t-tests were performed for statistical analysis of the data.

Figure 3. Immature DENV particles mature upon FcγIIR-mediated cell entry.

(A) K562 cells were infected with DENV or DENV-immune complexes in the presence or absence of furin inhibitor (25 µM). As a positive control for compound activity, wild-type DENV-infected cells were treated with an additional dose of furin inhibitor at 24 hpi to impede virus maturation and consequently the production of infectious virions (++). Virus particle production was determined as described in the legend to Figure 1B. Data are expressed as means and SD for three independent experiments; (n.d.) denotes “not detectable”. (B) K562 cells were infected with DENVprMΔ90 at MOG 100 in the presence of increasing concentrations of prM antibody 70–21. As control, the infectivity of wild-type virus (generated from the infectious clone pD2/IC-30P) at MOG 100 is shown. Virus production was assessed as described in the legend to Figure 1B. (C) prM antibodies do not affect cleavage maturation of DENV particles. Purified [³⁵S]methionine-labeled immature particles with and without enhancing concentrations of anti prM mAb 70–21 were incubated with furin at pH 6 for 16 h. Next, viral protein composition was analyzed by non-reducing SDS-polyacrylamide gel electrophoresis.

Figure 4. FcR-mediated entry of immature DENV does not enhance the number of progeny virions produced per cell.

K562 cells were infected with immature (prM) DENV-2 at MOG 100 in the absence or presence of 40 ng/ml of 70–21 antibody and with wild-type virus at MOG 10 and 100. (A) At 43 hpi, the cells were fixed, stained intracellularly with Alexa-647-coupled anti-E antibody 3H5.1, and subjected to flow-cytometric analysis. Mock-infected cells and an irrelevant murine IgG2a antibody were used as controls. (B) Mean fluorescent intensity of cells infected with immature virus (MOG 100) and wild-type DENV (MOG 10) (C) Titration of the virus particles released at 43 hpi from infected K562 cells using plaque assay. Data are expressed as means of at least three independent experiments. The error bars represent standard deviations (SD).

Figure 5. prM-specific antibody 70–21 enhances infectivity of wild-type DENV in a furin-dependent manner.

Cells were infected with wild-type (wt) DENV-2 at MOG 100 in the presence of increasing concentrations of anti-prM 70–21. Virus particle production was measured at 43 hpi by plaque assay on BHK-15 cells. (A) K562 cells, (B) U937 cells, (C) PBMCs. (D) Enhancement of infection is dependent on endogenous furin activity. K562 cells were infected with DENV with and without prior opsonization with 4000 ng/ml 70–21 in the presence or absence of furin inhibitor as described in the legend to Figure 3. Data are expressed as means of at least three independent experiments. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”; * denotes significance (p<0.05) analyzed using Two-tailed Student's t-tests.

Figure 6. Immature DENV becomes infectious in the presence of DENV-immune sera.

U937 cells were infected with immature (prM) DENV-2 at MOG 100 in the presence of 10-fold sequential dilutions of dengue patients sera. Virus particle production was measured at 43 hpi by plaque assay on BHK-15 cells. Sera of two patients showed significant enhancing activity towards immature DENV virions at a 10⁴ sera dilution. The error bars represent standard deviations (SD); (c.s.) denotes “control serum”; (n.d.) denotes “not detectable”; * denotes significance (p<0.05) analyzed using Two-tailed Student's t-tests.