Tomato ringspot nepovirus (ToRSV) encodes two polyproteins that are processed by a 3C-like protease at specific cleavage sites. Analysis of ToRSV cleavage sites identified previously and in this study revealed that cleavage occurs at conserved Q/(G or S) dipeptides. In addition, a Cys or Val is found in the -2 position. Amino acid substitutions were introduced in the -6 to +1 positions of two ToRSV cleavage sites: the cleavage site between the protease and putative RNA-dependent RNA polymerase, which is processed in cis, and the cleavage site at the N-terminus of the movement protein, which is cleaved in trans. The effect of the mutations on proteolytic processing at these sites was tested using in vitro translation systems. Substitution of conserved amino acids at the -2, -1, and +1 positions resulted in a significant reduction in proteolytic processing at both cleavage sites. The effects of individual substitutions were stronger on the cleavage site processed in trans than on the one processed in cis. The cleavage site specificity of the ToRSV protease is discussed in comparison to that of related proteases.
Rapid apple decline disease (RAD) has been affecting orchards in the USA and Canada. Although the primary cause for RAD remains unknown, viruses may contribute to the incidence or severity of the disease. We examined the diversity and prevalence of viruses in orchards affected by RAD in the Okanagan and Similkameen Valleys (British Columbia, Canada). Next-generation sequencing identified 20 previously described plant viruses and one viroid, as well as a new ilarvirus, which we named apple ilarvirus 2 (AIV2). AIV2 was related to subgroup 2 ilarviruses (42–71% nucleotide sequence identity). RT-PCR assays of 148 individual leaf samples revealed frequent mixed infections, with up to eight viruses or viroid detected in a single tree. AIV2 was the most prevalent, detected in 64% of the samples. Other prevalent viruses included three ubiquitous viruses from the family Betaflexiviridae and citrus concave gum-associated virus. Apple rubbery wood virus 1 and 2 and apple luteovirus 1 were also readily detected. The thirteen most prevalent viruses/viroid were detected not only in trees displaying typical RAD symptoms, but also in asymptomatic trees. When compared with reports from orchards affected by RAD in Pennsylvania, New York State, and Washington State, regional differences in relative virus prevalence were noted.
Formation of plant virus membrane-associated replication factories requires the association of viral replication proteins and viral RNA with intracellular membranes, the recruitment of host factors and the modification of membranes to form novel structures that house the replication complex. Many viruses encode integral membrane proteins that act as anchors for the replication complex. These hydrophobic proteins contain transmembrane domains and/or amphipathic helices that associate with the membrane and modify its structure. The comovirus Co-Pro and NTP-binding (NTB, putative helicase) proteins and the cognate nepovirus X2 and NTB proteins are among the best characterized plant virus integral membrane replication proteins and are functionally related to the picornavirus 2B, 2C, and 3A membrane proteins. The identification of membrane association domains and analysis of the membrane topology of these proteins is discussed. The evidence suggesting that these proteins have the ability to induce membrane proliferation, alter the structure and integrity of intracellular membranes, and modulate the induction of symptoms in infected plants is also reviewed. Finally, areas of research that need further investigation are highlighted.
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