Role of the Amphipathic Peptide of Semliki Forest Virus Replicase Protein nsP1 in Membrane Association and Virus Replication

Abstract

Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G 245 STLYTESRKLLRSWHLPSV 264 , has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A؉L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A؉L256A only yielded virus encoding L255A؉L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein.

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae

“…Recent research on two members of the Alphavirus-like superfamily, Semliki Forest virus (SFV, genus Alphavirus; family Togaviridae) and brome mosaic virus (BMV, genus Bromovirus, family Bromoviridae), has revealed that the MTR domain contains sequences responsible for punctate structure formation (den Boon et al, 2001;Spuul et al, 2007;Liu et al, 2009). Because GRSPaV also belongs to the Alphavirus-like superfamily, we predicted that the MTR domain of its replicase polyprotein might also contain sequences capable of forming punctate bodies in plant cells.…”

“…To test if the replicase polyprotein of GRSPaV contains a functionally similar amphipathic a-helix sequence involved in forming punctate structures and in membrane association, we first performed hydropathy analyses of the MTR domain using the Protean program (DNASTAR), which revealed several candidate regions. The region encompassing amino acid residues 165-187 (PRVISTGARNLFLHDEIHYWSIS) seemed to be the most promising for further analyses, in part because it had a sufficient length to allow formation of an amphipathic a-helix as hypothesized to be an IPM anchor for monotopic membrane association in SFV (Spuul et al, 2007). Importantly, key amino acid residues that are consistently identified in IPM anchors, namely R, F and W (Sapay et al, 2006), are also present in this region.…”

“…These data suggest Hence, association of GRSPaV replicase polyprotein with an intracellular membrane would have to either involve monotopic membrane association through an amphipathic a-helix as an in-plane anchor (Sapay et al, 2006) or be mediated through a yet-unidentified integral membrane protein provided by the cell. The former scenario would be reminiscent of the membrane association of the replicase protein of SFV (Spuul et al, 2007) and BMV (Liu et al, 2009). It was shown that amino acid residues 392-409 in the SFV MTR domain are responsible for forming punctate bodies, and for associating with the ER, endosomes and lysosomes in HeLa cells (Spuul et al, 2007).…”

“…The former scenario would be reminiscent of the membrane association of the replicase protein of SFV (Spuul et al, 2007) and BMV (Liu et al, 2009). It was shown that amino acid residues 392-409 in the SFV MTR domain are responsible for forming punctate bodies, and for associating with the ER, endosomes and lysosomes in HeLa cells (Spuul et al, 2007). In contrast, a region (residues 245-264) in the MTR domain of BMV, a plant-infecting virus of the Alphavirus-like superfamily, determines punctate body formation but not membrane association (den Boon et al, 2001;Liu et al, 2009).…”

“…We designate this region a 'subdomain' to distinguish it from the shorter predicted membrane-binding sequence. Our choice to use a larger region than the putative amphipathic sequence segment (aa 132-207 versus 147-187) was based on the observation that a single copy of the amphipathic sequence from SFV, when fused to GFP, failed to target to the correct location (Spuul et al, 2007), presumably owing to the fact that the sequence was too short, which would interfere with protein targeting and membrane association. As shown in Fig.…”