A Study On The Complement System Biology Essay

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The complement system is an important part of the innate immunity and an effector component of the first line of immune defense against a variety of pathogens including viruses (1,2). It comprises of about 30 proteins and functions by initiating a cascade of reactions eventually leading to phagocytosis, inflammation and lysis of the target, and augmentation of the adaptive immune responses. The three major pathways known to activate the complement system are - 1) the classical pathway: triggered by antigen-antibody complex or recognition of pathogen-associated molecular patterns by host pattern recognition molecules such as C-reactive protein, serum amyloid P and SIGN-R1 (3-7), 2) the alternative pathway: triggered by hydrolysis of C3 that forms the initial fluid phase C3-convertase , 3) the lectin pathway: triggered by binding of mannose binding lectin (MBL) or ficolins and activation of the associated serine proteases (MASPs). All these pathways converge at the C3 activation step: the formation of C3-convertases, C4b,2a in the classical & lectin pathways, and C3b,Bb in the alternative pathway which cleave the C3 molecule into anaphylatoxin C3a and opsonin C3b. The C3b generated then interacts with the C3-convertases to form the C5 convertases, which initiates activation of the terminal pathway and MAC (membrane attack complex) formation to lyse target cells (1,8).

Although the complement system is typically described as the first line of immune defense against pathogens, a critical look at its functional repertoire suggests that the system is evolved to perform clearance of detrimental substances from the body. For example, (i) attachment of the complement component C3 to the pathogen surface leads to engulfment of the foreign pathogens, (ii) formation of the membrane attack complex (MAC, C5b-9) on the pathogen surface results in direct lysis of the pathogens, (iii) release of anaphylatoxic peptides (C3a and C5a) as a result of complement activation produces the local inflammatory response against pathogens, (iv) its activation enhances the pathogen-specific adaptive immune responses, (v) it prevents immune precipitation, and help solubilization and clearance of immune complexes from the circulation, (vi) it is also involved in negative selection of self-reactive B cells (9) and (vii) it helps in clearance of cellular debris and apoptotic cells.

As the complement system has evolved to perform a wide variety of tasks mentioned above, it is required to recognize a wide assortment of existing structures and also have plasticity to recognize the newly emerging structures. This very feature however may lead to recognition of even self structures leading to damage of normal host tissues and cells (10). Studies performed in this direction have shown that this is indeed true and complement components involved in activation process do not discriminate between the self and non-self and hence the host cells are equally in danger of destruction by autologous complement activation. Thus, to keep the harmful effects of complement proteins at bay, host cells are protected by a family of proteins called regulators of complement activation (RCA). The members of this family are both plasma proteins like- factor H, C4-binding protein (C4bP) (11-13) and membrane bound proteins like- decay accelerating factor (DAF; CD55), membrane cofactor protein (MCP; CD46) and complement receptor-1 (CR1;CD35). Structurally, these regulators are formed by complement control protein (CCP) domains, which are bead like structures linked together with linkers of 2-7 residues. NMR and crystal structures show that each CCP domain folds into a  barrel structure and contain four cysteines as invariant residues that form two disulphide bridges. The mechanism of regulation of RCA involves cleavage of C3b and C4b to non-activating components (cofactor activity) and/or dissociation of the C3/C5 convertases (decay-accelerating activity) (14,15). Apart from the RCA proteins, regulation at the cell surface is also effectively accomplished by CD59 that inhibits MAC formation. Because complement regulation is essential for protecting self tissues from autologous complement, defect in the above regulators are associated with various pathological disorders such as paroxysmal nocturnal hemoglobinuria (PNH) (16), age-related macular degeneration (AMD) (17,18), atypical hemolytic uremic syndrome (aHUS) (19) and membranoproliferative glomerulonephritis type II (MPGN2) (20).

Viruses, arguably the most thriving pathogens, are obligate intracellular parasites due to lack of metabolic machinery. They are thus exposed to a plethora of host immune defenses including the complement system, which is both capable and efficient in recognizing and eliminating the virions as well as virus infected cells (21). Thus, complement exerts a strong selective pressure on viruses. To counteract this, viruses have evolved diverse complement evading strategies. The stratagems employed by viruses against complement activation comprise - (i) mimicry of host complement inactivation strategy by encoding homologs of host complement regulatory proteins (e.g., pox and herpesviruses) (2,8,22,23), (ii) conscription of host cell surface complement regulators like DAF, MCP, CD59, or fluid phase regulators, like-factor H, C4BP (e.g. poxviruses, herpesviruses, retroviruses, orthomyxoviruses, paramyxoviruses, flaviviruses and togaviruses) (22,23) and (iii) deliberate use of host complement receptors for hushed entry into cells (e.g., herpesviruses, paramyxoviruses, picornaviruses, adenoviruses and flaviviruses) (22,23).

Herpesvirus saimiri (HVS), a member of the γ-herpesvirus family, infects T-cells and is a natural resident in squirrel monkey (24). Though it does not cause any disease in its natural host, infection in other New world primates like tamarins, common marmosets and owl monkey causes acute peripheral T- cell lymphoma within less than 2 months (25). In addition, this virus is able to transform simian and human T-cell in vitro (26,27). Previous studies have shown that like Kaposi's sarcoma-associated herpesvirus (KSHV) and -herpesvirus 68, HVS also encodes a functional homolog of human complement regulators composed of 4 CCP domains (28). Further, it has been proposed that the protein exists in soluble as well as membrane bound form as a result of post-transcriptional processing of mRNA (28,29). In addition, unlike other viruses, it also encodes a homolog of CD59 (30,31).

The first functional characterization of HVS complement control protein homolog (CCPH) showed that the surface expressed CCPH has the ability to inhibit the deposition of C3d on the target cell (32). Data from our lab have shown that like membrane CCPH, sCCPH also has the ability to inhibit the deposition of C3b on the surface of erythrocytes during complement activation (33). Further, we also showed that it regulates complement by mediating cleavage of C3b and C4b by acting as cofactor for factor-I (cofactor activity) and by accelerating the dissociation of C3-convertases (decay-accelerating activity) (33). More recent data from our lab have revealed that while CCP2 domain alone is able to display the limited cofactor and decay activity, all the four CCP domains are required for its optimal activity (34). Although the functional characterization and mapping of functional domains in sCCPH using deletion mutagenesis was performed earlier, these functional domains were not identified in the context of the whole molecule, and no efforts were made to fine map its functional sites. In the present study, I therefore, sought to identify the functionally important domains by using monoclonal antibodies, and functionally important residues by employing loss-of-function and gain-of-function mutagenesis.

2. Review of literature

2.1 Herpesviruses and their classification

There is a long list of herpesviruses which infect humans, non-human primates, and various other animals. In fact herpesviruses have been isolated from most animal species, and about 200 herpesviruses have been identified thus far. All herpesviruses are enveloped viruses containing large DNA. The International Committee on Taxonomy of Viruses (ICTV) is addressing the herpesviruses taxonomy since 1971. Initially, herpesviruses were grouped into three subfamilies based on their biological properties (Roizman et al. 1973) but in 1992, Roizman et al. pointed out some misclassification in it and thus subfamilies were divided into genera with the use of genetic and molecular data (Roizman et al.1992). In the latest release of ICTV (2011), Herpesviridae family has been divided into three subfamilies: Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae. It is important to point out here that one species, Iguanid herpesvirus 2, has not been included in any of the subfamilies.

