Complement System As A Viral Target

Published Date: 02 Nov 2017

Abstract

Viruses are constantly under the peril of complement assault as they are efficiently recognized and neutralized by the complement system. In addition, the complement system is also known to enhance the virus specific B and T cell responses which play critical role in controlling viral infections. It is, therefore, important that viruses subvert and escape the complement-mediated attacks for their survival. In this chapter, we provide an overview of how complement system help in controlling viruses and what elusive strategies viruses have developed to evade these defenses.

Introduction

Viruses are amongst the most successful pathogens to have co-existed with the hosts by maintaining a precarious balance between the two for their successful existence []. Being predatory in nature, viruses are constantly in the pursuit of survival and thus, there exists a constant struggle for endurance between the viruses and their hosts: viruses pursue host for their propagation and the hosts on the other hand defy the viral intrusions owing to their well-developed and interconnected network of innate and adaptive immune defense mechanisms.

The complement system is one of the most phylogenetically ancient and pivotal innate immune defense mechanisms of the host, which emerged at least 1000 million years ago [Nonaka and Kimura, 2006]. Although the system was discovered as heat labile components of plasma necessary for antibody-mediated killing of bacteria, over the years it has emerged as a truly multitasking system that not only functions as an innate arm of the immunity, but also plays instructive role in generation of adaptive immune responses and participates in clearance of immune complexes and apoptotic cells []. Because complement possesses distinctive ability to recognize known as well as newly emerging non-self structures, it possesses the unprecedented capability to protect against varied pathogens including viruses. This is clearly evident from the system’s ability to neutralize diverse DNA [] as well as RNA [] viruses, including the recently emerged pandemic H1N1 virus (unpublished observation).

In contrary to the significant function of the complement system against viruses, it has gained diminutive importance as an antiviral defense system unlike other soluble innate immune barriers such as interferons and interleukins [fields virology and other virology books]. A likely reason for this is that mostly complement deficiencies have been shown to be associated with bacterial infections and not the viral infections [refs]. It could be argued that non-association of viral infection with complement deficiencies is largely due to the lack of systematic study on association between complement deficiencies and vulnerability to viral infections. Another reason could be that the system is a vital antiviral defense and therefore viruses have developed mechanisms to subvert the complement assault [ref]. This argument is well supported by many studies from other as well as our laboratory, which provide evidence that viruses have developed diverse strategies to target the complement system [refs]. Here, we highlight the mechanisms of complement-mediated control of viral infections and cover the ingenious elusive strategies devised by viruses to annul the complement-mediated assaults.

I) Role of complement in combating viruses

The complement system is evolved to perform immune surveillance in the host, which raises the question: how does the complement system recognize viruses? It is now evident from a significant body of knowledge that complement possesses the ability to recognize viruses with as well as without the help of pattern recognition molecules, which then culminates into complement activation and neutralization of virus particles via three major pathways – the classical, alternative and lectin pathways (Fig. 1). Based on the current literature on initiation of complement activation, it can be said that virus recognition by molecules such as antibodies (IgM, IgG3 and IgG1), C-reactive protein (CRP) [], serum amyloid P (SAP), specific intracellular adhesion molecule-grabbing nonintegrin (SIGN-R1) [] and C1q [] can lead to activation of the classical pathway, while recognition by lectins such as mannose binding lectin (MBL) and ficolins (L, M and H) can lead to activation of the lectin pathway. Among these, recognition of viruses by antibodies and C1q has been shown to be neutralized by the classical pathway [], while recognition of viruses by MBL has been shown to be neutralized by lectin pathway [].

Although the above repertoire of molecules allows the complement system to recognize a large variety of viruses it does not ensure the recognition of all of them. The system therefore is also equipped with a fail-safe mechanism, which allows labeling of viral particles as non-self without the help of a recognition molecule. In this mode of virus recognition, C3b is deposited onto viruses owing to continuous low level C3 activation in fluid phase by the alternative pathway initial C3-convertase [C3(H2O),Bb,P]. Once labeled with C3b, viruses are prone to complement-mediated neutralization as a result of activation of the alternative pathway on their surface. A prerequisite for such type of activation however is that, the viral surface should be devoid of complement regulators. A comprehensive list of viruses neutralized by various pathways is provided in Table 1.

Complement-mediated neutralization of viruses

It is clear from the above discussion that recognition of viruses by the complement system results in their neutralization. Thus the next obvious question is, which mechanisms complement utilizes to neutralize the viruses? Up until now, four mechanisms have been identified for complement-mediated neutralization of viruses: i) aggregation, ii) opsonization, iii) phagocytosis and iv) virolysis.

