Role of cytokines in poxvirus host tropism and adaptation

Abstract

Poxviruses are a diverse family of double-stranded DNA viruses that cause mild-to-severe disease in selective hosts, including humans. Although most poxviruses are restricted to their hosts, some members can leap host species and cause zoonotic diseases and, therefore, are genuine threats to human and animal health. The recent global spread of monkeypox in humans suggests that zoonotic poxviruses can adapt to a new host, spread rapidly in the new host, and evolve to better evade host innate barriers. Unlike many other viruses, poxviruses express an extensive repertoire of self-defense proteins that play a vital role in the evasion of host innate and adaptive immune responses in their newest host species. The function of these viral immune modulators and host-specific cytokine responses can result in different host tropism and poxvirus disease progression. Here, we review the role of different cytokines that control poxvirus host tropism and adaptation.

Current Opinion in Virology 2022, 57:101286

This review comes from a themed issue on Antiviral strategies

Edited by Lieve Naesens and Bruno Canard

For complete overview about the section, refer “Antiviral strategies (2023)”

Available online 22th November 2022

https://doi.org/10.1016/j.coviro.2022.101286

1879–6257/© 2022 Elsevier B.V. All rights reserved.

Introduction

Poxviruses are large double-stranded DNA (dsDNA) viruses infecting insects and various vertebrate species. They belong to the Poxviridae family of viruses and are further classified into two subfamilies: the Entomopoxvirinae, infecting insects, and the Chordopoxvirinae, infecting vertebrates. Poxviruses that infect a wide range of vertebrate species are grouped into 18 genera based originally on their serological reactions, but more recently by their genomic features [1]. Among these poxviruses, members of the genera orthopoxvirus include many of the commonly recognized human pathogens such as variola virus (VARV), the causative agent of smallpox, cowpox virus (CPXV), and monkeypox virus (MPXV). Outside the orthopoxvirus genus, examples of poxviruses with human tropism include molluscum contagiosum virus and tanapox virus (TPV). Most poxviruses have evolved within a small number of host species with which they share co-evolutionary history, however, in lab culture, they can frequently infect cells from different host species. This broader cellular infectivity, compared with more limited host specificity, is mainly due to the lack of requirement for selective receptor proteins on target cells. Unlike most other mammalian viruses, poxviruses rely on relatively ubiquitous cellular surface molecules and exploit multiple host and virus-encoded proteins required for cell binding, fusion, and entry processes 2, 3, 4. At the cellular level, since poxviruses can bind and enter most mammalian cells in vitro, tropism is largely determined by the viruses' ability to modulate diverse intracellular antiviral pathways activated in response to virus sensing and infection. However, at the host organism level, the innate antiviral pathways activated by different virus-induced cytokines play a major role in determining the poxvirus tropism 5, 6. Here, we specifically discuss the recent progress in understanding how these crucial host cytokines regulate poxvirus tropism and adaptation.

The linear dsDNA genome of poxviruses ranges from 130 to 375 thousand base pairs and encodes between 130 and 300 open-reading frames. The central region of the genome is highly conserved among poxviruses and includes many dozens of essential genes required for transcription, replication, and virion assembly. The two ends of the linear viral genome are much more variable and encode host-interactive genes that control host range, help evade host immune responses, and other functions to control cellular responses. In addition, the function of these viral genes (referred to as host range genes) is closely linked to the successful replication of poxviruses in cultured cells originating from different tissues and hosts. However, the role of many poxvirus-specific host range genes uniquely required for host tropism and evasion of host immune responses is not well characterized from different poxviruses [7].

