Apoptosis and autophagy coordinately shape vector tolerance to arbovirus infection

ABSTRACT

Apoptosis and macroautophagy/autophagy, two important processes for host intracellular immunity against virus infection, are commonly deployed by arbovirus to promote its survival within an arthropod vector. Numerous studies have focused upon either apoptosis or autophagy initiated by arbovirus infection, with limited insight into the mechanisms underlying orchestration of vector immune tolerance to arbovirus. Using plant tomato yellow leaf curl virus (TYLCV)-infected whiteflies we identified a master regulator, PEBP4, that can be hijacked by the TYLCV coat protein (CP) and in turn synchronously activates apoptosis and autophagy. Once TYLCV invades a whitefly cell, the viral CP captures membrane-localized PEBP4, stabilizing PEBP4 association with RAF1. The triple complex blocks a MAPK phosphorylation cascade and triggers apoptosis. Simultaneously, virus CP competes for PEBP4 binding, promoting the disassociation of PEBP4 from ATG8 and initiating autophagy. The activation of autophagy degrades invasive TYLCV, whereas apoptosis increases the viral load by antagonizing autophagy. Amplification of autophagy intensity can eliminate arbovirus load but enhances whitefly fitness cost. In contrast, suppression of autophagy causes the viral load to exceed the vector capacity, which is also detrimental to whitefly survival. Thus, only mild intracellular immunity balanced by apoptosis and autophagy permits long-term coexistence between vectors and arboviruses.

Abbreviations

CP: coat protein; MAPK: mitogen-activated protein kinase; PEBP: phosphatidylethanolamine binding protein; TYLCV: tomato yellow leaf curl virus.

KEYWORDS:

• Apoptosis
• arbovirus
• autophagy
• immune tolerance
• PEBP

Arbovirus infection typically triggers a series of acute immune reactions in plant or animal hosts with noticeable disease symptoms. Remarkably, it is persistently preserved by arthropod vectors without causing evident fitness cost, suggesting a unique pattern of immune tolerance employed by insect vectors that warrants their vectoral role in nature. Apoptosis and autophagy can regulate arbovirus infection, circulation, and maintenance within insect vectors, leading to a species-specific effect on arbovirus load. Numerous lines of research, however, have confirmed that apoptosis and autophagy synergistically rather than separately determine cell fate upon virus infection. Therefore, a deep understanding of the balanced interplay between apoptosis and autophagy will aid in the illustration of how arbovirus escapes vector intracellular immunity.

Apoptosis and autophagy are essential conserved degradation mechanisms, which participate in the removal of unwanted metabolites, invaders, or even the whole cell. Similarly, once infected by arbovirus, the activation of degradation processes in arthropod vectors containing numerous proteases and hydrolases eliminates arbovirus particles, minimizing physiological burdens generated by virus genome replication and protein expression. By contrast, a virus-induced degradation process could break the integrity of the midgut and salivary gland barriers to form potential tunnels that facilitate arbovirus entry into hemolymph and gain access to saliva. It is therefore thought that the magnitude of apoptosis and autophagy induced by arbovirus, along with mutual temporal antagonistic effects, shape the vector’s tolerance to arbovirus.

A recent study identified a phosphatidylethanolamine-binding protein (PEBP) that can be hijacked by the coat protein (CP) of tomato yellow leaf curl virus (TYLCV) to simultaneously activate apoptosis and autophagy in whitefly [1]. The PEBP family was originally named because of its phosphatidylethanolamine-binding function, and relevant research sharply increased since PEBP1 was demonstrated as a suppressor of RAF1 kinase that can regulate apoptosis. Soon after, other PEBP family members with the function of regulation of apoptosis via multiple signaling pathways, i.e., MAPK, NFKB, and TBK1, were also reported. In addition, PEBP1 can interact with Atg8-family proteins, a hallmark of autophagosome formation, via its highly conserved PE-binding domain to suppress autophagy. The results indicated that PEBP acts as a potential flexible regulator in balancing apoptosis and autophagy within the cell, resulting in an optimal intracellular environment to carry TYLCV.

