Inflammatory reactions play an important role in the early nonspecific protection of the organism against the viral infection. They are induced rapidly to limit the virus dissemination during the first hours and days upon infection while the full-fledged adaptive immune response is being formed. It is known that the complement system and the cytokines, such as tumor necrosis factor (TNF), interleukin-1β
), gamma-interferon (γ
-IFN), and chemokines, play the key role in inducing the inflammatory reactions [118
]. In addition, several other mediators influence either directly or indirectly the development of the inflammatory process [116
]. Therefore, poxviruses potentially need several genes whose protein products are able to act as inhibitors of various stages of inflammation development to suppress efficiently the inflammatory response.
The first viral gene whose product represses inflammatory response to infection was found in CPXV and termed SPI-2 (B12R
for CPXV-GRI, see ) [122
]. As noted above (see Section 3
), SPI-2 inhibits caspase 1 activity and thus prevents the processing of pro-IL-1β
and its secretion from the infected cell, suppressing, as a result, the induction of local inflammatory reactions. In addition, SPI-2 inhibits the production of inflammatory mediators (leukotrienes) in the arachidonic acid metabolism [123
]. Furthermore, as discussed above, SPI-2 is also involved in suppressing apoptosis of the infected cell. Thus, this protein evidently plays an important role in determining orthopoxvirus pathogenicity in vivo
. It should be noted that amino acid sequences of SPI-2 variants present in VARV, MPXV, and CPXV are somewhat different [32
]. In the case of VACV-COP, the gene encoding this protein is damaged ().
It was shown experimentally that the VACV-WR gene B15R
encodes a secreted glycoprotein acting as a soluble IL-1β
]. The production of this soluble receptor prevents the development of systemic reactions (such as fever) in VACV-infected mice [125
]. It was shown that a VACV-WR strain with disrupted B15R
had increased virulence in mice (when administered intranasally) [124
]. A further analysis showed that VACV strains associated with a higher frequency of postvaccination complications in humans lack IL-1β
-binding activity [125
]. These data agree well with the fact that the respective gene in VARV is disrupted (by fragmentation) ().
Thus, we may hypothesize that VARV suppresses production and secretion of IL-1β by infected cells but does not inhibit the effect of extracellular IL-1β synthesized by other cells of the body. This suggests that VARV is capable of suppressing local inflammatory reactions due to SPI-2 production in the region of virus replication; however, it does not inhibit the systemic reactions, as it is unable to synthesize IL-1β-binding protein. Decrease in the local inflammatory reactions may assist a more active virus replication, while uncontrolled development of the systemic reactions weakens the overall resistance of the organism to infection. A concurrent development of these reactions is likely to boost the pathogenic effect of the viral infection on the host organism. In the case of MPXV and CPXV, both genes in question are native ().
Orthopoxviruses, in particular, VACV-WR, but not the less virulent VACV-COP strain, also encode an IL-18-binding protein (), which is secreted from the cell and suppresses the activity of proinflammatory IL-18 [126
Similarly to other cytokines, TNF performs multiple functions [118
]. In particular, as noted above, it is a key cytokine inducing inflammation in the infected host along with IL-1β
and IL-18. It was shown that VARV-IND gene G2R
encodes CrmB protein homologous to type II TNF receptor [127
]. An orthologous TNF-inhibitory protein called M-T2 is an important secreted virulence factor of the rabbit myxoma virus (a poxvirus of the genus Leporipoxvirus
). Its VARV analogue G2 apparently has similar properties. An important difference between VARV and VACV is that the latter possesses no genes encoding TNF receptor analogues. In the CPXV genome, we detected five genes of the TNF receptor family [23
]. Four of them have TNF-binding activity [128
An analysis of amino acid sequences of CrmB isologs detected numerous species-specific differences. Using a baculuvirus expression system, we obtained individual CrmB proteins of VARV, MPXV, and CPXV and showed that their ability to suppress the activity of human, mouse, and rabbit TNFs differs considerably. Only CrmB-VARV inhibits human TNF activity with high efficiency [132
]. Presumably, this is a result of evolutionary adaptation of the viral receptors to the ligands of their hosts.