Host-restricted and zoonotic infection of poxviruses

One of the hallmarks of poxviruses is their host-restricted infections at the organismal level in vivo. Almost every vertebrate species can be infected with a selected member of poxviruses, some of which cause disease and some cause only subclinical infections. Genome sequencing of these poxviruses has identified a few viral genes unique to that poxvirus, and functional studies of such genes suggest that they have acquired host-specific functions [8]. For example, recently identified C7L-like host range gene M159 in MYXV-Tol (myxoma virus isolate Toledo), a member of Leporipoxvirus and known to cause disease in European rabbits, is critical for the recent species leap that now causes lethal disease in hares ( Figure 1). In culture, the recombinant knockout construct of MYXV-Tol lacking M159 can no longer productively infect neither a hare cell line nor primary hare PBMCs (peripheral blood mononuclear cells) [9••]. On the other hand, MYXV-Lau (myxoma virus isolate Lausanne), which causes myxomatosis in European rabbits and lacks Tol-M159, cannot infect this hare cell line nor primary hare PBMCs. However, the construction of a recombinant MYXV-Lau expressing just the Tol-M159 gene now allowed MYXV-Lau to replicate in both immortalized and primary hare cells. Although the host cell target(s) of M159 are yet to be identified, these results suggest that even the genetic acquisition of a single viral host range protein function can dramatically alter the tropism of recipient poxvirus. Similarly, C7L-like host range proteins from other poxviruses contribute to their host and cellular tropism [10]. Functional and structural studies of these C7-like proteins demonstrated that some of them bind and antagonize host sterile alpha-motif domain-containing 9 protein to overcome type-I IFN-mediated host restriction 11, 12, 13•. Thus, poxvirus-encoded proteins known as host range factors can dictate which cells, tissues, or hosts they can productively infect.

Figure 1.

Myxoma virus (MYXV) species leap from rabbits to hares. MYXV-Lau causes myxomatosis only in European rabbits (Oryctolagus cuniculus). However, during a recombination event, MYXV-Lau acquired a genomic cassette with a C7-like host range gene called M159 from an unknown poxvirus. This new MYXV isolate called MYXV-Tol can now cause myxomatosis-like disease in Iberian hares (Lepus granatensis) and European rabbits.

Apart from the virus-encoded proteins, host-specific factors and immune functions critically impact poxvirus tropism. The very well-studied poxvirus for host specificity is ectromelia virus (ECTV), a mouse-specific orthopoxvirus with a very narrow rodent-specific host range in nature. ECTV causes high mortality in susceptible mice strains, including BALB/c, DBA/2, A/J, and C3H, whereas C57BL/6, AKR, and I29 strains are much more resistant to the disease known also as mousepox 14, 15. In ECTV-resistant mice, multiple genetic loci have been identified and are referred to as restriction factors. For example, Ly49H (also called resistance to mousepox-1, Rmp-1) maps to the natural killer gene complex (NKC) and activates NK cells to control early virus replication in C57BL/6 mice, but is lacking in BALB/c mice [16]. Cytokines such as type-I IFN, IL-12, and IL-18 play essential roles in mediating this inherent genetic resistance to mousepox. These studies revealed that host-specific cytokine responses largely contribute to the tropism of poxviruses [17].

Unlike ECTV, some other orthopoxviruses are naturally capable of leaping from their reservoir host species to cause zoonotic diseases, including MPXV, CPXV, vaccinia virus (VACV)-like and Akhmeta virus. These viruses naturally circulate in wild and domestic animals, where they may or may not induce disease, have a broader host range, and often cause disease outbreaks in humans and other animals. For example, MPXV infections have been reported in various rodents, such as mice, rats, rabbits, hamsters, woodchucks, jerboas, porcupines, prairie dogs, hedgehogs, and several nonhuman primate species 18, 19. Although humans are considered accidental hosts, MPXV has now become the major zoonotic poxvirus for humans since the eradication of smallpox [20••]. Similarly, CPXV reservoirs are exclusively rodents in nature, but many wild animals can become accidental hosts, including cats, dogs, elephants, diverse zoo animals, and nonhuman primates from where humans can acquire an infection. Thus, poxviruses that can naturally leap into multiple host species are believed to have the further potential to acquire additional host-adapted mutations or acquired novel host regulatory proteins that can antagonize cytokine responses in diverse hosts. Apart from orthopoxviruses, members of capripoxvirus and parapoxvirus genera that normally infect farm animals can also infect humans after direct contact transmission, suggesting that these viruses encode host range proteins that can modulate human cytokine responses [21].

How cytokine-mediated innate immune responses regulate poxvirus host-specific infections and tropism

Cytokines and interferons (IFNs) are extracellular signaling molecules that play a key role in mediating an early immune response against invading pathogens, including viruses, and are essential components of host defense. In most cases, cytokine(s) activate protective responses that can provide complete clearance of viral infection. These cytokines, which can be either anti-inflammatory or pro-inflammatory, eventually clear the virus-infected cells by activating diverse mechanisms, including inflammation. These critical cytokines include IFNs, tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-12, IL-18, as well as multiple chemokines. Furthermore, these cytokines alone or in combination with each other, can further activate a network of downstream signaling pathways and stimulated genes such as interferon-stimulated genes (ISGs) and TNF-stimulated genes (TSGs).

