Functional characterization of the vaccinia virus I5 protein

The I5L gene is one of ~90 genes that are conserved throughout the chordopoxvirus family, and hence are presumed to play vital roles in the poxvirus life cycle. Previous work had indicated that the VP13 protein, a component of the virion membrane, was encoded by the I5L gene, but no additional studies had been reported. Using a recombinant virus that encodes an I5 protein fused to a V5 epitope tag at the endogenous locus (vI5V5), we show here that the I5 protein is expressed as a post-replicative gene and that the ~9 kDa protein does not appear to be phosphorylated in vivo. I5 does not appear to traffic to any cellular organelle, but ultrastructural and biochemical analyses indicate that I5 is associated with the membranous components of assembling and mature virions. Intact virions can be labeled with anti-V5 antibody as assessed by immunoelectron microscopy, indicating that the C' terminus of the protein is exposed on the virion surface. Using a recombinant virus which encodes only a TET-regulated copy of the I5V5 gene (vΔindI5V5), or one in which the I5 locus has been deleted (vΔI5), we also show that I5 is dispensable for replication in tissue culture. Neither plaque size nor the viral yield produced in BSC40 cells or primary human fibroblasts are affected by the absence of I5 expression

RNA–protein interactions promote asymmetric sorting of the ASH1 mRNA ribonucleoprotein complex

In Saccharomyces cerevisiae, ASH1 mRNA is localized to the tip of daughter cells during anaphase of the cell cycle. ASH1 mRNA localization is dependent on four cis-acting localization elements as well as Myo4p, She2p, and She3p. Myo4p, She2p, and She3p are hypothesized to form a heterotrimeric protein complex that directly transports ASH1 mRNA to daughter cells. She2p is an RNA-binding protein that directly interacts with ASH1 cis-acting localization elements and associates with She3p. Here we report the identification of seven She2p mutants—N36S, R43A, R44A, R52A, R52K, R63A, and R63K—that result in the delocalization of ASH1 mRNA. These mutants are defective for RNA-binding activity but retain the ability to interact with She3p, indicating that a functional She2p RNA-binding domain is not a prerequisite for association with She3p. Furthermore, the nuclear/cytoplasmic distribution for the N36S and R63K She2p mutants is not altered, indicating that nuclear/cytoplasmic trafficking of She2p is independent of RNA-binding activity. Using the N36S and R63K She2p mutants, we observed that in the absence of She2p RNA-binding activity, neither Myo4p nor She3p is asymmetrically sorted to daughter cells. However, in the absence of She2p, Myo4p and She3p can be asymmetrically segregated to daughter cells by artificially tethering mRNA to She3p, implying that the transport and/or anchoring of the Myo4p/She3p complex is dependent on the presence of associated mRNA