PLoS Pathog. 2014 Apr 10;10(4):e1004027.

Complement-related proteins control the flavivirus infection of Aedes aegypti by inducing antimicrobial peptides

Xiaoping Xiao1, Yang Liu1, Xiaoyan Zhang3, Jing Wang1, Zuofeng Li3, Xiaojing Pang1, Penghua Wang4 and Gong Cheng1, 2*

1 Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, P.R. China, 100084

2 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, P.R. China, 310003

3 School of Life Sciences and Technology, Tongji University, Shanghai, P.R. China, 200092

4 Section of Infectious Diseases, Yale University School of Medicine, New Haven, CT, USA, 06511.

* Corresponding to gongcheng@mail.tsinghua.edu.cn

Address correspondence to: Gong Cheng Ph.D., School of Medicine, Tsinghua University, Beijing, P.R. China, 100084. Phone: (+86)-10-62788494; Fax: (+86)-10-62788494; E-mail: gongcheng@mail.tsinghua.edu.cn.

 

Abstract

The complement system functions during the early phase of infection and directly mediates pathogen elimination. The recent identification of complement-like factors in arthropods indicates that this system shares common ancestry in vertebrates and invertebrates as an immune defense mechanism. Thioester (TE)-containing proteins (TEPs), which show high similarity to mammalian complement C3, are thought to play a key role in innate immunity in arthropods. Herein, we report that a viral recognition cascade composed of two complement-related proteins limits the flaviviral infection of Aedes aegypti. An A. aegypti macroglobulin complement-related factor (AaMCR), belonging to the insect TEP family, is a crucial effector in opposing the flaviviral infection of A. aegypti. However, AaMCR does not directly interact with DENV, and its antiviral effect requires an A. aegypti homologue of scavenger receptor-C (AaSR-C), which interacts with DENV and AaMCR simultaneously in vitro and in vivo. Furthermore, recognition of DENV by the AaSR-C/AaMCR axis regulates the expression of antimicrobial peptides (AMPs), which exerts potent anti-DENV activity. Our results both demonstrate the existence of a viral recognition pathway that controls the flaviviral infection by inducing AMPs and offer insights into a previously unappreciated antiviral function of the complement-like system in arthropods.

PMID: 24722701

 

Supplement:

   Many mosquito-transmitted flaviviruses, such as West Nile virus, Japanese encephalitis virus, dengue virus (DENV) and yellow fever virus (YFV), are etiologic agents of severe human diseases, including hemorrhagic fever, biphasic fever, encephalitis, and meningitis. The majority of these diseases occur in endemic tropical and subtropical regions, nonetheless, increase of international travels and fast urbanization have expanded their prevalence in new territories. Unfortunately, there are no vaccines or therapeutics available for most of the mosquito-borne flaviviruses. A better understanding of mosquito-flavivirus interaction might provide novel strategies for limiting arboviral transmission in nature.

The antiviral machineries of blood-sucking arthropods, such as mosquitoes, ticks, and sand flies, are quite different from those of mammals. Arthropods lack an immunoglobulin-based adaptive immune response. Thus, the innate immune system of arthropods plays a central role in the antiviral processes of these organisms. The complement system functions during the early phase of infection and directly mediates pathogen elimination. The recent identification of complement-like factors in arthropods indicates that this system shares common ancestry as an immune defense mechanism in both vertebrates and invertebrates (1-3). Thioester (TE)-containing proteins (TEPs) share high similarity with mammalian C3, which have been identified as important immune players in many arthropods (3-6).

 

cg fig1

Figure 1. The AaMCR/AaSR-C immune axis restricts the flavivirus infection by inducing antimicrobial peptides. (1) viral recognition: AaSR-C recognizes the flaviviral surface protein and subsequently recruits AaMCR to form a protein-virus complex; (2) AMP induction: the conjugation of AaSR-C and AaMCR with viruses triggers an immune signalling pathway to stimulate AMPs expression; and (3-4) viral inactivation: the positive charges of AMPs can electrostatically or hydrophobically associate with the components of the viral surface, subsequently resulting in the flaviviral inactivation.

