Recent Challenges in Understanding Henipavirus Immunopathogenesis: Role of Nonstructural Viral Proteins

KEYWORDS:

• emerging infectious diseases
• highly pathogenic virus
• innate immunity
• interferon

Hendra virus (HeV) and Nipah virus (NiV) are closely related recently emerged zoonotic paramyxoviruses, belonging to Henipavirus genus. These largely unknown viruses are unique among paramyxoviruses in that they cause a severe disease in an unusually broad range of animals, as well as in humans [1]. Since the initial spillover of HeV from fruit bats of the Pteropus family to horses in Australia, and NiV from bats to pigs in Malaysia, Henipaviruses have caused almost annual outbreaks over the last two decades. Utilization of highly conserved cell surface molecules (ephrin B2 and B3) as entry receptors facilitates their transmission to various species. Henipaviruses induce severe pneumonia and encephalitis with a mortality rate in humans between 50 and 100%, making them one of the most deadly viruses known to infect humans. Multiple rounds of person-to-person transmission are observed in NiV outbreaks in Bangladesh [2], thus further extending the risk of infection in humans. Several species of fruit bats, widely distributed in Australia, Southeast Asia and Africa [3], appear to be the Henipavirus reservoir, maintaining a permanent risk of new outbreaks. Being able to cause severe zoonosis with serious health and economic problems, without efficient treatment yet available, Henipaviruses are considered as a potential agent for bioterrorism and classified as biosecurity level 4 pathogens.

The viral nonstructural proteins

HeV and NiV share similar entry and replication strategies, and display high similarities in their sequences (specific genes sharing 70–88% nucleotide homologies) [4]. These large enveloped ssRNA viruses of negative polarity produce six structural and three nonstructural proteins: V, W and C. All nonstructural proteins are derived from the P gene, which codes for the phosphoprotein P. As in other paramyxoviruses, this P gene contains an editing site, which can introduce G residues in the initial sequence and leads to a reading frameshift in the course of RNA translation [5]. The resulting V (+1G at the editing site) and W proteins (+2G at the editing site) share their N-terminal part with the P protein, but have their own specific C-terminal part. Thus, V and W proteins share some similar properties with P protein through their common N-terminal part, but in addition have their own properties, through their specific C-terminal parts. For example, the nonstructural proteins have different intracellular distribution: the V protein is cytosolic, whereas W is nuclear and they show differences in their ability to interact with the innate immune system. Moreover, due to the existence of an alternative open reading frame inside the P gene sequence, the third nonstructural Henipavirus C protein is produced and therefore, the C protein does not share any amino acid homology with the V, W and P protein [5].

Interaction with the host innate immune system

The Henipavirus nonstructural proteins can block the host innate immune response, thus becoming virulence factors responsible for high pathogenicity of these viruses. Unlike HeV and NiV, the third recently discovered member of Henipavirus genus, Cedar virus, lacks the editing site in its P gene and thus cannot produce V nor W protein, and does not cause clinical disease in tested animal models such as ferrets and guinea pigs [6]. Interactions between Henipavirus nonstructural proteins and innate immune signaling pathway proteins allows viral escape from cellular pathogen recognition and are thus important for Henipavirus pathogenicity. Some of these interactions have been already described in human cells, nevertheless, the mechanisms involved are not completely understood and other unknown cell targets may exist.

The RIG-I-like receptors (RLRs) are cytosolic receptors that recognize dsRNAs, leading to the triggering of innate immunity. The viral RNA recognition by the RLRs sets off signaling pathways, leading to the production of type 1 IFN-α and -β (IFN-I), as well as other proinflammatory cytokines [7]. IFN-I is involved in triggering the antivirus cell defense and further production of proinflammatory cytokines, allowing recruitment of cells of the immune system and development of the adaptive immune response. In the cytosol, the Henipavirus V protein interacts directly with the MAD5 protein [8,9] and indirectly with the RIG-I protein [10], thus inhibiting RNA recognition by these two RLRs. In the nucleus, the NiV W protein blocks the nuclear transduction signal, disrupting the phosphorylated form of the IRF3 [11]. In agreement, Cedar virus has been shown to induce much higher IFN-β production than HeV, possibly due to the absence of V protein [6].