Virion morphology is the primary criterion for inclusion or exclusion of entries into the family herpesviridae. The viruses included in this family are mostly spherical shaped of approximately 200 nm diameter (range 120-260 nm) and are composed of 4 important components: the core, the capsid, the tegument, and the envelope. The core: it contains the viral DNA in the form of a torus. The genomes are single copy of linear double-stranded DNA, and are densely packed into capsid. The size of herpesviruses genome studied to date ranges from 125 to 240 kbp, with the most elaborately characterized genomes being made up of about 70 to 165 genes. The capsid: it is an icosahedrons molecule held together by triplexes, comprising 162 capsomers, 150 of which are hexon and 12 pentons with 6 and 5 copies, respectively. Its external diameter is about 100 nm. The tegument: surrounding the capsid is the tegument, which contains 30 or more viral proteins and are structurally very poorly defined. It is believed that these proteins help the virus to adapt to host environment by shutting down the host protein synthesis, augmenting viral gene expression, and inhibiting host defense proteins. The envelope: it has a trilaminar appearance and seems to be made from pieces of altered cell membrane. Typically it contains many glycoproteins, which vary from virus to virus.

Unlike morphology, serology defines the herpesviruses in closely related groups. Typically, the enveloped glycoproteins are targeted by the subset of neutralizing antibodies which are major serological tools. Natural host of the virus is usually restricted and is a single species, though occasionally there can be inter-species transfers. In experimental models, certain members of alphaherpesvirinae can cross the species barrier and infect variety of other species, but beta and gammaherpesvirinae are very much restricted. While in their host, herpesviruses rarely cause severe symptoms of infection, and these are limited to individuals which are either immunocompromised or are very young. These viruses are known to be transmitted by aerosol and mucosal contacts.

Table 1.1 Classification of herpesviruses: subfamily and genus level identification of representative members (A.J.Davison 2005)

Formal name

Common name

Abbreviation

Subfamily Alphaherpesvirinae

Genus - Simplexvirus

Human herpesvirus 1

Human herpesvirus 2

Ateline herpesvirus 1

Bovine herpesvirus 2

Simiriine herpesvirus 1

Genus - Varivellovirus

Human herpesvirus 3

Bovine herpesvirus 1

Bovine herpesvirus 5

Subfamily Betaherpesvirinae

Genus - Cytomegalovirus

Human herpesvirus 5

Cercopithecine herpesvirus 8

Genus - Roseolovirus

Human herpesvirus 6

Human herpesvirus 7

Subfamily - Gammaherpesvirinae

Genus - Lymphocryptovirus

Human herpesvirus 4

Genus - Rhadinovirus

Bovine herpesvirus 4

Ateline herpesvirus 2

Human herpesvirus 8

Saimiriine herpesvirus 2

Murid herpesvirus 4

Cercopithecine herpesvirus 17

Herpes simplex virus (Type) 1

Herpes simplex virus (Type) 2

Spider monkey herpesvirus

Bovine mamillitis virus

Herpesvirus tamarinus

Varicella-zoster virus

Infectious bivine rhinotracheitis virus

Bovine encephalitis virus

Human cytomegalovirus

Rhesus monkey cytomegalovirus

Epstein-Barr virus

Movar virus

Herpesvirus ateles

Kaposi sarcoma-associated herpesvirus

Herpesvirus saimiri

Murine gammaherpesvirus 68

Rhesus rhadinovirus

HHV-1, HSV-1

HSV-2, HHV-2

AtHV-1

BoHV-2

SaHV-1

VZV, HHV-3

BHV-1, BoHV-1

BHV-5, BoHV-5

HCMV, HHV-5

RhCMV, CeHV-8

HHV-6

HHV-7

EBV, HHV-4

BHV-4, BoHV-4

HVA, AtHV-2

KSHV, HHV-8

HVS, SaHV-2

MHV-68, γ-HV-68

RRV, CeHV-17

2.1.1 Herpesviru saimiri

Herpesvirus saimiri (HVS) is a classical prototype of gamma 2 herpesviruses or rhadinoviruses which also includes human herpesvirus 8 or Kaposi's sarcoma-associated herpesvirus. HVS is a T-lymphotropic virus whose natural host appears to be the squirrel monkey (Saimiri sciureus). It has been seen that HVS causes latent infection in the squirrel monkeys, but with no apparent disease. Unlike in squirrel monkeys, it is known to cause fatal disease in marmosets and owl monkeys [].

Like other rhadinoviruses, HVS genome codes for several homolog of cellular counterpart including D-type cyclin, a G-protein coupled receptor, an interleukin-17, a superantigen homolog and the inhibitors of complement activation and apoptotic pathways. It has been demonstrated that the cellular homolog genes preserve the respective function. Though these functions are primarily not required for the transformation and pathogenesis of the virus, they are considered important for the pathogenic persistence in its natural host. The HVS strains have been sub-grouped into A, B and C based on the sequence divergence in the left non- repetitive terminal coding region essential for the pathogenicity and T-cell transformation. Among the different subgroups, subgroup C includes the most oncogenic forms and strain C488 from this group is capable of transforming human T-cell line to a stable growth. The antigen specificity and other essential functions of the parental T-cell clone are maintained in transformed cell. Based on preserved phenotypic function of the transformed cell, HVS provides excellent tool for T-cell immunology and as a gene delivery vector.

(i) Presence and infectivity

HVS is commonly and abundantly found in squirrel monkeys which are natural resident of South American rain forest. Though it infects squirrel monkeys via saliva within first two years of their life, it does not cause disease in this animal and establishes life long persistence (Melendez et.al 1968). Infection of HVS in different species can cause distinct results (Fleckenstein and Desrosiers 1982). While HVS is non-pathogenic to its natural host, it causes acute peripheral T-cell lymphoma in other new world primates such as Tamarins (Saguinus spp), common marmosets (callithrix jacchus) and owl monkeys (Aotus Trivirgatus) (Melendez et.al 1969, Wright et.al 1976) within two months of its experimental infection. For experimental infection, usually, intramuscular or intravenous injections are performed with a typical viral load of 106/ml of tissue culture. Even the isolated DNA can cause tumor when injected intramuscularly to the susceptible species (Fleckenstein 1978a). Different monkeys however differ in susceptibility to different subgroups. For example, tamarins are susceptible to all subgroup viruses whereas common marmosets are not susceptible to subgroup B viruses (Fleckenstein and Desrosiers 1982). The lymphoma in cynomolgus monkeys show wide spread infiltrates with different sizes of blast (Alexendra et.al 1997; Kannape et.al 2000a), which infiltrate into lymphatic organs like spleen, lymph nodes, Waldeyer's ring, and other organs (e.g., intestine, pancreas, liver, lungs and salivary glands).

