The p33 auxiliary replicase protein of Cucumber necrosis virus targets peroxisomes and infection induces de novo peroxisome formation from the endoplasmic reticulum

Abstract

Tombusviruses replicate on pre-existing organelles such as peroxisomes or mitochondria, the membranes of which become extensively reorganized into multivesicular bodies (MVBs) during the infection process. Cucumber necrosis virus (CNV) has previously been shown to replicate in association with peroxisomes in yeast. We show that CNV induces MVBs from peroxisomes in infected plants and that GFP-tagged p33 auxiliary replicase protein colocalizes with YFP(SKL), a peroxisomal marker. Most remarkably, the ER of CNV infected Nicotiana benthamiana 16C plants undergoes a dramatic reorganization producing numerous new peroxisome-like structures that associate with CNV p33, thus likely serving as a new site for viral RNA replication. We also show that plants agroinfiltrated with p33 develop CNV-like necrotic symptoms which are associated with increased levels of peroxide. Since peroxisomes are a site for peroxide catabolism, and peroxide is known to induce plant defense responses, we suggest that dysfunctional peroxisomes contribute to CNV induced necrosis.

Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection

“…CNV RNA may also contain sequences that facilitate the assembly process, which would contribute to the preferential encapsidation efficiency (see below). In addition, as has been suggested for the encapsidation of BMV RNA, the presence of the replicase could enhance the specificity of encapsidation (25,48,49), a process which, in the case of CNV, would not take place in chloroplasts, since CNV replicase does not enter chloroplasts; rather, it is targeted to peroxisomes (50). As the total number of reads corresponding to encapsidated host RNAs was relatively low and the CNV preparation used for the analyses whose results are presented in Fig.…”

“…Nonspecific encapsidation of chloroplast RNAs may occur in the absence of a functional replicase, as has been previously suggested for BMV (25,48,49), whereas more specific encapsidation may occur in its presence in the cytoplasm. Also, CNV is replicated within spherules derived from peroxisomes in the cells of N. benthamiana (50). Should encapsidation take place in the vicinity of replication, the predominant RNA is likely to be viral RNA, and thus, virions would contain a low level of host RNA species.…”

Cucumber Necrosis Virus Recruits Cellular Heat Shock Protein 70 Homologs at Several Stages of Infection

“…Four-to six-week-old N. benthamiana plants were coagroinfiltrated with pNbHsc70-2/pBin(ϩ) and pTBSVp19/ pBin(ϩ) or with pGFP/pBin(ϩ) and pTBSVp19/pBin(ϩ) using cultures adjusted to an OD 600 of 1.0. Leaf samples were analyzed at 3 dpai using a Leica TCS SP2-AOBS microscope as described previously (72).…”

Tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast

“…In addition to the viral-coded p33 RNA chaperone and p92 pol RNA-dependent RNA polymerase (RdRp), which are essential components of the TBSV VRC (Stork et al, 2011; White and Nagy, 2004), TBSV also recruits a number of host proteins based on extensive proteomics screens (Li et al, 2008b; Li et al, 2009; Mendu et al, 2010; Nagy and Pogany, 2010; Serva and Nagy, 2006). Interestingly, genome-wide and global proteomics screens in S. cerevisiae identified ~20 host genes involved in lipid biosynthesis and metabolism affecting tombusvirus replication and recombination, suggesting that tombusvirus replication depends on active lipid biosynthesis and extensive remodeling of membranes (Jiang et al, 2006; Li et al, 2008b; Li et al, 2009; Mendu et al, 2010; Nagy and Pogany, 2010; Panavas et al, 2005; Rochon et al, 2014; Serva and Nagy, 2006; Serviene et al, 2006; Serviene et al, 2005). …”