C1q binding to dengue virus decreases levels of infection and inflammatory molecules transcription in THP-1 cells

Abstract

Dengue virus infection elicits a spectrum of clinical presentations ranging from asymptomatic to severe disease. The mechanisms leading to severe dengue are not known, however it has been reported that the complement system is hyper-activated in severe dengue. Screening of complement proteins demonstrated that C1q, a pattern recognition molecule, can bind directly to dengue virus envelope protein and to whole dengue virus serotype 2. Incubation of dengue virus serotype 2 with C1q prior to infection of THP-1 cells led to decreased virus infectivity and modulation of mRNA expression of immunoregulatory molecules suggesting reduced inflammatory responses.

Keywords: C1q; Classical complement pathway; Dengue; Dengue fever; Dengue hemorrhagic fever; Viral persistence.

Fig. 1

C1q binds to full recombinant Envelope proteins of all Dengue Virus (DENV1-4) serotypes (A–D, respectively) but not to Bovine Serum Albumin (BSA; negative control). Within each serotype, binding of C1q occurs preferentially to the portion of ENV containing DI/II (A, B and D), except for serotype 3 (C). ELISA plates were coated with DENV ENV full length, ENV DI/II and ENV DIII (aa 1–413, aa 1–296 and aa 297–413, respectively; Table S1) fragments and BSA, and incubated with C1q concentrations ranging from 1 up to 40 µg/ml. Results obtained are representative of 5 independent experiments. Statistical analysis was done using GraphPad program and Student’s paired, 2 tails t-test. Values were considered significant when p<0.05.

Fig. 2

Dengue viruses (DENV2) bind to C1q. DENV2 bind to 2H2 antibody and to C1q, but not to BSA. Ct+, positive control used was an extract of Vero Cells infected with DENV2. Amplicon size, 189 bp. To capture the viruses, wells were coated with 2H2 antibody, C1q and BSA and incubated with DENV2. Viral RNA was extracted by Trizol and DENV2 presence was confirmed by PCR using specific primer sequences for this virus. Results obtained are representative of 3 independent experiments.

Fig. 3

Incubation of DENV2 with C1q leads to a decrease in infection and in the levels of transcription of cellular factors. (A) DENV2 strain 16681 viruses were incubated either with vehicle (DENV), C1q and factor D at 37°C for 1 hour prior to infection of THP-1 cells and the level of infection was measured by RT-PCR 24 hours later. Cells infected with DENV2 incubated with C1q, but not with factor D, had a significant decrease in infection of around 30%. DENV2 viruses were incubated with the complement molecules in their physiological concentration, i.e., the concentrations they are normally found in human blood, 10 µg/ml and 750 ng/ml for C1q and factor D, respectively (Nascimento et al., 2009b). (B) Levels of transcription of several cellular factors are altered by infection of THP-1 with DENV2 previously incubated with C1q. DENV2 strain 16681 viruses were incubated either with vehicle (DENV) or C1q at 37°C for 1 hour prior to infection of THP-1 cells and the level of transcription of several proteins involved in viral clearance was measured by RT-PCR 24 hours later. Levels of CD14 (involved in monocyte maturation), CD86 (involved in the induction of a T cell response) and DC-SIGN (a cellular receptor involved both in viral infection and modulation of innate immune response) show, are significantly decreased in cells infected with virus pre-incubated with C1q. Results obtained are representative of 4 independent experiments. Statistical analysis was done using GraphPad program and Student’s paired, 2 tails t-test. Values were considered significant when p<0.05.