It was recently shown that orthopoxvirus TNF-binding protein CrmB possesses a further biological activity; that is, it has high affinity to certain chemokines critically involved in attracting dendritic cells, B-, and T-lymphocytes to the inflammation focus [134
]. Its immunomodulatory activity is determined by the unique C-terminal domain termed SECRET (smallpox virus-encoded chemokine receptor). The amino acid sequence of this domain has no homology to any vertebrate protein or to any other known viral chemokine-binding protein. De novo
modeling of the spatial structure of the SECRET domain showed that it might be a structural homologue of the secreted CC-chemokine-binding protein G3 of VARV (), in spite of the low similarity of their amino acid sequences [135
Chemokines are chemoattractant cytokines, which control migration and effector functions of leukocytes, thereby playing an important role in development of inflammatory response and protection against pathogens [136
]. It was demonstrated that VACV strain Lister at the early stages of infection produced a protein secreted from the cells in large amounts [137
], which bound a wide range of CC chemokines and inhibited their activities [138
]. This gene is damaged in many other VACV strains. Presumably, isologs of this protein (G3 in VARV-IND) of various orthopoxvirus species have different functions, as analysis of their amino acid sequences detected considerable species-specific distinctions [3
The VACV protein A41 and its orthopoxvirus isologs also are secreted glycoproteins that efficiently and selectively bind to certain CC and CXC chemokines preventing chemokine-induced leukocyte migration to the infection locus [139
]. This chemokine-binding protein probably is essential for virus propagation, since it is conserved in all orthopoxvirus species studied ().
Interestingly, all orthopoxviruses in question also produce a soluble γ
-IFN-receptor, which can modulate the host's inflammatory response to infection [141
]. The protein B9 of VARV-IND and its isolog produced by VACV-COP contain a considerable number of amino acid substitutions [144
]. Probably, these species-specific differences in the structure of viral γ
-IFN-binding protein are related to the difference in VARV and VACV virulence.
In addition to the above genes, orthopoxviruses also carry a gene of a complement-binding protein (C3L
in VACV-COP) [145
], one of whose functions may be regulation of inflammation. The complement system comprises over 20 blood plasma proteins. Antiviral functions of the complement systems include virus neutralization, lysis of infected cells, and enhancement of inflammatory and adaptive immune response [2
The VACV protein C3, named VCP, secreted from infected cells and controlling the reaction of complement activation comprises four short degenerated repeats of approximately 60 amino acids each (short consensus repeat, SCR) characteristic of the protein family of complement activation regulators (RCA) [146
]. It is considered that the gene encoding VCP originated initially due to incorporating a part or the complete coding sequence of a protein belonging to RCA family of the host into the viral genome followed by adaptation (alteration) of the gene in question to perform the functions necessary for the virus [147
]. The X-ray structural analysis showed that SCR sequences of VCP form a series of discrete tightly linked compact domains [148
VCP is a unique multifunctional viral protein functionally resembling as different RCA proteins as factor H, membrane-bound cofactor protein, type I complement receptor, and decay-accelerating factor (DAF). Firstly, VCP binds complement components C3b and C4b; secondly, it blocks different stages of the complement cascade and inhibits both the classical and the alternative complement pathways; thirdly, it blocks complement-driven virus neutralization activated by antiviral antibodies, and, finally, binds heparin-like molecules on the surface of endothelial cells, blocking the binding of chemokines and preventing signal transduction for chemotaxis [119
]. The model of CPXV-infected mice showed that VCP suppresses inflammatory response in vivo
VCPs of VARV, CPXV, and VACV contain four SCRs each. We have revealed the unique structure of the MPXV VCP [3
]. Due to premature termination of synthesis, the protein sequence is truncated and the C-terminal SCR-4 is deleted in Central African MPXV strains, whereas Western African MPXV strains lack the gene for VCP completely [3
]. Possibly this deletion or truncation of the gene for VCP prevents effective inhibition of inflammatory response by MPXV and therefore the specific feature of human monkeypox clinical course, distinguishing it from smallpox, is lymphadenitides.
Amino acid sequences of VACV and VARV VCPs differ at 12 positions. A baculovirus system was used to produce individual VCPs of VARV and VACV [152
]. It was shown that VCP of VARV is a significantly more efficient inhibitor of human complement than its VACV counterpart. This observation further supports the concept that viral soluble receptors are evolutionary adapted to the host's ligands.
To sum up, orthopoxviruses possess a multigene system controlling at different stages development of the host's inflammatory reactions. Orthopoxviruses display species-specific distinctions not only in the set of these genes but also in their structure, and as a result in targeted activities of the encoding proteins.