During the early days of HVS research, experiments were performed using transformed T-cell lines, which were derived form leukemias or tumors of subgroup A or B virus infected tamarins (Fleckenstein and Desrosiers 1982). However, during the course of time, these cell lines stopped producing virus particles, e.g., cell line 1670 (Markzynska et.al 1973; Fleckenstein and Desroseiers 1982) and 70N2 (Falk et.al 1972b). Nevertheless, common marmoset and tamarin T-cells can be transformed by HVS for stable growth (Chou et.al 1995; Desrosiers et.al 1986). These HVS transformed cell lines are semi permissive in nature; they release virus particles. HVS subgroup C strain such as C488 can specifically transform human T-cell to stable growth in vitro (Biesinger et.al 1992). But, unlike semi permissive T-cell line from new world monkeys, human T-cell transformed with C488 do not produce virus particles (Biesinger et.al 1992; Fickenscher et.al 1996a, 1997). Similarly, transformation of T cells from old world monkeys like macaques, which are closer to human, result in a weak virus particle producing T-cell line (Alexendra et.al 1997; Kannape et.al 2000a). Although there are no reports of rodents infected with HVS, non-permissive infection and tumor induction was described in New Zealand white rabbits with variable efficiency (Ablashi et.al 1985).

Fig.1. Electron microscopy of herpesvirus saimiri. Left micrograph, fully enveloped mature HVS particle and nucleocapsid within inclusion body in cytoplasm of infected cells; Middle micrograph, HVS nucleocapsid seen within cytoplasm of infected cells; and Right micrograph, fully enveloped mature extracellular HVS particle with electron dense viral DNA. (Figure adapted from Fickenscher et al. [ref]).

(ii) Genomic composition and replication

The rhadinoviruses have genomes which are categorized as M type (M-DNA) as they have intermediate density in CsCl gradients. The M-DNA splits into two types of DNA with different densities on CsCl and hence named as low density L-DNA (Low G+C content), which contains all the viral genes, and high density H-DNA (High G+C content), which contains terminal repetitive region without the coding capability. In Greek, the word "rhadino" means fragile and hence γ2-herpesviruses are termed as rhadinoviruses (Roizman et.al 1992).

Fig.2. Genome depiction of Herpesvirus saimiri with its open reading frames and important genes indicated.

In the HVS A1 strain sequenced by Albrecht et al. [ref], there are multiple tendem repeats of 1444 bp with 70.8% G+C in H-DNA. The long unique L-DNA however, is made up of 112930 bp with 34.5% G+C (Albrecht et al. 1992a; Fleckenstein and Desrosiers, 1982). Both ends of the linear virion genome are capped by different numbers of H-DNA which result in variable size of total M-DNA genome. The highly oncogenic subgroup C strain (C488), sequenced by Ensser et al. [ref], contains 113027 bp long L-DNA flanked by two distinct repeat units of 1318 and 1458 bp. The longer repeat is 140 bp larger than the shorter repeat unit. Again, due to different number of terminal H-DNA segments, the M-genome has variable size ranging from 130-160 kbp with approx size of 155 kbp (Ensser et al. 2003). The L-DNA genome of HVS possesses at least 76-77 open reading frames and 5 to 7 U-RNAs (Albrecht et al 1992a; Hor et al. 2001; Ensser et al. 2003). These gene blocks belong to typical herpesvirus genes and are highly conserved in herpesvirus families (Gompels et al. 1988; Albrecht and Fleckenstein, 1990). The transforming oncogenes and viral homolog of cellular genes constitute the interspersed or flanking genes blocks which are usually not found in other herpesviruses families. Though most of the genes are conserved in different HVS strains, there are variations reported at the left end of HVS L-DNA in the region of glycoprotein gene orf51 and R transactivator gene orf50 (Biesinger et al. 1990; Thurau et al. 2000; Hor et al. 2001; Ensser et al. 2003).

The replication mechanism is not completely understood for rhadinoviruses in general and HVS in particular. The origin of lytic replication in strain HVS A11 was mapped to the untranslated region which is upstream of the thymidylate synthase gene (Lng and Fleckenstein, 1990; Schofield, 1994). In strain C484, the plasmid maintenance was described by the putative origin in the left-terminal region of L-DNA (Kung and Medveczky, 1996), but this is not conserved in different HVS strains, and is not required for viral replication or episomal persistence (Ensser et al., 1999; Medveczky et al., 1989). Herpesvirus saimiri is known to persist as stable non integrated episome with high copy number in transformed human T-cells (Biesinger et.al 1992). However, there is no information on the viral factors involved, and genetic correlation of plasmid like origin of replication.

Randall et al. in 1985 have shown that unlike herpes simplex virus, infection by HVS in tissue culture is asynchronous. Thus, classification of immediate early (IE) genes was difficult in HVS and data obtained was mostly using inhibition of protein synthesis by cycloheximide. Nuclear phosphoprotein of 52 kDa is encoded by IE gene ie57 (Hoyle et.al 1990; Nicholas et.al 1988). This protein shows homology with simplex virus gene ICP27/IE63 and performs similar function of post-translational regulation by repressing spliced form of gene while stimulating expression of unspliced form (whitehouse house et.al 1998a). In addition, ie57 also helps in RNA export (Goodwin et.al 199) and in redistribution of nuclear components of splicing machinery (Cooper et.al 199). Thus, it appears that ie57, a post-transcriptional regulator, is the only replication regulatory IE gene present in HVS.

A delayed early orf50 has been identified as a strong viral transactivator (Nicholas et.al 1991) and interestingly it bears homology to R transactivator of EBV. In HVS, owing to its differential splicing, orf50 codes for a large protein ORF 50A and smaller C-terminal variant ORF 50B (Whitehouse et.al 1997a). Among these, the C-terminus variant contains the transactivation domain and binds to the TATA-binding protein in the basal transcriptional complex. The genomic orf50 region encoding ie57 is highly divergent (Thurau et al., 2000; Ensser et al., 2003). There are no reports of HVS or HVA encoding homolog to bZip/Zta of KSHV or EBV (Sinclair, 2003). Like the latent nuclear antigen LANA of KSHV, the ORF73 protein of HVS strains A11 and C488 localizes to the host cell nucleus and also can associate with chromosomal DNA of host cell (Hall et al., 2000; Schafer et al., 2003). Further, studies performed suggest that transition between rhadinoviral latency and lytic replication can be controlled by HVS ORF73.

(iii) Cellular homolog encoded by HVS

Several homologs to cellular gene are found in the intronless viral genes of rhadinoviruses like HVS and KSHV, which indicate the role of reverse transcription during the capture process. Although, some of the genes are common to many rhadinoviruses including EBV, there are a few cellular gene homologs unique to specific viruses. Thus, the events of the uptake of cellular genes seem to be evolutionarily infrequent. The cellular homologs found in the virus can be broadly classified into two major groups: (i) cellular growth control or nucleotide metabolism genes, and (ii) adaptive or innate immunemodulatory genes.