Aggregation: Viruses are known to be neutralized by antibodies as a result of aggregation, which occurs owing to decrease in the total number of infectious virus units. A similar mode of neutralization has also been shown to occur due to complement activation. Studies on polyoma virus [] and more recently on influenza [] and simian virus 5 [] have shown that complement markedly enhances the aggregation of these viruses when they are coated with antibodies. Interestingly, this aggregation was shown to be dependent on the presence of complement components up to C3. Because C3b is monovalent in nature, it is not clear what induces C3b-dependent crosslinking of these viruses. It is however likely that C3b-binding polymeric molecule(s) present in serum produce this aggregation.

Opsonization: Activation of the complement system on the viral surface results in coating of the surface with complement components. Notably, such coats are thick enough to be observed under the electron microscope []. It is conceivable that such coating can inhibit the virus attachment process due to steric hindrance. It is however also probable that opsonization hinders post-attachment steps like entry, uncoating, DNA/RNA transport to the nucleus or early gene expression. Examples where complement coating without virolysis has resulted in virus neutralization include complement-mediated neutralization of Newcastle disease virus [], HSV-1 [], HTLV-1 [], HIV-1 [], influenza [], West Nile virus [], Dengue virus [Cell host mic 2010] and glycoprotein C (gc)-null herpes simplex virus-1 and -2 []. Of these examples, in particular in HSV-1 and WNV, opsonization has been shown to affect the post-entry steps.

Phagocytosis: Opsonization of pathogens by complement components C3b and C4b, and further inactivation of C3b into iC3b and C3d result in a stable labeling of the viral surface with complement fragments. These fragments then can serve as ligands for recognition by various complement receptors on phagocytic cells, e.g., CR1 (CD35), CR2 (CD21), CR3 (CD11b/CD18), CR4 (CD11c/CD18) and CRIg resulting in engulfment of viral particles by phagocytes. Consequently, complement coated viral particles are recognized and engulfed by phagocytes. Such type of neutralization has been shown only in case of HSV [] and Japanese encephalitis virus [] due to lack of focus in this area.

Virolysis: Enveloped viruses are susceptible to complement-mediated virolysis as a result of activation of the terminal complement cascade and insertion of the membrane attack complex (C5b-9) into their envelopes. Many viruses however are known to evade virolysis owing to incorporation of the host complement regulator CD59 into their envelope (discussed later in the review). Viruses that have been shown to be susceptible to complement-mediated virolysis include alphaviruses, herpesviruses, coronaviruses, retroviruses and paramyxoviruses (reviewed in [cooper and nemwrow 1983 and kalyani rev]).

Complement-mediated enhancement of acquired immunity

The complement system is known to bridge the innate and acquired immunity []. The subsequent question therefore is does complement enhance antiviral immunity? It is now well appreciated that the system is capable of enhancing both virus-specific B as well as T cell responses []. Early studies performed in this direction utilized decomplemented animals (obtained by injecting cobra venom factor; CVF) as well as C5-deficient strains of mice to address this question. It was evident from these studies that presence of complement limits viral infection. Infection studies performed using influenza [], rabies [], and sindbis viruses [] in C5-deficient mice and/or CVF-treated animals showed increased viral load in the target organ and higher mortality. Whether lack of virus control was due to direct effect of complement on viruses or due to indirect effect of complement on the acquired immune responses was not clear in these studies. Because it was known that antibodies help neutralize viruses as a result of classical pathway activation, efforts were also made to examine the protective role of complement-fixing antibodies during viral infection. Such antibodies showed protection in mouse models of yellow fever virus (YFV) [] and Dengue 2 virus (DEN-2) [] infection.

Later studies performed utilizing complement-knockout mice led to a better understanding of the role of complement in boosting antibody and cell-mediated immune responses. It was observed that mice deficient in complement components (C3 and C4) or receptor (CD21/CD35) challenged with HSV produced a reduced IgG response to the virus and this was due to failure of memory B-cell generation in these mice []. The memory B-cell generation nonetheless was not altered in C3-/-, C4-/- and CD21/CD35-/- mice when they were challenged with vesicular stomatitis virus (VSV) or lymphocytic choriomeningitis virus (LCMV). The authors therefore proposed that the route of infection and replication capacity of the virus at the site of infection dictates whether complement system is required for enhancing the B cell response []. Later, Diamond and his colleagues further examined the role of complement in the development of acquired immunity during flavivirus infection. In particular, they showed that mice deficient in C3 or CD21/CD35 generate reduced virus-specific IgM and IgG response and are more susceptible to lethal infection []. Further, they also observed that different complement pathways participate differently in priming the adaptive immune response, thus alternative pathway deficiency results in reduction of T-cell responses, while classical and lectin pathway deficiencies result in reduction in B- as well as T-cell responses [].