Apart from host cytokines, poxviruses also encode diverse immune modulatory factors that can counteract the antiviral responses activated by different cytokines, including ISGs and TSGs, to determine the host-specific tropism and disease caused by different poxviruses. For example, VACV has been shown to be relatively resistant to IFN responses in cells from different species and also can confer resistance to IFN to other viruses such as vesicular stomatitis virus 22, 23. On the other hand, MYXV-Lau, a leporipoxvirus, exhibits resistance to IFN only in rabbit cells, but the virus is relatively sensitive to the IFN-induced antiviral state in human or mouse cells [24]. This species-specific anti-IFN function is mainly because MYXV-Lau host range factors such as dsRNA-binding vaccinia E3-like protein M029 have specialized in inhibiting restriction factors present in rabbits but not in other species that are yet to be identified [25]. For most of the cytokines that function to protect against viruses, poxviruses have co-evolved encoded proteins that dampen their functions at a different level. They encode self-defense proteins that can directly target the cytokine and prevent it from receptor binding or other interactions, proteins that function as a decoy receptor that also directly binds cytokines and thus competes with the natural receptor, and proteins that can inhibit or modulate the downstream intracellular signaling networks activated by different cytokines [7]. In most cases, whether these virus-encoded proteins targeting different cytokines and their network subsequently determine the tropism of poxviruses is yet to be studied in greater detail [24]. Several reviews have focused on this topic of how viruses counteract different cytokines, and it is beyond the scope of this mini-review 23, 26, 27, 28, 29•, 30, 31.

• i)Interferons:

  IFNs are the key cytokines that are rapidly produced and released from the cells in response to virus infection or by sensing virus-induced ligands such as pathogen-associated molecular patterns or damage-associated molecular patterns. Subsequently, the released IFNs bind to IFN receptors on the surface of target cells to trigger signaling pathways that activate the expression of hundreds of inducible genes known as ISGs 32, 33. There are three types of IFNs, namely type-I, type-II, and type-III IFNs, which have many subtypes. Some ISGs can be upregulated by all IFNs, while others are upregulated by selective IFNs. For example, Interferon Regulatory Factor 1 (IRF1) is upregulated preferentially by IFN-alpha and not by IFN-gamma [34]. This type of selective IFN-induced response is vital for cell, tissue, or host-specific innate immune responses and generation of an antiviral state within responsive cells, thereby controlling selective virus infection and spread 35, 36. Thus, one can predict that the orchestration of the expression of host-specific ISGs can alter the cellular or host tropism of different viruses, including poxviruses. Poxvirus-related functions of some of the key ISGs, such as ISG15, protein kinase R, and 2′,5′-oligoadenylate synthetases, are well characterized 37, 38, 39. However, many more remain to be studied in greater detail. As mentioned before, in the case of poxviruses, most of our knowledge about the role of IFNs in tropism has come from studies on ECTV using different strains of mice and genetic knockouts of C57BL/6 mice [14]. In ECTV-resistant C57BL/6 mice, apart from different genetic loci, potent NK, cytotoxic T lymphocytes (CTLs), and IFNɣ responses are generated against ECTV infection at higher levels than in ECTV-susceptible BALB/c mice [15]. Further studies in C57BL/6 mice with genetic deficiencies in innate immune pathways such as TLR9–MyD88–IRF7 and STING–IRF7/NF-kB confirmed that inefficient production of type-I IFNs will increase mortality in C57BL/6 mice [40]. Crosstalk between IFN-I and NF-κB pathways also confers resistance to lethal poxvirus infection [41]. A recent study demonstrated that IFN-I response is required in a cell-type-specific manner: C57BL/6 mice lacking IFNR in NK cells and monocytes become sensitive to disease caused by ECTV [42••]. In addition, at the cellular level, different DNA sensing pathways such as cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) can trigger the production of type-I IFNs in selected cell types and thus regulate poxvirus cellular tropism 23, 43, 44•, 45. Using ECTV, it was shown that bone marrow-derived cells play a major role in cGAS-dependent IFN production and protection against ECTV [45]. However, ECTV-encoded protein Schlafen (vSlfn) has been identified as the primary inhibitor of the cGAS–STING pathway, without which the virus is severely attenuated [44•]. Apart from ECTV, using VACV and mice knocked out for various innate sensing molecules, the role of IFNs in protection against infection and pathogenesis has been extensively documented 23, 27.