Cell cycle control and genes of nucleotide metabolism

For nucleotide metabolism, orf2 codes for dihydrofolate reductase (DHFR) (Ensser et.al 1999), while orf70 codes for thymidylate reductase (Bodemer et.al 1984). In addition, the large tegument proteins coded by orf3 and orf75 reflect homology with formyl-glycineamide ribotide amidotransferase (FGARAT) (Albrecht et.al 2000). These enzymes might play role in DNA synthesis and hence viral replication (Helmut Fickenscher and Bernard Fleckenstein 2001). Orf72 of the virus codes for Cyclin D (Jung et.al 1994), which unlike cellular cyclin D, is not inactivated by cyclin-dependent kinase inhibitor p16, p21 and p27 (Swanton et.al 1997). This de-regulation might be helpful in compelling the cells to go in S phase and thereby enhance viral replication in permissive cells (Swanton et.al 1997).

Apoptosis inibitor

In HVS, the product of orf71 is a viral FLICE (FADD-like interleukin converting enzyme) apoptosis inhibitory protein (vFLIP), which interacts with cellular FADD (Fas-associated death-like domains) and FLICE via hemophilic interaction of their death-effector domains (Helmut Fickenscher and Bernard Fleckenstein 2001) to block the formation of the death-signal-induced complex and thus prevent FLICE (caspase 8) activation. The vFLIP has been shown to partially protect OMK cells from Fas-dependent apoptosis at the late stage of infection (Thome et.al 1997). Another ORF, orf16, shows homology with BcL-2 domains BH1 and BH2.

Genes for viral cytokines

The cellular homolog encoded by orf13 is IL-17, which is a CD4+ T-cell specific cytokine. It is important to point out here that studies on orf13 led to the identification of its cellular homolog CTLA-8 (Rouvier et al., 1993). Both cellular IL-17 and viral IL-17 are functionally identical (Yao et al., 1995; Fossiez et al., 1998). But, deleting orf13/vIL-17 from HVS C488 did not change its replication or oncogenicity (Knappe et al., 1998a). Apart from the vIL-17 which is unique to HVS, the G-protein coupled receptor common to rhadinoviruses is also found in HVS. The viral IL-8 receptor (IL-8R) encoded by orf74 is grouped into low affinity B type of IL-8R (Ahuja and Murphy,1993; Murphy, 1994; Nicholas et al., 1992).

Genes encoding homolog of murine superantigens

The viral IE gene IE14/vSag displays protein sequence homology with superantigen of mouse mammary tumor virus (MMTV), and also with murine mls superantigens (Thomson and Nicholas, 1991). Binding studies have confirmed that expressed IE14/vSag binds to MHC II molecules and stimulates T-cell proliferation yet, no evidence of selective advantage specific to Vb families are found, which is typical to superantigens (Yao et al., 1996; Duboise et al., 1998a), norr after HVS infection and transformation (Knappe et al., 1997). Gene deletion studies have indicated that this gene is not essential for replication and its role in transformation of human and simian T-cells in vitro and pathogenicity is controversial (Knappe et.al 1997, 1998b; Duboise et al., 1998a).

Homolog of complement regulatory protein

Like other gammaherpesviruses, HVS is also known to encode for a C3-convertase regulator. It is a gene product of orf4 and has been named as complement control protein homolog (CCPH) as it inhibits complement []. Analysis of orf4 transcripts showed that it encodes two transcripts as a result of alternative splicing of the gene: a longer of 1.7 kb and a shorter of 1.5 kb. The longer form encodes a regulator containing transmembrane region (membrane form; mCCPH), while the shorter form encodes a regulator lacking this region (secreted form; sCCPH) []. The present study focuses on the secretory form of the protein (sCCPH). Unlike other gammaherpesviruses, HVS also encodes a protein homologous to CD59 (product of orf15). Functional studies have shown that HVS-CD59 inhibits MAC formation []. Details on the structure and function of these proteins are described elsewhere (Section xx).

2.2 The complement system: Overview

The complement system is one of the most ancient and efficient mechanisms of immune defense employed by the host against pathogens (Hoffmann et al., 1999*). The existence of this system in living beings came long before the existence of immunoglobulins: the presence of C3-like molecules in non-vertebrate deuterostomes has been demonstrated by Al-Shariff et.al in sea urchins [] and Nonaka et al. in ascidians [], and in proterostomes like mosquito by Levashina et al. []. In addition, presence of lectin-associated serine protease in ascidians has also been demonstrated by Sekine et al. []. Thus, it is clear that the system has emerged at least 700 million years ago and has co-evolved with other innate immune mechanisms.

The word complement was originated when it was found that serum components complement the lysis of bacteria by antibody []. A simple and classic experiment by Jules Bordet in 1895 revealed that fresh serum enhances lysis of vibrios in the presence of heated immune serum (*). This study pointed out that serum contains heat labile factors that act against bacteria (pathogens). Subsequent, studies established that these factors are nothing but a group of heat labile proteins which help "amboceptors" or antibodies in early clearance of pathogens from blood circulation and hence the factors were rightly named as complement by Paul Enrich in late 1890's. These studies drew attention of scientific community and led to serial and sequential studies on identification and purification of complement components. We now know that the system comprises of more than 30 proteins (*) which include soluble as well membrane bound proteins.

Evolution of the complement system seems to have happened with a goal of clearance of detrimental substances from the body as the system appears to sense "danger signals" and act either directly or indirectly to eliminate the harmful entities. For example, i) attachment of the complement component C3 to the pathogen surface leads to pathogen engulfment by phagocytes, ii) formation of the membrane attack complex (MAC, C5b-9) on the pathogen surface results in their direct lysis, iii) release of anaphylatoxic peptides as a result of complement activation generates local inflammatory response against the pathogen, iv) complement prevents immune precipitation, and help solubilization and clearance of immune complexes from the circulation, v) it helps in clearance of apoptotic cells, vi) it also helps in selection of appropriate antigens for a humoral response by tagging them with C3 [], vii) it is involved in negative selection of self-reactive B cells [], and viii) it promotes the development of Th17 cells through synergistic interaction with toll-like receptor signaling and interleukin-6 production (PNAS plus), which play important role in controlling infection.

Apart from its above mentioned well investigated roles, recent studies have also implicated the role of complement in various biological processes like liver regeneration [], collective cell migration during development [], and activation of Wnt signaling to promote aging related phenotypes [], to name a few. Because the complement system has evolved to perform these wide varieties of tasks and its inappropriate activation is potentially hazardous to host cells [], it is important that it is well controlled. Consistent with this, activation of the complement system is effectively controlled on the host cell surface as well as in body fluids.

Figure 3. Complement-activation pathways. The complement system is activated by three main pathways: classical pathway (CP),lectin pathway (LP) and alternative pathway (AP). Activation of any of the three pathways results in the formation of C3 convertases (C4b,2a in case of CP/LP, and C3b,Bb in case of AP), which cleave the central C3 to form C3b, a major opsonin. Deposition of C3b on the virus targets it for neutralization by opsonization, aggregation, phagocytosis and lysis. Attachment of C3b to C3 convertases leads to the formation of C5 convertases (C4b,2a,3b or C3b2Bb,P), which cleave C5into C5a and C5b. The newly formed C5b if associated immediately with C6 starts the assembly of membrane attack complex C5b-9 resulting in virolysis. The cleavage products C3a and C5a are major anaphylatoxins that induce proinflammatory responses, chemotaxis and immune stimulation. The complement-activation pathways are tightly regulated at several steps by the regulators (human regulators are marked in blue, while viral inhibitors are marked in red).