Because complement deficiencies in mice were associated with exacerbated influenza disease [], efforts were also made to dissect the effect of complement on T cells. It was shown that C3 deficiency, but not CD21/CD35 deficiency, caused reduced priming of CD4+ and CD8+ cells in lung-draining lymph nodes and as a result reduced the recruitment of virus-specific CD4+ and CD8+ effector T cells in the lung []. Very recently, it has been shown that defective priming in influenza-infected C3 deficient mice is due to defect in DC-mediated transport of viral antigen to the draining lymph node []. Whether this is also due in part to direct effect of C3a and C5a on T cells is not clear at present. Importance of C3 in induction of virus-specific CD8+ T cells was also observed during LCMV infection though the mechanism remains unresolved []. In yet another study, it was shown that efficient generation of CTL response against HIV requires opsonization of virus with complement []. Importance of complement was also shown during ectromelia virus infection, wherein it was evident that genetic absence of C3, C4 and factor B causes earlier dissemination of virus and increased virus titer in the target organs [].

II) Complement evasion by viruses: diverse strategies

The last few decades have seen identification of many diverse strategies adopted by viruses to counteract the host complement. These include molecular mimicry of the host complement regulators, molecular piracy of the host complement regulators and utilization of complement receptors for cellular entry. In this section, we specify/detail how these strategies help viruses to thwart the complement attack.

Molecular mimicry as evasion strategy

The central step in complement activation is the cleavage of C3 by C3-convertases. This results in labeling of pathogens by C3b, which are then treated as "foreign" by the host complement system. On the host cells, this step is tightly regulated by a series of complement regulators belonging to a family of proteins termed as regulators of complement activation (RCA) []. Large DNA viruses, such as pox and herpes viruses, have been shown to encode homologs of RCA proteins []. It is believed that these viral regulators have been integrated into the viral genome during co-evolution as a result of horizontal gene transfer []. Sequence variations in the viral RCA (vRCA) proteins point towards diversification of these homologs after their acquisition. In addition to these, viruses also encode of homologs of non-RCA complement regulators as well as novel complement regulators with no structural similarity to human complement regulators to subvert the host complement attack.

The human RCA family members are composed of characteristic structures known as short consensus repeats (SCRs) or complement control protein (CCP) domains []. These elongated modules are arranged in tandem and vary in number from 4 to 59. Typically, CCP domain consists of approximately 60 amino acids that are organized into eight or less antiparallel -strands wherein the structure is stabilized by four invariant cysteines which link up through two disulfide bonds in 1-3 and 2-4 arrangement. The domain also contains an invariant tryptophan which is buried inside the small hydrophobic core [Norman et al JMB 1991 219(4):717-25]. Interestingly, this arrangement allows exposure of most side chains to the solvent resulting in a larger surface area compared to the proteins of similar molecular weight [muzammil rev]. Functionally, the RCA proteins regulate C3-convertases by accelerating their irreversible decay (decay-accelerating activity; DAA) as well as by acting as cofactor in factor I-mediated inactivation of C3-convertase subunits C3b and C4b (cofactor activity; CFA). The vRCA proteins mimic the human RCA proteins both structurally and functionally. They however vary in length from 2-8 CCPs [ref], though 2 CCP containing viral regulators have not yet been functionally characterized.

RCA homologs of poxviruses: The only subfamily that has been found to encode orthologs of complement regulatory elements within Poxviridae is the Chordopoxvirinae. A large number of viruses belonging to this sub-family encode vRCA into their genome as listed in Table 2. These viruses belong to genera Orthopoxvirus (e.g., vaccinia, variola, monkeypox, ectromelia, cowpox, camelpox and buffalopox), Capripoxvirus (e.g., goatpox virus, lumpy skin disease virus and sheep pox virus), Leporipoxvirus (e.g., myxoma and rabbit fibroma virus), Suipoxvirus (swinepox virus), Yatapoxvirus (e.g., yaba-like disease virus, yaba monkey tumor virus and tanapox virus) and Cervidpoxvirus (e.g., deerpox virus). Interestingly they exhibit greater than 90% sequence similarity to each other. Amongst these, vaccinia virus complement control protein (VCP), small-pox inhibitor of complement enzyme (SPICE), monkey pox inhibitor of complement enzymes (MOPICE), and ectromelia virus inhibitor of complement enzyme (EMICE) have been studied by various groups including ours, which has led to significant insight into pathogenesis of these viruses [ ].