In addition to RLRs, the Toll-like receptors (TLRs) are transmembrane proteins that also recognize viral RNAs and trigger signaling pathways. The TLRs 3, 7 and 8 are especially involved in the recognition of single or double stranded viral RNAs [7]. As IRF3 is a part of the TLR signaling pathway in addition to the RLR pathway, interaction of W protein with IRF3 also disturbs TLR pathway signaling [11]. Moreover, NiV C protein interacts with a cytosolic kinase, which is responsible for the phosphorylation of IRF7 [12]. Since this phosphorylation is essential for nuclear transduction, the NiV C protein consequently disturbs IFN-I production. Therefore, by interacting with intracellular signaling pathways, Henipaviruses disrupt the sensors of the innate immunity in human cells and, thus, production of IFN-I.

Blocking of the danger signal

The transmembrane IFN-I receptor (IFNAR) initiates IFN-I signaling in cells via the activation of STAT proteins [13]. The IFNAR signaling pathway leads to the activation of a panel of interferon-stimulated genes, which allow cells to engage its antiviral defense state, with the production of antiviral proteins (such as the protein kinase R capable of virus cycle disruption) and therefore provides the most important antiviral protection of uninfected cells known thus far. The NiV P and V proteins confine the STAT proteins in the cytosol as a high molecular weight complex, preventing their nuclear translocation and the activation of the interferon-stimulated genes [14]. In addition, the W protein interacts with the STAT proteins in the nucleus to sequestrate them and interfere with their recycling towards the cytosol [15]. Therefore, by acting at different levels on the innate immune signaling pathways, the nonstructural proteins enable virus escape from the host cellular defenses.

However, numerous gray areas still persist

Despite initial steps made towards a better understanding of the interactions between HeV and NiV nonstructural proteins and the innate immune signaling pathways, Henipaviruses still present many gray areas. Numerous results were obtained in a protein transfection context, in which the proteins are expressed in much higher quantities than during natural infection. Differing results in the ability of Henipaviruses to block IFN-I signaling were reported [16–18], which could be partly explained by the differences between the experimental protocols used and the analyzed cell types. Indeed, in some endothelial cells, the W protein seems to remain in the cytosol, where it may not be able to play its key role [19]. Development of reverse genetic systems, which allow the generation of recombinant viruses bearing specific mutations in nonstructural proteins, have contributed to better understanding of the role of these proteins during infection [16,20]. Both distinct and overlapping roles of NiV nonstructural proteins in modulating an antiviral response in human endothelial cells were shown [20] and NiV C protein was demonstrated to have an important function in the regulation of proinflammatory responses [18]. Nevertheless, it is highly likely that additional cell targets of NiV and HeV nonstructural proteins still remain to be elucidated.

Lessons from differential virus–host interactions among distinct species

Although Henipavirus provokes serious diseases in their spillover hosts, they seem to be asymptomatic in their natural hosts, fruit bats. The interactions between Henipaviruses and bat innate immunity are far from being understood. Although IFN-I signaling was shown to play a critical role in protecting mice from Henipavirus infection [21], both IFN-I and IFN type III were inhibited by HeV and NiV in bat cells, suggesting that bats control Henipavirus infection by a yet unidentified mechanism [22]. Surprisingly, the same authors found that interferon signaling remains effective in human cells [17], which is difficult to align with the high pathogenicity of NiV. Recently, a new Henipavirus was reported in rats in China, whereas until now the Henipaviruses have not been reported in wild animals other than fruit bats [23]. This new discovery makes the study of Henipavirus pathogenicity in natural hosts even more important.

Faced with these questions, studies are in progress to try to understand the host–pathogen relationship in different species and differences in pathogenicity. Additional analysis is essential to better comprehend the basis of high Henipavirus pathogenicity. Further elucidation of virus-host interactions will provide new insights from which to develop novel therapeutic strategies against these highly lethal emergent viruses.

Disclaimer

The funding bodies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Financial & competing interests disclosure

This work was supported by INSERM, ANR MIE and Astrid. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

This work was supported by INSERM, ANR MIE and Astrid. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.