AP: Alternative pathway; C3: Complement component; CP: Classical pathway;

DAF: Decay-accelerating factor LP: Lectin pathway.

The complement activation pathways

The defense barricade of complement system is formed by three major pathways namely- classical, alternative and lectin pathways. In addition, complement activation has also been reported by other less characterized pathways such as i) C2-bypass pathway wherein MBL/MBLassociated serine proteases (MASPs) were shown to activate C3 [J Clin Inves 2006; 116: 1425-1434], ii) activation of lectin pathway by pIgA [J Immunol 2001; 167: 2861-2868], and iii) direct cleavage of C3 [] and C5 [] by thrombin. Activation of all these pathways lead to formation of C3-convertases, which cleave C3 molecule and initiate the activation of terminal complement cascade leading to formation of membrane attack complex C5b-9.

The classical pathway

Activation of the classical pathway is initiated primarily by the antigen-antibody complex. In addition, the pathway is also activated by direct interaction of C1q, C-reactive protein (CRP) (**), serum amyloid protein (SAP) and specific intracellular adhesion molecule-grabbing non-integrin receptor1 (SIGN-R1) with microbial surface. Thus essentially, irrespective of the recognition molecule involved, C1q binds to the recognition unit and then causes activation of other components of the C1 complex, i.e., C1r and C1s. First, binding of C1q results in autoactivation of C1r, which is wrapped around C1q along with C1s, and this involves cleavage of Arg446-Ile447 bond in the protease domain. The active C1r then cleaves and activates C1s which in turn cleaves C4 to C4a and C4b. The larger fragment C4b generated tethers itself covalently to the activating surface by its TED domain and presents a site for C2 binding. The serine protease of C1s of C1 complex then cleaves the C4b bound C2 leading to the formation of C3 convertase C4b2a.

The lectin pathway

The relatively more recent and less studied pathway is the lectin pathway however, like the alternative pathway, it is also phylogenetically old and plays an important role in pediatric immune defense at the time when passive immunity achieved from maternal antibodies are not available and adaptive immunity is not mature enough. This pathway is known to be triggered in humans by five pattern recognition molecules namely, mannose binding lectins and three ficolins (H, L and M ficolins), and collectin 11 (CL11 or CL-K1), which recognize terminal mannose expressed on the surface of pathogens. Like C1q in the classical pathway, here, the pattern recognition molecules identified above form a complex with MBL-associated serine proteases (MASP-1, MASP-2 and MASP-3), MAp44 and MAp19 (alternatively splice product of MASP-2). Binding of the recognition molecule-MASP complex to the pathogen surface causes activation of MASPs leading to the cleavage of C4 followed by C2 cleavage resulting in formation of C3-convertase C4b,2a. Up until now it was believed that MASP-2 autoactivates and then cleaves C4 and C2 to form C3-convertase. The role of MASP-1 was not taken into consideration though it was shown to auto activate and cleave C2 (but not C4). A recent study however showed that activation of MASP-2 is strictly dependent on MASP-1 and about 60% of the C2 cleaved during activation of lectin pathway is mediated by MASP-1 [PNAS]. It is thus clear that presence MASP-1 is essential for activation of the lectin pathway.

The alternative pathway

The third pathway of complement which does not require any recognition molecule for its activation is the alternative pathway. This pathway being the most ancient pathway has unique methodology of its own wherein a low level spontaneous hydrolysis of complement component C3 by water molecule (generation of C3(H2O) or tick over process) leads to its association with a serum serine protease factor B (homologous to C2) in the presence of magnesium ions. Once in association with C3(H2O, fB undergoes conformational change which renders it for cleavage by factor D into 30 kDa Ba fragment and 60 kDa Bb fragment. The larger Bb fragment remains in association with C3(H2O) to form the initial alternative pathway C3-convertase, C3(H2O)Bb that cleaves C3 to form C3b while the smaller fragment Ba goes into systemic fluid. The newly generated C3b attaches to the activating surface (pathogens) and engages fB to form C3 convertase C3b,Bb resulting in positive feed-back loop. Though alternative pathway is initiated spontaneously, its activation can occur as a result of C3b deposition mediated by the classical or the lectin pathways. Thus it provides an amplification loop for the other two pathways causing deposition of millions of C3b molecules in minutes. The half-life of alternative pathway C3-convertases is very short (90s) but in vivo it is stabilized by a serum protein called properdin that increases the stability of the convertase upto 5-10 folds. More recent studies suggest that properdin not only stabilizes the alternative convertase, but also can act as initiator molecule by recognizing certain pattern on activating surface [ref]. Studies in mice and in vitro data have clearly shown the ability of properdin to recognize bacteria and altered self (apoptotic T-cells) and initiate the alternative pathway. Factor D is synthesized as a pro-factor D and its activated form is essential for the cleavage of factor B. The mechanism by which pro-factor D is converted to factor D was not clear until recently. A recent study however revealed that MASP1, the serine protease of lectin pathway, converts pro-factor D into mature factor D. These studies clearly indicate that different complement pathway do not work in isolation rather there is continuous cross-talk between them.

Activation of terminal complement cascade

As discussed above, activation of all the three pathways leads to formation of C3-convertase (C4b,2a in CP/LP and C3b,Bb in AP), which uses C3 as a substrate and cleaves it into C3a and C3b. The newly generated C3b can either form a new AP C3 convertase by attaching to the activating surface or can attach to the already formed C3-convertase to form C5-convertase enzyme (C4b,2a,3b or C3b,Bb,C3b). This C5-convertase subsequently cleaves complement protein C5 which results into a smaller anaphylatoxin fragment C5a and a larger fragment C5b; the C5b fragment initiates the assembly of pore forming membrane attack complex. It is now clear that cleavage of C5 to C5b induces a conformational change in the molecule leading to exposure of a labile binding site (half-life: 2 min) (Cooper and Mu¨ ller-Eberhard, 1970), which is accessed the C6 molecule. Next, C7 binds to this complex resulting in the formation of C5b-7 (trimer) which binds to the membrane, as the complex turns lipophilic owing to binding of C7 (Preissner et al., 1985; Stewart et al., 1987). Binding of C8 (a heterotrimeric molecule) to the lipophilic complex through C8 (Brannen and Sodetz, 2007; Stewart et al., 1987) initiates the membrane insertion event, where C8 penetrates the lipid bilayer (Steckel et al., 1983). The complex C5b8 now acts as receptor for C9 and facilitates oligomerization of 10-15 C9 molecule which leads to perforation of membrane and lysis of the target cell (Podack et al., 1982; Tschopp, 1984; Tschopp et al., 1985). Very recently Hadders et al [ref] have proposed a model for pore formation by MAC based on the crystal structure of C5b6 and cryo-electron microscopy reconstruction of soluble C5b-9. Their data suggests that MAC resembles the bacterial cholesterol-dependent cytolysins (CDCs) pores, which is different than the current model of pore formation by MAC based on perforin (Law et al., 2010). Apart from pore formation and lysis of cells, it has been reported that TCC stimulates activity in T helper cell polarization, [].