The first vRCA to be discovered was VCP. It was identified when an attenuated strain of the vaccinia virus was found to have a major deletion towards the left end of the genome and one of the ORF encoded by the deleted region matched with human RCA proteins [Kotwal & Moss, 1988]. Later, it became clear that it was able to abrogate the antibody-dependent complement-mediated neutralization of the virus and play a role in vaccinia virus pathogenesis [ ]. Analysis of VCP ORF (C21L) showed that it is a four CCP module containing protein, which are connected by short linkers of 4 amino acids that provide flexibility to the protein []. Structural studies showed that each of its module fold to form a 6--strand structure [Wiles et al., 1997; Henderson et al., 2001].

Infection of cells with vaccinia virus showed that it is a major secretory protein of the virus, which is produced at the late stage of infection []. Its functional characterization demonstrated that the secreted VCP is capable of regulating complement activity [] owing to its decay-accelerating activity against classical (CP) and alternative pathway (AP) C3 convertases []. Later, it was recombinantly expressed in Pichia pastoris and further functional characterization which revealed that it also possesses cofactor activity against C3b as well as C4b []. Being a soluble protein, it was thought that VCP was able to inhibit complement only in solution, but subsequent studies demonstrated the ability of VCP to bind to heparin sulfate proteoglycans [ ] as well as the viral protein A56 [ ] which permits anchorage on the cell surface, thus providing a basis that VCP can additionally protect infected cells from the complement attack.

Understanding the functioning of any protein requires detailed understanding of its interaction with the target protein(s). For VCP, this was achieved by utilizing a multi-prong approach that included SPR studies, monoclonal antibodies (mAbs), deletion mutagenesis and chimeric proteins. The SPR study revealed that unlike human complement regulators, interaction of VCP with human complement proteins C3b and C4b follows followed a simple 1:1 binding model and possessed very fast on- and off-rates for the target proteins. These results therefore suggested that fast recycling of the viral regulator increases its effectiveness as inactivator. Further, it was also established that like human regulators, these interactions were also highly dependent on ionic strength [Sahu et al., 1998; Bernet etal., JVI 2004]. In an another study, binding of truncation mutants of VCP to C3b and C4b suggested that the first three CCP domains were sufficient for binding, but optimal binding was rendered by all the four CCPs []. The truncation truncated mutants also became handy to map the functional domains in VCP. The results pointed out that the cofactor activity was facilitated by the first three CCPs, while the minimum region required for the classical pathway decay acceleration activity was CCP1 and 2, but CCPs 3 and 4 were necessary for the absolute activity. Though this and other studies [Rosengard et al., 1999; Smith et al., 2003; Mullick et al., 2005] identified the minimum domains required for the functional activity, they failed to identify the domains critical for interacting with factor I during cofactor activity, and that required for dissociation of the catalytic subunit from C3 convertases during decay accelerating activity. These queries were subsequently answered by examining the activities of domain swap mutants wherein CCP modules of VCP were swapped with homologous modules of the human regulators DAF and MCP as they possess only decay and cofactor activity, respectively. It was explicit from the results that CCP1 of VCP imparts decay of the catalytic subunit and CCPs 2 and 3 recruit factor I for CFA [Ahmad et al., 2010].

