Dichorhaviruses Movement Protein and Nucleoprotein Form a Protein Complex That May Be Required for Virus Spread and Interacts in vivo With Viral Movement-Related Cilevirus Proteins

Abstract

Brevipalpus -transmitted viruses (BTVs) belong to the genera Dichorhavirus and Cilevirus and are the main causal agents of the citrus leprosis (CL) disease. In this report, we explored aspects related to the movement mechanism mediated by dichorhaviruses movement proteins (MPs) and the homologous and heterologous interactions among viral proteins related to the movement of citrus leprosis-associated viruses. The membrane-spanning property and topology analysis of the nucleocapsid (N) and MP proteins from two dichorhaviruses revealed that the MPs are proteins tightly associated with the cell membrane, exposing their N- and C-termini to the cytoplasm and the inner part of the nucleus, whereas the N proteins are not membrane-associated. Subcellular localization analysis revealed the presence of dichorhavirus MPs at the cell surface and in the nucleus, while the phosphoproteins (P) were located exclusively in the nucleus and the N proteins in both the cytoplasm and the nucleus. Co-expression analysis with the MP, P, and N proteins showed an interaction network formed between them. We highlight the MP capability to partially redistribute the previously reported N-P core complex, redirecting a portion of the N from the nucleus to the plasmodesmata at the cell periphery, which indicates not only that the MP might guide the intracellular trafficking of the viral infective complex but also that the N protein may be associated with the cell-to-cell movement mechanism of dichorhaviruses. The movement functionality of these MPs was analyzed by using three movement-defective infectious systems. Also, the MP capacity to generate tubular structures on the protoplast surface by ectopic expression was analyzed. Finally, we evaluated the in vivo protein–protein interaction networks between the dichorhavirus MP and/or N proteins with the heterologous cilevirus movement components, which suggest a broad spectrum of interactions, highlighting those among capsid proteins (CP), MPs, and Ns from citrus leprosis-associated viruses. These data may aid in understanding the mixed infection process naturally observed in the field caused by distinct BTVs.

Membrane Association and Topology of Citrus Leprosis Virus C2 Movement and Capsid Proteins

“…Computer analysis from deduced amino acid sequences of the CiLV-C2 p29 and MP was performed using several computer tools. In the analysis, we also included the deduced amino acid sequences of the CSNV NSm and TMV 30K MP, which have been shown to be peripheral membrane proteins [ 18 , 19 ], as well as for the Lep, CiLV-C MP and GFP, two integral membrane-, and one non-membrane-associated proteins, respectively [ 7 , 19 , 24 ]. Highlighted, they revealed the presence of three hydrophobic regions (HR) for CiLV-C2 MP that encompass the residues 79–97 (HR1), 113–131 (HR2), and 167–185 (HR3), and two for p29: 3–21 (HR1) and 178–196 (HR2) ( Figure 1 and Table S1 ).…”

“…High-speed ultracentrifugation was performed to separate the plant leaf lysed extract, containing the abovementioned proteins, into pellet (P30) and supernatant (S30) fractions. Treatment with Na 2 CO 3 is known to render microsomes into membrane sheets, releasing soluble luminal proteins [ 20 ], while urea treatment should release all polypeptides bound to the membrane, except for the integral membrane proteins [ 21 , 24 ]. We observed that the HA-tagged p29 and MP remained mostly associated with the P30 membranous fraction after Na 2 CO 3 treatment ( Figure 2 A, P30 78% for p29, and P30 99% for MP), suggesting that these proteins are tightly associated with the membrane.…”

“…The open question is to understand why this model diverges from the rest of the MPs assigned to the 30K superfamily and if this feature results or not in a biological fitness or benefit. It is worth mentioning that, although both CiLV-C and CiLV-C2 MPs have been proven to be functional to rescue the defective alfalfa mosaic virus (AMV), turnip crinkle virus (TCV), and TMV cell-to-cell movement-mutants, including the systemic transport of AMV [ 10 , 24 ], the cileviruses do not move systemically in their natural or experimental plant hosts [ 4 ]. It has been hypothesized that members of this genus evolved from an ancestor arthropod virus that became capable of infecting plants after acquiring the movement protein from one or more plant virus(es) [ 4 ].…”

“…At 3 days post-infiltration (dpi), approximately 1.5 g of N. benthamiana leaves expressing the tested proteins were ground in lysis buffer (20 mM de HEPES, pH 6.8; 150 mM potassium acetate; 250 mM mannitol; 1 mM MgCl 2 , and 50 µL of protease inhibitor cocktail for plant cell, Sigma–Aldrich, St. Louis, MO, USA). The homogenate was clarified by low centrifugation at 3000× g for 10 min at 4 °C, then the obtained supernatant was ultracentrifuged at 40,000× g for 40 min at 4 °C to yield the soluble (S30) and the crude (P30) microsomal fraction as reported previously [ 7 , 24 ]. Next, the P30 membrane-rich fraction was resuspended in lysis buffer, divided in six equal parts, and subjected to the chemical treatments with lysis buffer, 100 mM Na 2 CO 3 (pH 11), 2, 4, or 8 M urea, and held for 30 min on ice.…”

Unravelling the involvement of cilevirus p32 protein in the viral transport

“…We also tested this ability for all other encoded cilevirus proteins all being unable to restore the AMV transport, resulting in infection of individual cells. Additionally, the CiLV-C p32 was also able to complement in trans the movement-deficiency phenotype of a turnip crinkle virus ( TCV) and tobacco mosaic virus (TMV) mutants in N. benthamiana plants 48 , 49 , indicating the capacity of cilevirus MP to rescue the local movement of viruses that differ in movement transport mechanisms.…”