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Abstract: Positive-stranded RNA viruses subvert subcellular membranes to built viral replicases complexes (VRCs) in infected cells. Tombusviruses use peroxisomal membranes for the assembly of their VRCs and they can efficiently switch to the endoplasmic reticulum membrane in the absence of peroxisomes. In this paper, we show that the ER-resident Sec39p vesicular transport protein is critical for the formation of active VRCs in yeast model host. Repression of Sec39p expression in yeast or in plants resulted in greatly reduced tombusvirus accumulation. Moreover, the purified tombusvirus replicase from Sec39p-depleted yeast cells showed low in vitro activity. Also, tombusvirus RNA replication was poor in cell-free extracts or in isolated ER membranes from yeast with repressed Sec39p expression. The tombusvirus p33 replication protein was mislocalized to the ER when Sec39p was depleted in yeast. Overall, Sec39p is the first peroxisomal biogenesis protein characterized that is critical for tombusvirus replication in yeast and plants. [Copyright &y& Elsevier]

Abstract: Replication of plus-strand RNA viruses depends on lipids present in cellular membranes. Recent genome-wide screens have revealed that eight phospholipid biosynthesis genes affected the replication of Tomato bushy stunt virus (TBSV) in yeast model host. To test the importance of phospholipids in TBSV replication, we studied one of the identified genes, namely INO2, which forms a heterodimer with Ino4, and is a transcription activator involved in regulation of phospholipid biosynthesis. Deletion of INO2, or double deletion of INO2/INO4, reduced TBSV replication and inhibited the activity of the viral replicase complex. In addition, the stability of the viral replication protein is decreased as well as the localization pattern of the viral protein changed dramatically in ino2∆ino4∆ yeast. Over-expression of Opi1, a repressor of Ino2 and phospholipid biosynthesis, also inhibited TBSV RNA accumulation. In contrast, over-expression of Ino2 stimulated TBSV RNA accumulation. We also observed an inhibitory effect on Flock house virus (FHV) replication and the reduced stability of the FHV replication protein in ino2∆ino4∆ yeast. These data are consistent with the important role of phospholipids in RNA virus replication. [Copyright &y& Elsevier]

Abstract: Replication of Tomato bushy stunt virus (TBSV) RNA takes place on the cytosolic membrane surface of peroxisomes in plants and in yeast, a model host. To identify the host proteins involved in assisting the peroxisomal localization of the tombusvirus p33 replication protein, we tested if p33 could bind directly to yeast proteins involved in peroxisomal transport in vitro. This work has led to the demonstration of Pex19p–p33 interaction via pull-down and co-purification experiments. Pex19p was also detected in the tombusvirus replicase after protein cross-linking, suggesting that Pex19p transiently binds to the replicase as could be expected from a transporter. To validate the importance of Pex19p–p33 interaction in TBSV replication in yeast, we re-targeted Pex19p to the mitochondria, which resulted in the re-distribution of a large fraction of p33 to the mitochondria. The expression of the mitochondrial-targeted Pex19p inhibited TBSV RNA accumulation by 2–4-fold in vivo and reduced the in vitro activity of the tombusvirus replicase by 80%. These data support the model that Pex19p is a cellular transporter for localization of p33 replication protein to the host peroxisomal membranes. [Copyright &y& Elsevier]

Abstract: Replication of tombusviruses and other plus-strand RNA viruses depends on several host factors that are recruited into viral replicase complexes. Previous studies have shown that eukaryotic translation elongation factor 1A (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. In this paper, we show that methylation of eEF1A by the METTL10-like See1p methyltransferase is required for tombusvirus and unrelated nodavirus RNA replication in yeast model host. Similar to the effect of SEE1 deletion, yeast expressing only a mutant form of eEF1A lacking the 4 known lysines subjected to methylation supported reduced TBSV accumulation. We show that the half-life of several viral replication proteins is decreased in see1Δ yeast or when a mutated eEF1A was expressed as a sole source for eEF1A. Silencing of the plant ortholog of See1 methyltransferase also decreased tombusvirus RNA accumulation in Nicotiana benthamiana. [Copyright &y& Elsevier]

Abstract: Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during replication. Instead, these viruses might use RNA chaperones as shown here for the essential p33 replication protein of Tomato bushy stunt virus (TBSV). In vitro experiments demonstrate that the purified recombinant p33 promotes strand separation of a DNA/RNA duplex. In addition, p33 renders dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNAs. We also demonstrate that the RNA chaperone activity of p33 facilitates self-cleavage by a ribozyme in vitro. In addition, purified p33 facilitates in vitro RNA synthesis on double-stranded (ds)RNA templates up to 5-fold by a viral RNA-dependent RNA polymerase. We propose that the RNA chaperone activity of p33 facilitates the initiation of plus-strand synthesis as well as affects RNA recombination. Altogether, the TBSV RNA chaperone might perform similar biological functions to the helicases of other RNA viruses with much larger coding capacity. [Copyright &y& Elsevier]