Smallpox inhibitor of complement enzymes (SPICE) is another important poxviral homolog encoded by the highly virulent variola virus []. Because of its potent activity against human complement, it can serve as an excellent model of structural craftsmanship for complement inhibition. The protein was studied by multiple groups to comprehend the molecular basis of complement inactivation. Initial study by Rosengard et al., [ ] demonstrated SPICE to be 100- and 6-fold more efficient than VCP in inactivating human C3b and C4b, respectively. This was followed by a study by Sfyroera et al [ ] wherein SPICE was shown to be 75- and 1000-fold more potent than VCP in inhibiting classical and alternative pathway, respectively. Subsequently, in yet another study by Liszewski et al [JI 2006], SPICE was demonstrated to contain higher decay activity against the classical pathway C3 convertases than VCP. It was therefore very clear that SPICE possesses enhanced complement regulatory activity against human complement than VCP. Thus, the obvious question to be asked was what dictates this robust activity of SPICE? Since SPICE differs from VCP by only 11 amino acids, any one or more of these 11 residues in SPICE that were distinct from VCP were liable for this enhanced function. Utilizing an electrostatic modeling approach and mutagenesis experiments, Sfyroera et al (2005) predicted and showed the influence of a two residue substitution in VCP (E108K and E120K) in enhancing its C3b binding and C3b cofactor activity. What remained missing in the study was determining the contributions of these and other residues in C4b cofactor activity and decay acceleration activities. A systematic analysis of the contribution of each of the 11 variant amino acids of SPICE towards its complement regulatory activities indicated that substitution of four residues (H98Y/S103Y/E108K/E120K) are enough to make VCP as potent as SPICE and this was primarily due to interaction of these residues with factor I []. Notably, all these four residues were also found to dictate the human specificity []. Intriguingly, consistent with the species tropism of vaccinia virus, in our recent report we showed that VCP is a potent inhibitor of the bovine complement []. Importantly, we also demonstrated that species selectivity in VCP and SPICE is primarily dictated by the presence of oppositely charged residues in their middle domains [ref].

Other poxvirus RCA homologs that have been studied in detail are the monkey pox inhibitor of complement enzymes (MOPICE) encoded by monkey pox viruses [Liszewski et al., JI 2006; Chen et al., Virology, 2005] and ectromelia virus inhibitor of complement enzyme (EMICE) encoded by ectromelia virus [Moulton et al., JVI 2010]. MOPICE is composed of three CCP domains and a truncated fourth domain. Despite truncation, it has been shown to bind to both C3b and C4b and also possess cofactor activities against C3b and C4b. It however lacks both the CP and AP DAA. Interestingly, the presence of MOPICE is seen only in the more virulent strains of Congo basin and not in the less virulent strains of West Africa [Chen et al., Virology, 2005]. Recent in vivo studies however do not consider MOPICE as the only factor responsible for augmented virulence of Congo basin strain []. Unlike MOPICE, EMICE is a 4 CCP module containing protein and shares high homology with all the poxviral inhibitors of complement: it closely resembles VCP with 18 aa variation and 2 aa deletion, MOPICE with 19 aa difference and 1 aa deletion, SPICE with 26 aa difference and 2 aa deletion [Moulton et al, 2010]. Amongst all the poxviral inhibitors of complement enzymes, EMICE exhibits greatest divergence in CCP1. Functionally, recombinant EMICE regulates complement activation by factor-I mediated inactivation of C3b and C4b as well as decay of the classical pathway C3-convertase []. Because inhibitory activities of SPICE and VCP are consistent with the species tropism of variola and vaccinia, it would be interesting to determine whether MOPICE and EMICE also function in species specific manner.

RCA homologs of herpesviruses:

Need to add

Note: Add EMICE in neutralization. Moulton et al., JVI, 2010, 84(18): 9128-9139.

Moresoever, studies of Isaacs et al (1992) demonstrated that VCP was able to abrogate the antibody-dependent complement mediated neutralization of the virus [ ]. The first evidence for in vivo role of VCP OR role of VCP in pathogenesis was further established by studies with vaccinia virus lacking VCP which resulted to attenuated lesions in rabbits [ ]. A recent study by the same group compared the pathogenesis of wild type and VCP knockout virus in mice using an intradermal model of the virus infection [Girgis et al., JVI 2011]. Attenuation of VCP knockout vaccinia virus in wild type mice compared to C3-/- mice was correlated with accumulation of T cells in the vicinity of infection, enhanced neutralizing antibodies and reduced viral titers. These studies showed that VCP plays a role in virulence by reducing both antibody and T cell responses, thus providing valuable insight into how poxviruses modulate complement in pathogenesis/virulence and demonstrates that VCP can modulate the adaptive immunity.

CONCLUSION: Notwithstanding/despite the unknown etiology of many viral diseases, increasing studies provide evidences about critical association of complement activation with disease progression and pathogenesis. Such studies have also revealed a delicate balance between viral pathogenesis and the complement system. Viruses have ingenuisly decoded the human regulators and encoded orthologous homologs.

Immune evasion and immune modulation

CONCLUSION: Notwithstanding/despite the unknown etiology of many viral diseases, increasingly studies provide evidences about critical association of complement activation with disease progression and pathogenesis.

It is apparent from the above account that viruses are constantly under the peril of complement assault. Yet, most of them thrive well in the host and cause diseases to humankind and animals. It is, therefore, apparent that viruses have developed elusive strategies to evade host complement during co-evolution with their hosts.

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