Proteolytic Processing of Plant Proteins by Potyvirus NIa Proteases

Abstract

The NIa protease of potyviruses is a chymotrypsin-like cysteine protease related to the picornavirus 3C protease. It is also a multifunctional protein known to play multiple roles during virus infection. Picornavirus 3C proteases cleave hundreds of host proteins to facilitate virus infection. However, whether or not potyvirus NIa proteases cleave plant proteins has so far not been tested. Regular expression search using the cleavage site consensus sequence [EQN]xVxH[QE]/[SGTA] for the plum pox virus (PPV) protease identified 90-94 putative cleavage events in the proteomes of Prunus persica (a crop severely affected by PPV), Arabidopsis thaliana and Nicotiana benthamiana (two experimental hosts). In vitro processing assays confirmed cleavage of six A. thaliana and five P. persica proteins by the PPV protease. These proteins were also cleaved in vitro by the protease of turnip mosaic virus (TuMV), which has a similar specificity. We confirmed in vivo cleavage of a transiently expressed tagged version of AtEML2, an EMSY-like protein belonging to a family of nuclear histone readers known to be involved in pathogen resistance. Cleavage of AtEML2 was efficient and was observed in plants that co-expressed the PPV or TuMV NIa proteases or in plants that were infected with TuMV. We also show partial in vivo cleavage of AtDUF707, a membrane protein annotated as lysine ketoglutarate reductase trans-splicing protein. Although cleavage of the corresponding endogenous plant proteins remains to be confirmed, the results show that a plant virus protease can cleave host proteins during virus infection and highlight a new layer of plant-virus interactions. Importance Viruses are highly adaptive and use multiple molecular mechanisms to highjack or modify the cellular resources to their advantage. They must also counteract or evade host defense responses. One well-characterized mechanism used by vertebrate viruses is the proteolytic cleavage of host proteins to inhibit the activities of these proteins and/or to produce cleaved protein fragments that are beneficial to the virus infection cycle. Even though almost half of the known plant viruses encode at least one protease, it was not known whether plant viruses employ this strategy. Using an in silico prediction approach and the well-characterized specificity of potyvirus NIa proteases, we were able to identify hundreds of putative cleavage sites in plant proteins, several of which were validated by downstream experiments. It can be anticipated that many other plant virus proteases also cleave host proteins and that the identification of these cleavage events will lead to novel antiviral strategies.

Re-examination of nepovirus polyprotein cleavage sites highlights the diverse specificities and evolutionary relationships of nepovirus 3C-like proteases

“…The case has been made that evolution of 3C- and 3CL-Pros is driven at least in part by evolutionary pressures dictated by the cleavage of host proteins [ 74 ]. Recently, cleavage of several plant proteins by potyvirus NIa 3CL-Pros was confirmed experimentally [ 82 ]. It can be anticipated that the 3CL-Pros of nepoviruses also target plant proteins.…”

“…Cleavage of similar families of plant proteins by nepoviruses could be anticipated, given the similar infection cycles and shared hosts and symptomatology of many nepoviruses [ 29 ]. The identification of plant proteins cleaved by potyvirus 3CL-Pros was facilitated by a bioinformatic interrogation of plant proteomes using the consensus cleavage site sequence of the plum pox virus 3CL-Pro [ 82 ]. It is hoped that clarification of nepovirus 3CL-Pros specificities will also help identify plant protein targets of these proteases.…”

In-silico comparative modeling and interaction studies of PRSV proteins - Accelerated towards dissection of structure based evolutionary divergence and functional interaction of virus within the host

“…The highly adaptive nature of viruses and the numerous molecular modes they adopt enables them to take over the host cellular machinery to their benefit. At the same time, they must also avoid host defense responses activated in response to virus invasion, to ensure complete infectivity [3].…”

“…The highly adaptive nature of viruses and the numerous molecular modes they adopt enables them to take over the host cellular machinery to their benefit. At the same time, they must also avoid host defense responses activated in response to virus invasion, to ensure complete infectivity [3]. Thus, the evolving strategies of the invading virus and the resultant host defense elicitation can be fully understood only when the evolutionary dynamics of virus at nucleotide as well as protein structural level are fully explored.…”

Identification of viral genes involved in pepper mottle virus replication and symptom development in Nicotiana benthamiana

“…The nuclear inclusion a (NIa) protease of potyviruses has been identified as a multiprotein that plays various roles in virus infection ( Xiao et al., 2022 ). The tobacco etch virus (TEV) NIa has RNA binding activity; it can interact with NIb protein and contributes to viral accumulation ( Schaad et al., 1996 ; Daròs and Carrington, 1997 ).…”