Dissecting the Subcellular Localization, Intracellular Trafficking, Interactions, Membrane Association, and Topology of Citrus Leprosis Virus C Proteins

Abstract

Citrus leprosis (CL) is a re-emergent viral disease affecting citrus crops in the Americas, and citrus leprosis virus C (CiLV-C), belonging to the genus Cilevirus, is the main pathogen responsible for the disease. Despite the economic importance of CL to the citrus industry, very little is known about the performance of viral proteins. Here, we present a robust in vivo study around functionality of p29, p15, p61, MP, and p24 CiLV-C proteins in the host cells. The intracellular sub-localization of all those viral proteins in plant cells are shown, and their co-localization with the endoplasmic reticulum (ER), Golgi complex (GC) (p15, MP, p61 and p24), actin filaments (p29, p15 and p24), nucleus (p15), and plasmodesmata (MP) are described. Several features are disclosed, including i) ER remodeling and redistribution of GC apparatus, ii) trafficking of the p29 and MP along the ER network system, iii) self-interaction of the p29, p15, and p24 and hetero-association between p29-p15, p29-MP, p29-p24, and p15-MP proteins in vivo. We also showed that all proteins are associated with biological membranes; whilst p15 is peripherally associated, p29, p24, and MP are integrally bound to cell membranes. Furthermore, while p24 exposes an N-cytoplasm-C-lumen topology, p29, and p15 are oriented toward the cytoplasmic face of the biological membrane. Based on our findings, we discuss the possible performance of each protein in the context of infection and a hypothetical model encompassing the virus spread and sites for replication and particle assembly is suggested.

Citrus Leprosis Virus C Encodes Three Proteins With Gene Silencing Suppression Activity

“…In our previous study, we showed that p15 is localized into the nucleus of N. benthamiana epidermal cells with unknown function (Leastro et al, 2018). Here, we clearly identified the p15 RSS activity using four different methods.…”

“…All proteins, except the p61, were detected using a monoclonal anti-HA antibody (Figure 2B). In a previous study, we also failed to detect the p61 protein by Western blot assay (Leastro et al, 2018). To confirm that the necrotic phenotype observed with the AMV construct carrying the p61 protein correlated with its expression, P12 leaves with an RNA 3 variant containing a frameshift version of the p61 gene were inoculated.…”

“…The first segment (RNA1) has two ORFs that code for a replication-associated protein containing conserved domains of methyl transferase, helicase, and RNA dependent RNA polymerase, and the capsid protein (p29)" (Locali-Fabris et al, 2006;Pascon et al, 2006;Leastro et al, 2018). The second segment (RNA2) encodes four proteins: (i) p15, a small protein for which a specific function needs to be determined, but potentially involved with virus replication as suggested by its involvement in the formation of vesicles thought ER remodeling (Leastro et al, 2018); (ii) p61, which exhibits features of glycoprotein (Kuchibhatla et al, 2014;Leastro et al, 2018) with biological activity on remodeling the ER system and redistributing the Golgi apparatus (Leastro et al, 2018); (iii) p32 which has the function of a movement protein (MP) (Leastro et al, unpublished); and (iv) p24, an integral membrane protein with the ability to form vesicle-like spherical structures in association with the ER, which strongly suggests the involvement of this protein in viral replication and assembly (Leastro et al, 2018). Those features, in addition to membrane topology presented for the p24 protein (Leastro et al, 2018) combined with its homology to virion membrane proteins of plant and arthropod viruses (Kuchibhatla et al, 2014;Leastro et al, 2018) suggest a potential structural role as a matrix protein.…”

Sequence Relationships of RNA Helicases and Other Proteins Encoded by Blunervirus RNAs Highlight Recombinant Evolutionary Origin of Kitaviral Genomes

“…Other proteins show only rather short amino sequence motifs allowing motif-based sequence comparisons with some kitaviral polypeptides ( Supplementary Table 2). Particularly, p31 protein ( Figure 1B) contains a rather long C-terminal hydrophobic segment similar to that of cilevirus p61, which exhibits some features of glycoprotein (Leastro et al, 2018), whereas p30 protein includes FIGURE 1 | Genomes and sequence analysis of blunerviruses. (A) Genome structure of the blueberry necrotic ring blotch blunervirus.…”

Passion Fruit Green Spot Virus Genome Harbors a New Orphan ORF and Highlights the Flexibility of the 5′-End of the RNA2 Segment Across Cileviruses

“…ORFs p15 and p61 are separated by a stretch of ∼1000 nts known as the intergenic region (IR). CiLV-C proteins can form homo-and heterodimers, and associate with plant cell membranes producing severe remodeling of the endoplasmic reticulum and the Golgi complex (Leastro et al, 2018).…”