Unlike mammalian PEBPs, whitefly PEBP4 protein contains an extra pre-domain in front of the conserved PE-binding domain. This unique domain is not only required for plasma membrane localization of whitefly PEBP4 but guarantees a species-specific regulatory mechanism without interfering with the fundamental roles served by other PEBP family members. Although members of human PEBPs have contrasting effects on the RAF1-MAP2K/MEK-MAPK/ERK signaling pathway, the phosphorylation status of PEBPs is a switch signal for its association status with multiple pathways. We confirmed in whitefly that TYLCV CP interacts with PEBP4 to suppress MAPK phosphorylation, whereas knockdown of PEBP4 can enhance MAPK phosphorylation, suggesting TYLCV CP possibly blocks the phosphorylation sites of PEBP4 and prevents the dissociation from RAF1. CP subsequently activates apoptosis most probably through unbalancing the expression of anti- and pro-apoptotic factors (Figure 1(A)).

Figure 1. PEBP4 coordinates intracellular immune balance in whitefly to coexist with TYLCV infection. (A) Dual-activation effects of PEBP4 on whitefly apoptosis and autophagy. (B) Autophagy intensity is dynamically shaped by CP-PEBP4 and apoptosis. P, phosphorylation; PE, phosphatidylethanolamine; dashed line, dissociation; gray whitefly, reduced survivorship. This figure was created with BioRender.com.

Similar to human PEBP1, whitefly PEBP4 arrests ATG8 to suppress autophagy. Otherwise, ATG8 is liberated and conjugated with PE (lipidation), as part of autophagosome initiation and autophagy activation. We discovered that TYLCV CP competes for whitefly PEBP4 binding with ATG8, suggesting whitefly PEBP4 had a higher affinity for TYLCV CP, rather than ATG8. Intriguingly, whitefly consists of more than 200 protein members of the PEBP family with predicted conserved PE-binding domains, which far exceeds any other arthropod species. Given that all PEBP-family proteins contain the conserved the PE-binding domain, it remains unclear whether other PEBPs have a compensatory effect on prevention of ATG8 liberation. Mutual antagonism is the most prominent situation, even though other scenarios exist between apoptosis and autophagy when they are simultaneously activated in the same cell. No matter what the scenario is, one of them could eventually dominate the cell fate. In whitefly, PEBP4 causes the synchronous activation of apoptosis and autophagy in the same infected cell, which provides a molecular basis for balancing homeostasis between them. Our pharmacological assays and RNAi experiments clearly revealed that autophagy, rather than apoptosis, determines the final TYLCV load in whitefly, while apoptosis fine-tunes the viral load by suppressing autophagy. To precisely control virus load, whitefly can regulate the anti-viral autophagy by a positive feedback loop of CP-PEBP4-ATG8 or via the antagonistic effect of apoptosis (Figure 1(B)). As TYLCV in continuously ingested, whitefly activates autophagy to fight against arbovirus infection, while coactivation of apoptosis dynamically suppresses autophagy to prevent arbovirus elimination. Such mechanisms are developed for insect vectors that can optimize the co-existence with arbovirus without obvious physiological burden.

Immune tolerance is commonly utilized by vectors to minimize the detrimental effects of immunopathology caused by arbovirus. It is widely accepted that, at least empirically, viruliferous vectors present little or even no pathological lesions relative to a virus-infected host, but increasing evidence shows that chronic symptoms occur in insect vectors after acquiring arbovirus. We demonstrated that whitefly survivorship is reduced in situations where suppression of autophagy leads to overload of TYLCV, or where over-activation of autophagy results in an extra physiological cost in whitefly. Thus, these findings indicate that only a mild and balanced intracellular immune response can allow arbovirus to persist in the vector population.

In summary, this work identified a vector protein, PEBP4, that balances apoptosis and autophagy to enable co-existence with arbovirus. The homeostasis achieved by simultaneous activation of apoptosis and autophagy warrants the vectoral role of whitefly. A more detailed understanding of vital tolerance regulators and the mechanism of immune homeostasis regulation in other arthropod vectors and the arbovirus system needs to be considered. Such information could refine precise approaches for pest and disease control that disturb the mutualism between vector and arbovirus from the perspective of immune tolerance, which in turn increases the fitness cost of the insect vector with virus retention.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDPB16).