Regulation of complement activation

The snag of complement system lies in the fact that its activation proteins do not discriminate between self and non-self and hence the host cells are equally in danger of destruction by autologous complement activation. Thus, there must be regulation at the cell surface as well in fluid phase to keep the harmful effect of complement proteins at bay. Consistent with this contention, the host cells are protected by a family of proteins called regulators of complement activation (RCA). The proteins in this family are both soluble like- factor H, C4-binding protein (C4bP) (**) and surface bound like - decay accelerating factor (DAF; CD55), membrane cofactor protein (MCP; CD46) and complement receptor-1 (CR1; CD35). It is conceivable that effective regulation would require regulation at various levels of complement activation. Thus, complement regulation is also known to occur at the early and late stages of complement activation, which include regulation by C1-inhibitor (acts as protease inhibitor during initiation of enzymatic cleavage by - C1r, C1s, MASP1 and MASP2), CD59 (inhibits MAC formation) and carboxypeptidase N (inactivates anaphylatoxins) (**).

Regulation at initiation level:

The circulatory serine protease inhibitor C1 inhibitor efficiently inhibits classical and lectin pathway of complement activation by preventing the activation of C1r and C1s in the fluid phase and by inactivating MASP-1 and MASP-2. More recently identified complement C2 receptor inhibitor trispanning (CRIT) is a membrane bound inhibitor of the classical pathway. This inhibitor receptor is found on a wide range of tissues including hematopoietic cells and inhibits activation by binding to C2 and hampering its cleavage by C1s. In addition, MAp44 is another newly identified regulator of the lectin pathway which competitively inhibits binding of MASP-2 to MBL and ficolins [ref].

Regulation at the C3- and C5-convertase level:

In general, most of the regulatory proteins inhibit complement activation at the convertase level. Principally, they utilize two mechanistic approaches to restrain the convertase activity. The first approach is to dissociate the convertase components, leading irreversible decay of the enzymes. The convertases of both the classical (CP) and e alternative pathways (AP) are aptly and irreversibly dissociated by regulators of complement activation (RCAs) proteins like decay accelerating factor (CD55; decays AP and CP convertases), factor H (decays only AP convertases), factor H like protein 1 (decays only AP convertases) and CR1 (decays both AP and CP convertases). The other approach of regulation of convertases is inactivation of convertase subunits C3b and C4b by supporting the protease factor I. Here, RCA proteins serve as essential cofactors for factor I. This inactivation aims at minimizing the number of active C3b/C4b molecule by cleaving them into inactive forms. There are various proteins that serve as cofactors for factor I which are found both in fluid phase and membrane bound form. The fluid phase cofactors are C4-binding protein (C4BP; supports C4b inactivation), factor H (supports C3b inactivation), and membrane bound cofactors are MCP (CD46; supports C3b and C4b inactivation) and CR1 (CD35; supports C3b and C4b inactivation).

Figure 4. Complement regulatory activities of RCA proteins. (A) C4b cofactor activity: the viral or host RCA binds to C4b, the subunit of CP/LP C3-convertase, and acts as a cofactor for its cleavage and inactivation by factor I into C4c and membrane-bound C4d. (B) C3b cofactor activity: the viral or host RCA binds to C3b, the subunit of AP C3-convertase, and acts as a cofactor for its cleavage and inactivation by factor I into C3f and membrane-bound iC3b. (C) CP/LP decay-accelerating activity: the viral or host RCA binds to the bimolecular CP/LP C3-convertase (C4b,2a) and irreversibly dissociates it into its subunits. (D) AP decay-accelerating activity: the viral or host RCA binds to the bimolecular AP C3-convertase (C3b,Bb) and irreversibly dissociates the convertase into its subunits. Abbreviations - AP: Alternative pathway; C3: Complement component 3; CP: Classical pathway; LP: Lectin pathway; RCA: Regulators of complement activation.

Although factor H is a fluid phase regulator, it can also regulate complement activation on cell surface by virtue of its ability to bind the glycosaaminoglycans and sulphated polysaccharides present on host tissue. Thus, it efficiently protects own cell from the damage of AP activation. The other AP C3/C5 convertase inhibitor functioning at the convertase level is the complement receptor of immunoglobulin superfamily (CRIg) which is a macrophage complement receptor and plays important role in scavenging of pathogens. Unlike other C3 convertase regulators, CRIg binds to C3b and inhibits the C3 and C5 convertases by blocking their interaction with their substrates i.e., C3 and C5. In a recent report, it has been suggested that human plasma protein β2-glycoprotein I (β2GPI) upon surface binding such as to apoptotic cells, acquires ability to bind C3/C3b and helps in subsequent degradation by factor H and factor I (blood paper).

Regulation at the MAC level:

Even if complement activation is not efficiently regulated at upstream of the cascade, there is regulation at the end of cascade, by inhibiting MAC formation. This feat is achieved by membrane bound regulator CD59, and fluid phase regulators vetronectin, clusterin and the most recently identified fH related protein 1 (FHR1) [ref]. The membranous protein CD59, is a GPI anchored glycoprotein and is a intrinsic regulator of complement activation at MAC level. It interferes with the MAC formation by binding to both C8 and C9 and limiting the polymerization of C9. Vitronectin and clusterin are chaperons which can bind to the C5b6 complex wherein they inhibit the rearrangement of loosely folded α-helix to form lipophilic β-barrels thereby blocking its lipid binding site and hence the insertion of C5b67 to the membrane is prohibited which ultimately inhibits MAC formation (cell paper on C5b6). The complement regulatory protein CFHR1 is a plasma protein, which shows significant sequence homology with fH but its regulatory mechanism is different from fH. This newly found regulator inhibits the alternative pathway at the C5 convertase and at the MAC level. It has been suggested that FHR1 prevents cleavage of C5 into C5a and C5b. In addition, FHR1 also binds to C5b67 and prevents its insertion and thus the MAC formation.

2.3 Complement and viruses

Viruses, the most successful pathogens, [and arguably the most intelligent-Malik: viruses do not have brain], are obligate parasites owing to lack of metabolic machinery and hence use host cells for their propagation and survival. The host immune systems including the complement system on the other hand are able to recognize both cell free virions and virus-infected host cells as potential threat and have the potential to eliminate them. This seesaw between host immune system and viruses have led to coevolution of viruses as well as the immune system. Viruses in particular have developed multiple strategies to evade the immune system. Since complement system is one of the important defense in controlling viruses, it has also became a viral target. The various stratagems employed by viruses against complement are- (i) imitating host complement inactivation strategy by encoding homologs of host complement regulatory proteins (e.g., pox and herpesviruses) (**) (ii) conscription of host cell surface complement regulators like-DAF, MCP, CD59, or fluid phase regulators, like-factor H, C4BP on their surface (e.g. poxviruses, herpesviruses, retroviruses, orthomyxoviruses, paramyxoviruses, flaviviruses and togaviruses) (**) and (iii) deliberate use of host complement receptors for hushed entry into cells (e.g., herpesviruses, paramyxoviruses, picornaviruses, adenoviruses and flaviviruses) (**).

