A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements

Abstract

SUMMARYViral movement proteins exploit host endomembranes and the cytoskeleton to move within the cell via routes that, in some cases, are dependent on the secretory pathway. For example, melon necrotic spot virus p7B, a type II transmembrane protein, leaves the endoplasmic reticulum (ER) through the COPII-dependent Golgi pathway to reach the plasmodesmata. Here we investigated the sequence requirements and putative mechanisms governing p7B transport through the early secretory pathway. Deletion of either the cytoplasmic N-terminal region (CR) or the luminal C-terminal region (LR) led to ER retention, suggesting that they are both essential for ER export. Through alanine-scanning mutagenesis, we identified residues in the CR and LR that are critical for both ER export and for viral cell-to-cell movement. Within the CR, alanine substitution of aspartic and proline residues in the DSSP b-turn motif (D 7 AP 10 A) led to movement of discrete structures along the cortical ER in an actin-dependent manner. In contrast, alanine substitution of a lysine residue in the LR (K 49 A) resulted in a homogenous ER distribution of the movement protein and inhibition of ER-Golgi traffic. Moreover, the ability of p7B to recruit Sar1 to the ER membrane is lost in the D 7 AP 10 A mutant, but enhanced in the K 49 A mutant. In addition, fluorescence recovery after photobleaching revealed that K 49 A but not D 7 AP 10 A dramatically diminished protein lateral mobility. From these data, we propose a model whereby the LR directs actin-dependent mobility toward the cortical ER, where the cytoplasmic DSSP b-turn favors assembly of COPII vesicles for export of p7B from the ER.

Dissecting the multifunctional role of the N‐terminal domain of the Melon necrotic spot virus coat protein in RNA packaging, viral movement and interference with antiviral plant defence

“…To evaluate the ability of the mutants analysed above to move from cell to cell, the same mutations were introduced into an infectious vector [pMNSV(Al/GFP)] in which the green fluorescent protein (GFP) and CP genes were linked in frame using spacer sequences encoding the Thosea asigna virus (TAV) 2A peptide (Fig. a) (Serra‐Soriano et al ., ). It is not known exactly how 2a peptides work, but they allow the splitting of the linked proteins during their translation (Luke and Ryan, ).…”

“…Cell‐to‐cell movement was examined as described previously by monitoring the formation of fluorescent foci by MNSV(Al/GFP) constructs (Navarro et al ., ; Serra‐Soriano et al ., ). Binary constructs were agroinfiltrated [optical density at 600 nm (OD 600 ) of 0.2] into fully expanded leaves from 2‐week‐old melon plants.…”

“…To measure viral cell‐to‐cell movement, a cloning strategy relying on the use of the BveII type IIs endonuclease was employed to introduce all the CP deletions and the R81A point mutation into the previously obtained pMNSV(Al)/GFP vector (Serra‐Soriano et al ., ) (see primers in Table S1).…”

The Vesicle-Forming 6K 2 Protein of Turnip Mosaic Virus Interacts with the COPII Coatomer Sec24a for Viral Systemic Infection

“…In the case of the Norwalk virus, the nonstructural protein p22, which inhibits COPII vesicle trafficking, contains the YX⌽ESDG motif (61). The DSSP domain in the N-and the lysine residue in the C-terminal tail of MNSV p7B are required for ER export (62). On the other hand, no clearly defined ER export motifs were identified for the TGBp3 of BaMV (17).…”

Estudio integral de los mecanismos de resistencia inducida. Inductores frente a estrés biótico y abiótico

“…The open reading frame (ORF) at the 5′ end terminates in an amber codon to yield two proteins involved in replication: p29 and p89. Cell-to-cell viral movement is supported by two proteins, p7A and p7B, encoded by two small centrally-located ORFs (Genoves et al, 2009(Genoves et al, , 2010Serra-Soriano et al, 2014). The ORF at the 3´end encodes coat protein (CP) p42, which is also involved in the systemic transport of the virus and is a symptom determinant.…”