 

The human complement system consists of more than 30 secreted and membrane-bound proteins. Invading pathogens are recognize by pattern receptors, including C1q, ficolin, and mannose-binding C-type lectin (MBL), subsequently triggering a complement cascade. However, the mechanism of pathogen recognition in insects may be quite different from that in mammals (3). Comparative genomic analyses have shown that no homologues of human C1q or ficolin exist in A. aegypti (Cheng G and Xiao XP, unpublished data). Moreover, the extracellular C-type lectins found in mosquitoes, which are the putative homologues of human MBL, have recently been shown to act as cellular receptors facilitating West Nile virus and DENV infection (7-8), rather than as antiviral pattern recognition receptors. In this study, we demonstrate that macroglobulin complement-related factors (AaMCR), a member of A. aegypti TEPs, is crucial for restricting flaviviral infections. Silencing of AaMCR or immuno-blockade of AaMCR impaired the antiviral ability of mosquitoes. However, AaMCR does not directly interact with DENV surface proteins, suggesting that the antiviral effect of AaMCR is mediated by a certain particular adaptor. To further explore the essential players in this MCR-based antiviral response, we identified 10 proteins containing complement control protein (CCP) domains and investigated their role in flaviviral infection in A. aegypti. Knockdown of a CCP gene AAEL006361 enhanced flaviviral infections in the mosquitoes most significantly. Sequence analyses suggested that the protein encoded by AAEL006361 contains 2 CCP domains at its N-terminus and is a homologue of Drosophila Scavenger receptor-C (SR-C), which is a well-known pattern recognition receptor of bacterial surface components that initiates an antibacterial phagocytic response (9). In accordance with these findings, our results show that AaSR-C is capable of binding DENV surface proteins. The virion-bound AaSR-C then recruits the complement-like factor AaMCR, which induces antiviral immune factors such as AMPs to control viral infection. Indeed, in mammals, most CCP-containing complement components function as regulators of the complement cascades. For example, factor H act as a negative regulator of the alternative complement pathway, and C4-binding protein (C4BP) functions as an inhibitor of complement C4 (10). This observation indicates the common distant complement system mechanism between mammals and arthropods. We therefore raised the proposed model for complement-like factors against flavivirus in mosquitoes (Figure 1).

Technical supplement:

Material introduced into mosquitoes via thoracic microinjection: The materials, such as double-strand RNA (dsRNA), antibodies, purified recombinant proteins or viruses, can be introduced into mosquitoes via dsRNA thoracic microinjection. In the procedure, the plastic microscope slides were pre-cooled on ice. A number of A. aegypti mosquitoes (usually 10-15 per slide) were placed onto the cold slides after thoroughly anesthetizing, and subsequently the mosquitoes were place the slides with the mosquitoes onto the frozen platform prepared for microinjection. The materials diluted by cold PBS was microinjecte into the thorax of the mosquitoes (Figure 2). The injected mosquitoes were dropped into sealed paper cups (16 oz), and then maintained under a standard condition for further investigation.

 

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Figure 2. Material introduced into mosquitoes via thoracic microinjection

 

Reference:

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  10. Leung E, Blom AM, Clemenza L, Isenman DE (2006) The complement regulator C4b-binding protein (C4BP) interacts with both the C4c and C4dg subfragments of the parent C4b ligand, evidence for synergy in C4BP subsite binding. Biochemistry 45: 8378-8392.

 

Acknowledgements: This work was supported by grants from the National Natural Science Foundation of China to G.C. (81301412 and 81422028), the National Key Basic Research Program of MOST to G.C. (2013CB911500), the Excellent Young Scientist Foundation of Beijing to G.C. (2013D009004000002), the Grand Challenges Explorations of Bill & Melinda Gates Foundation to G.C. (OPP1021992). G.C. is a Newton Advanced Fellow awarded by the Academy of Medical Sciences and the Newton Fund, and a Janssen Investigator of Tsinghua University. We thank the technical supports from the Core Facility of Center for Life Sciences and Center of Biomedical Analysis (Tsinghua University).

 

cg fig3Contact:

Gong Cheng Ph.D.

Principal Investigator

School of Medicine, Tsinghua University, Beijing, P.R. China, 100084.

Phone: (+86)-10-62788494; Fax: (+86)-10-62788494

E-mail: gongcheng@mail.tsinghua.edu.cn.

http://www.med.tsinghua.edu.cn/index.php/en/research/faculty/item/cheng-gong

 

 

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