Complement-mediated virus neutralization

It is well known that viruses activate the complement system and eventually get neutralized by them (xxx). All the three complement activation pathways have the ability to neutralize viruses with different recognition mechanism, but with common effector mechanism. Many successful viruses however have developed strategies to subvert complement and are also known to exploit it to infect cells. Viruses which activate classical pathway after recognition with antibodies include, bacteriophages (xxx), herpes simplex virus (xxx), Japanese encephalitis virus (xxx), vesicular stomatitis virus (VSV) (xxx), influenza virus (xxx) and hantavirus (xxx). Further, there are reports which indicate that C1q can directly bind to viral surfaces proteins and activate CP. For example, p15E of oncornavirus (xx), gp41 (xxx) and gp120 (xxx) of HIV-1 and gp21 of human T-cell leukemia virus type I [HTLV‑1] (xxx) are known to be directly recognized by C1q leading to CP activation. Structural glycoprotein motifs on the viral surfaces are recognized by MBL leading to activation of LP. Viruses which have been shown to activate LP include various oncolytic viruses (xxx), hepatitis C viruses (xxx), influenza virus (xxx), HSV-2 (xxx) and HIV (xxx). As activation of AP does not depend on any kind of recognition molecule, it can virtually be activated by any virus. Factors that influence activation include propensity of the viral surface to attach C3b and recruit factor H. Thus, presence of GAGs on the viral surface play important role in AP activation, as they can recruit factor H to regulate AP activation (discussed later in review of literature). Viruses which are known to activate AP include Epstein-Barr virus (EBV) (xxx), Sindbis virus (xxx), Sendai virus (xxx), simian virus 5 (xxx), VSV (xx), mumps virus (MuV) (xx) and measles virus (MeV) (xx).

The inability of viruses to control complement activation on their surface results in onset of effector mechanisms which ultimately eliminate viruses. The various effector mechanisms are: neutralization by aggregation and opsonization, neutralization by phagocytosis, and neutralization due to direct lysis.

Neutralization by aggregation & opsonization

Antibodies are known to cause aggregation of viruses which result in their neutralization due to reduction in infectious units available for infection. It is an established fact that like antibodies, complement components can also cause aggregation of viruses. It has been shown by Oldstone et al. that in the presence of specific antibodies, C1-C3 play critical role in aggregation and neutralization of a polyoma virus (xxx). More importantly, C3b deposition on viral surface was shown to be essential for this aggregation. Because C3b is not a multivalent protein, aggregation by the same is not a possibility. It was proposed by the authors that polyoma virus possesses a C3b receptor, which help aggregation. Complement-mediated aggregation and neutralization of viruses has also been shown in case of influenza and simian virus. However what induces aggregation is not clear in these cases. The multivalent complement proteins which can promote aggregation and neutralization include C1q, MBL and properdin. These however have not been to aggregate any viruses thus far.

(b) (c) (d)

Fig 5. Complement mediated neutralization of viruses; (a) opsonization-mediated by complement protein C3b, (b) Aggregation involving C1, (C) Direct lysis of virus via MAC and (d) Chemotactic movement of phagocytes guided by small fragments of complement C3a and C5a resulting in phagocytosis of opsonized viruses.

Viruses opsonized with complement proteins can also be neutralized without undergoing aggregation or virolysis. This condition causes coating of viral surfaces with complement proteins which produces steric inhibition and thus viral surface proteins/receptors are no longer available for host cell attachment hampering viral entry. Different complement proteins are known to mediate neutralization of different viruses. For example, C1q is known to neutralize HTLV-1 (xxx) and influenza (xxx), C1-C3 are known to neutralize HIV-1 (xxx) and West Nile virus (xxx), and C1-C5 are known to neutralize glycoprotein C (gC)-null HSV-1 and -2 (xx).

Neutralization by phagocytosis

Following complement activation, activated complement proteins like C3b and C4b are attached to virion surface. These complement components and their cleavage products, such as iC3b and C3d, can then be recognized by phagocytes by means of various complement receptors (CRs), such as CR1 (CD35), CR2 (CD21), CR3 (CD11b/CD18), CR4 (CD11c/CD18), and the more recently identified CR-Ig. Two viruses namely HSV (XXX) and Japanese encephalitis virus (xxx), have been shown to be neutralized by phagocytosis.

Neutralization by direct lysis

Complement activation ultimately culminates in the formation of the MAC, which after insertion into the membrane of enveloped viruses, causes disintegration of membrane, degeneration of the nucleocapsid and, finally, virion neutralization due to virolysis. It is well known fact complement mediates lysis of several viruses, such as herpesviruses, coronaviruses, alphaviruses, and retroviruses. There are reports on paramyxovirus, MeV (xxx) and, more recently studied on MuV (xxx), where it has been shown that these viruses also neutralized by virolysis. However, complement mediated neutralization of HIV-1 is under debate due to inconsistent results by various groups. Despite complement activation and deposition of complement proteins on viral surface, HIV-1 has been shown to resists virolysis in vitro. I was proposed that inhibition of virolysis is due to incorporation of host complement regulatory proteins on viral membrane (xxx). In contrast to this finding, other groups have shown that HIV-1 is indeed susceptible to complement mediated lysis in presence of neutralizing antibodies (xx). In a recent study, Huber et al. (xxx) have shown that during acute infection complement mediated lysis of HIV-1 in patient sera is inversely proportional to viral load which supports the earlier finding.

Complement evasion by viruses

Though complement inflicts paramount challenge to viruses following their entry into host, many of them not only survive rather use the host complement regulators to evade deleterious effect of the complement system. The commonly employed mechanism to evade complement attack are: i) encoding complement homolog, ii) recruiting host complement regulator, iii) silent entry into host cell with the help of certain complement proteins.

Encoding homolog of complement regulator, mimicry at molecular level

As discussed earlier in this section, host cell express complement regulators to protect from autologous complement mediated damage. These regulators belong to the family of regulators of complement activation (RCA) [ref] (xxxxx). Genome sequencing of viruses have reveled that two families of DNA viruses namely poxviridae and herpesviridae encode homologs of RCA proteins. It is believed that these cellular genes have been acquired by these viruses from their hosts by horizontal gene transfer. Analyses of the amino acid sequences of these viral homologs suggest that the acquired proteins have been modified to the present form.