  Type-I IFNs also play a major role in controlling the infection of MYXV in cells derived from mice, humans, and likely other vertebrate species. For example, in mouse primary embryonic fibroblasts, virus-mediated induction of type-I IFN through ERK and IRF3 signaling pathway completely inhibits MYXV replication. Mice that are genetically lacking STAT1 and thus defective in IFN signaling became susceptible to lethal MYXV infection after intracranial injection [46]. Thus, the ERK–IFN–STAT1 pathway contributes to the species-specific protection against MYXV infection in host species outside of lagomorphs. However, MYXV has evolved rabbit-specific strategies that can inhibit this highly conserved pathway to cause lethal disease in rabbits. This rabbit-specific specificity is likely related to the evolutionary time, estimated to be at least 10 million years, that MYXV has co-evolved with South American rabbits. It is anticipated that IFN signaling contributes to the selective tropism of most, if not all, chordopoxviruses. Apart from the natural innate protection against certain poxviruses, the host can also co-evolve with the virus to acquire genetically controlled innate immunity against the selective pressure from poxviruses. This is evident from the genomic sequencing of feral rabbits from Australia and Europe, and it was found that the evolution of resistance in European rabbits to MYXV is associated with enhanced innate antiviral immunity that was acquired in just the 70 years following the first release of MYXV into wild rabbit populations in the early 1950s [47]. MYXV, on the other hand, also evolved to overcome these newly acquired innate host defenses 48, 49. Thus, the ongoing dynamics of the virus-host battle can shape and reshape the host tropism of poxviruses.

• ii)TNF and TNF superfamily cytokines:

  TNF is a potent pro-inflammatory cytokine that plays an essential role in the host control of many viral infections, including poxviruses [26]. TNF and the TNF ligand superfamily members bind to TNF receptor (TNFR) superfamily members to trigger downstream TNFR signaling that leads to the induction of hundreds of TSGs. TNF and the TNF signaling network directly play key roles in protection against poxvirus infections. For example, C57BL/6 mouse strains that are naturally resistant to lethal ECTV infection produce high levels of TNF and potent immune responses than susceptible BALB/c strain that produces little TNF and only weak immune responses. However, C57BL/6 mouse lacking TNFRs or TNF (TNF^-/-) becomes much more susceptible to ECTV infection and disease 50, 51•. Furthermore, exogenous treatment with mouse TNF was shown to reduce ECTV replication and mortality in mice [52]. Similarly, in TNFR2-knockout C57BL/6 mice, infection with VACV resulted in higher viral loads in spleens and livers and defective viral clearance [53]. Like type-I IFN, TNF also plays a role in restriction of MYXV in primary human macrophages [54]. Treatment of human fibroblasts with IFN-β and TNF resulted in activation of a synergistic antiviral state that can completely restrict MYXV and other poxviruses such as VACV and TPV that infect humans [55]. These studies suggest that, apart from the independent action of individual cytokines, their cooperative action can also further alter the tropism of poxviruses and other viruses [6]. Indeed, the treatment of human cells with TNF plus IFN synergistically upregulates an additional set of downstream genes that neither cytokine alone will induce [55]. This strongly suggests that co-induction of multiple cytokines at the organismal level plays a key role in tropism within tissues and host organisms that is not well modeled by assessing individual cytokines in cultured cells.

