Tandem leader proteases of Grapevine leafroll-associated virus-2: host-specific functions in the infection cycle

Abstract

Several viruses in the genus Closterovirus including Grapevine leafroll-associated virus-2 (GLRaV-2), encode a tandem of papain-like leader proteases (L1 and L2) whose functional profiles remained largely uncharacterized. We generated a series of the full-length, reporter-tagged, clones of GLRaV-2 and demonstrated that they are systemically infectious upon agroinfection of an experimental host plant Nicotiana benthamiana. These clones and corresponding minireplicon derivatives were used to address L1 and L2 functions in GLRaV-2 infection cycle. It was found that the deletion of genome region encoding the entire L1-L2 tandem resulted in a ~100-fold reduction in minireplicon RNA accumulation. Five-fold reduction in RNA level was observed upon deletion of L1 coding region. In contrast, deletion of L2 coding region did not affect RNA accumulation. It was also found that the autocatalytic cleavage by L2 but not by L1 is essential for genome replication. Analysis of the corresponding mutants in the context of N. benthamiana infection launched by the full-length GLRaV-2 clone revealed that L1 or its coding region is essential for virus ability to establish infection, while L2 plays an accessory role in the viral systemic transport. Strikingly, when tagged minireplicon variants were used for the leaf agroinfiltration of the GLRaV-2 natural host, Vitis vinifera, deletion of either L1 or L2 resulted in a dramatic reduction of minireplicon ability to establish infection attesting to a host-specific requirement for tandem proteases in the virus infection cycle.

Fig. 1

(A) Diagrams of GLRaV-2 genome (top), full-length, GFP-tagged cDNA clone of GLRaV-2 (LR-GFP, middle) and GFP/GUS-tagged minireplicon (mLR-GFP/GUS, bottom). Functions of viral genes are shown above and below the diagram. L1 and L2, leader proteases; MET, HEL, and POL, methyltransferase, RNA helicase, and RNA polymerase domains, respectively; p6, 6-kDa movement protein; Hsp70h, Hsp70 homolog; p63, 63-kDa protein; CPm, minor capsid protein; CP, major capsid protein; p19, 19-kDa protein; p24, 24-kDa protein; GFP, green fluorescent protein; GFP/GUS, GFP fusion with β-glucuronidase; 35S, 35S RNA polymerase promoter of Cauliflower mosaic virus; RZ, ribozyme. (B) Diagrams of the mutations introduced into L1 and L2 (left) and corresponding phenotypes indicating processing activity, levels of GUS expression, and systemic infectivity. L2_HA, insertion of the triple hemagglutinin epitope into L2 (HA, white strip); M1, replacement of the L1 catalytic Cys residue with Ala (C₄₉₃A); M2, replacement of the L2 catalytic Cys residue with Ala (C₇₆₇A); ΔL1, deletion of the entire L1 coding region; ΔL2, deletion of the entire L2 coding region; ΔL1/2, deletion of the entire L1 and L2 coding regions; ΔNT1, deletion of the region encoding N-terminal, non-proteolytic domain of L1; ΔPro1, deletion of the region encoding C-terminal, proteolytic domain of L1.

Fig. 2

L1- and L2-mediated processing of the N-terminal part of the GLRaV-2 polyprotein generated in vitro. Lanes correspond to mutant variants shown in the Fig. 1B except for NC, no RNA control. (A) Immunoblot analysis of the in vitro translation products using HA-specific antibody (α-HA) to detect L2. Arrows at the right mark the following processing products: L1–L2, unprocessed fusion of L1 and L2; L2+, L2 fused to a part of MET; L2_HA, fully processed, HA-tagged L2. Numbers at the left show the mol. mass (kDa) of the protein markers. (B) Analysis of the ³⁵S-methyonine-labeled in vitro translation products. Designations are as in (A).

Fig. 3

Systemic transport of the LR-GFP variants harboring HA-tagged L2 following agroinoculation of N. benthamiana. Variants are marked as in Fig. 1B. (A) Images of the upper, non-inoculated leaves under epifluorescent stereoscope showing GFP expressed in veins. (B) Immunoblot analyses using anti-CP antibody (α-CP, top). Bottom panel, loading control showing Coomassie-stained Rubisco band on the membrane used for immunoblotting. (C and D) Lack of systemic infection in the plants agroinoculated using ΔNTD1 and ΔL1 variants revealed using epifluorescent microscopy (C) or immunoblotting (D).

Fig. 4

Sucrose density gradient separation and immunoblot analysis of the GLRaV2 virions using anti-CP antibody (α-CP, top) or anti-HA antibody (α-HA, bottom). Gradient fractions were numbered from the bottom of the gradient. The insets show HA-specific immunogold electron microscopy analysis (IGEM) of the combined fractions marked by white boxes; white circles highlight gold microspheres. Arrows mark positions of L2-Pro and CP.