[Malik: you may like to add Tables for every strategy]

Poxviral RCA homologs

Chordopoxvirinae is the only subfamily of family Poxviridae which has been found to encode complement regulatory homologs. There are a number of viruses in this subfamily, which encode for complement control homologs, but they belong to different genera like- Orthopoxvirus (e.g., vaccinia, variola, monkeypox, cowpox, camelpox and ectromelia), Suipoxvirus (swinepox), Leporipoxvirus (myxoma and rabbit fibroma virus) and Yatapoxvirus (yaba monkey tumor virus). Though the sequence homology with human RCA is only about 30% yet the sequence homology within this family is more than 91%. Among the several poxviral RCA homolog, the homolog of vaccinia, variola and monkeypox viruses have been characterized and studied in depth to reveal the complement inhibitory properties. Vaccinia virus encoded RCA homolog, known as VCP, is the first to be identified as complement regulatory molecule (XXX). VCP is one of the two major proteins secreted by vaccinia virus-infected cells. It is 263 amino acids long, and a four CCP-containing protein, encoded by the C21L gene of the viral genome. Structural studies revealed that each CCP module folds to form a compact 6--strand structure (XXXX). Initial functional characterization using VCP produced by the virus-infected cells showed that the molecule binds to C3b and C4b, and accelerates the decay of the CP, as well as AP, C3 convertases (XX). Due to the soluble nature of VCP it was generally thought that it is important for complement inhibition only in solution. Reports of VCP binding to heparin sulfate proteoglycan (XXX) and also to viral protein A56 (XXXX), which allow its anchorage to the cell surface clearly indicate that VCP can also protect the infected cells from complement mediated damage. The presence of VCP protects vacinia virus from antibody-dependent complement mediated neutralization (XXX). It has been reported that absence of VCP causes attenuated lesions in rabbits (XX). Extensive functional characterization studies have revealed that, VCP inhibits complement activation by serving as a cofactor during factor I-mediated cleavage of C3b and C4b, and also by enhancing the decay of C3-convertase. Since VCP binds to both C3b and C4b, many groups attempted to dissect the domain requirement of VCP for binding to these complement proteins (XXXXX). The binding data of deletion mutant indicated that three N-terminus module are enough for ligand binding, but optimum binding requires the presence of all the four domains. Though different domains facilitate different function, all the four domains were found to be essential for its optimal function (XX). Data from our lab have revealed that CCP2 and CCP3 provides putative factor I binding site, while N-terminal CCP1 enhances decay of convertases (XXX). A molecule analogous to VCP is also encoded by variola and is referred to as SPICE (smallpox inhibitor of complement enzymes) (XXX). Due to its invariant presence among all variola strains, SPICE was thought to have a possible role in pathogenesis. Another poxviral homolog of RCA is encoded by monkeypox virus and is known as MOPICE (monkeypox inhibitor of complement enzymes) (XXX). This homolog however is present only in the highly virulent strains of the Congo basin, and not in the less-virulent strains of West Africa (XXX). The contrasting and interesting difference between MOPICE and other poxviral homolog is that MOPICE has only three CCP in contrast to 4 CCP in other pox viral homologs; MOPICE is truncated at 4th CCP. Similar to VCP and SPICE, MOPICE also binds to C3b and C4b, and have cofactor activity while it does not have decay accelerating activity (XXX). Deletion data from our lab on VCP have indicated that absence of CCP4 in VCP causes severe loss in decay accelerating activity, thus it can be inferred that absence of 4th CCP in monkeypox is responsible for the absence of decay activity (XX). Efforts have also been made to characterize RCA homolog from cowpox virus. It was shown that the protein has a modulatory effect on complement-mediated host inflammatory responses in vivo and, hence, named as inflammation modulatory protein (IMP) (XX).

Fig 6. Structural representation of various pox and herpes viral RCAs (vRCAs). All vRCAs are structurally similar to human RCAs and are composed of characteristic structural domains called CCP modules. S/T indicate serine threonine rich region, while TM represent transmembrane domain. The predicted N-linked glycosylation sites have been represented by stick with diamond head and stick with oval head indicate predicted O-linked glycosylation sites. Domains required for various activities in sCCPH, VCP, and KAPOSICA have been identified [Malik identify domains properly, do not start your lines from the middle of the domains?????].

Herpesviral RCA homologs

Analogous to poxviruses, the subfamily of Herpesviridae that encodes RCA homolog is Gammaherpesvirinae. Among several herpesviruses encoding RCA homolog, herpesviruses whose RCA proteins have been characterized include Kaposi's sarcoma-associated herpesvirus (KSHV), herpesvirus saimiri (HVS), murine -herpesvirus 68 (-HV68) and rhesus rhadinovirus (RRV). In contrast to pox viral complement regulators where sequence identity is more than 90 %, herpesviral homolog exhibits sequence similarity that varies between 43 and 89%. It is therefore apparent that herpesviral RCA homolog are less identical and might have subtle differences in their function or mechanism of action. Unlike poxviral regulators, herpesvirual regulators have glycosylation sites and glycosylation is found in both soluble as well in membrane form. The ORF4 gene of HVS and KSHV encodes for a membrane- bound RCA homolog, while HVS also encodes soluble form due to differential splicing (xxx).The functional characterization of KHSV was performed by two different groups independently and simultaneously, thus the protein was named Kaposica (xxx), and KCP (xxx). Kaposica is composed of four CCP modules, a dicysteine motif, a serine/threonine (S/T)-rich region and a transmembrane domain (xxx). The protein has three putative N-glycosylation sites while the S/T region carries several O-glycosylation sites. Like poxviral regulators, Kaposica also possesses heparin binding sites which is located in its first CCP domain (xxxx), and this heparin binding site has been shown to be important for its binding to Chinese hamster ovarian cells through GAGs (xxx). Characterization of Kaposica for its function disclosed that similar to VCP and SPICE, it also interacts with both C3b and C4b, and act as cofactor for factor I-mediated inactivation of these proteins. Further, it also decays CP C3-convertases more efficiently than AP C3-convertases (xxxxx). Domain requirement characterization of Kaposica indicate that two N-terminal domains are critical for CP DAA, the internal domains CCP2 and CCP3 are vital for the cofactor activities. Nevertheless all the domains are important for its optimal activity (xxx). Very recent study from our lab has convincingly proved that positive charge in CCP1 and CCP4 of Kaposica plays critical role in its activity (xxx)

Apart from Kaposica, RCA homolog of murine -HV68 has also been characterized. Structurally, it has four CCP domains, and is expressed as both membrane-bound and soluble forms. It is believed that the soluble protein is generated as a result of proteolytic cleavage of the membrane protein (xxx). Functionally, it has been shown that -HV68 RCA efficiently inhibits murine C3 deposition on zymosan particles via CP, as well as AP, but the detailed mechanism is not clear (xxx). Role of -HV68 in pathogenesis has been studied in detail and has been found that lack of -HV68 RCA causes less infection (xxx).Other herpesviral RCA homologs that were characterized are those of HVS (xxx). In HVS, it has been shown that HVS-CCPH (sCCPH) possesses both cofactor and decay activity and all the 4 domains are required for its optimum functioning (xxxx).

Evasions by acquiring or inducing host complement regulatory proteins

Host cells are lined with various complement regulators on their surface, such as, DAF (CD55), MCP (CD46) and CD59. Thus, it is conceivable that during maturation by budding, viruses acquire one or more of these regulators which protect them from host complement. The well known examples for this strategy are herpesviruses, retroviruses, poxviruses, paramyxoviruses and orthomyxoviruses.

A study employing mAbs against human MCP and DAF demonstrated that the cofactor and decay activities of HCMV virions could be inhibited using these antibodies, suggesting that HCMV acquires complement regulators from host cell surface during budding and these regulators inturn provides protection against comp

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