• iii)Other cytokines:

  IL-18 is a pleiotropic pro-inflammatory cytokine belonging to the IL-1 superfamily. IL-18 plays an important regulatory role in both innate and acquired immune responses against diverse pathogens, including poxviruses [56]. IL-18 signals through membrane-bound IL18Rα and IL18Rβ, which then stimulates the production of IFNɣ from T-helper lymphocyte cells (Th1) and macrophages and also enhances the cytotoxicity of NK cells. IL-12 is a member of the heterodimeric cytokines, composed of two chains, IL12A (p35) and IL12B (p40). IL-12 signals through a heterodimeric receptor formed by IL12Rβ1 and IL12Rβ2 to activate NK and T cells to stimulate the production of antiviral cytokines such as IFNɣ and TNF [57]. IL-12 and IL-18 are important for the cell-mediated immune response against poxviruses [58]. C57BL/6 mice that are lacking either IL12p40 (IL12p40^-/-) or IL-18 (IL-18^-/-) or both cytokines (double-knockout IL12p40^-/-IL18^-/-) are becoming highly susceptible to ECTV infection [58]. In these mice, the Th1 cytokine response was diminished, but the Th2 cytokine response was enhanced. In addition, there were reduced cytotoxic NK cells and CTL responses, resulting in reduced proliferation of virus-specific CD8+ T cells compared with the wild-type mice [58]. Thus, IL12p40 and IL-18 play an important role in the activation of antiviral responses by upregulation of IFNɣ production and cell-mediated immune responses. The role of IL-12 and IL-18 in activating innate and adaptive immune responses against viruses was further tested using recombinant VACVs expressing either IL-12 or IL-18 alone or both cytokines together [59]. Expression of either IL-12 or IL-18 enhanced viral clearance from ovaries and spleen in virus-infected mice, which involved NK and T cells [59]. However, when both cytokines were expressed from the same virus, the Th1 response increased for virus clearance [59]. Thus, IL-12 and IL-18 synergistically activate the antiviral response. In another study, expression of IL-18 using VACV resulted in attenuation of virus replication but elicited improved CTL responses [60]. An IL-12-expressing MYXV construct was also attenuated in European rabbits, suggesting that these cytokines can alter the tropism of poxviruses in hosts, and profoundly regulated the viral disease manifestations [61].

Conclusions

Cytokines are the gatekeeper and among the first lines of host defense against invading pathogens, including poxviruses. On the other hand, successful viruses have demonstrated the ability to emerge, re-emerge, or persist in a host in a fashion that is linked to their ability to subvert or evade antiviral cytokine responses. The outcome of such host and virus interactions determines the overall tropism of the virus at the cellular, tissue, and host level. Among the DNA viruses, poxviruses are known to circulate in almost every vertebrate species and can cause disease in host-restricted manner. However, poxviruses are also known to leap species, occasionally re-emerge, and cause zoonotic infections. For example, MPXV was long thought to be a rodent poxvirus in Africa that only occasionally caused disease in humans as a dead-end infection with a secondary human-to-human attack rate of less than 10%. But the current worldwide MPXV epidemic revealed the potential for extended human transmission that is linked to human behavior rather than evolution of new genetic viral variants (which, of course, may still occur). But host species leaping of poxviruses can also be due to either acquiring novel genes or selecting mutations in key immune-evading proteins that allow dampening of the cytokine responses in the newly adapted host 62•, 63. For example, VARV, which caused smallpox and killed millions of people per year for centuries, may have jumped from an unknown precursor host species or reservoir thousands of years ago and then adapted to humans after the original host had gone extinct [64]. During its adaptation in humans, VARV has lost multiple genes from the most recent common ancestor, suggesting that mutation or gene loss can enhance host- specific virulence of poxviruses 65, 66••. After the successful eradication of smallpox by a very successful worldwide vaccination program, newer emerging poxviruses such as MPXV and CPXV are appearing as ‘new’ zoonotic viruses and are becoming a progressively bigger threat to human health. The current MPXV outbreak and spread in many countries in populations that are not vaccinated prove that they are still a global threat. Sequencing of the circulating MPXV clades suggests that the MPXV has acquired mutations in certain genes involved in regulating host responses, compared with the apparent precursor virus from West Africa, but it is unknown if any of these mutations are responsible for the apparent increases in human-to-human transmission 20••, 67•, 68. More functional studies can only reveal whether they newly acquired host immune regulatory functions. Understanding the role of cytokines and how poxviruses counteract them also has implications for the development of vaccines, antiviral drugs, use of poxviruses as a vaccine platform, expression vector, and oncolytic viruses for the treatment of cancers.

Funding

This work was supported by National Institutes of Health grant R01 AI080607 and R21 AI163910 to G.M. and M.M.R. The funders had no role in study design, data collection, and interpretation or the decision to submit the work for publication.

Conflict of interest statement

The authors declare no conflict of interest.

Data availability

No data were used for the research described in the article.