Membrane Topography of the Hydrophobic Anchor Sequence of Poliovirus 3A and 3AB Proteins and the Functional Effect of 3A/3AB Membrane Association upon RNA Replication

Abstract

Replication of poliovirus RNA takes place on the cytoplasmic surface of membranous vesicles that form after infection of the host cell. It is generally accepted that RNA polymerase 3D pol interacts with membranes in a complex with viral protein 3AB, which binds to membranes by means of a hydrophobic anchor sequence that is located near the C-terminus of the 3A domain. In this study, we used fluorescence and fluorescence quenching methods to define the topography of the anchor sequence in context of 3A and 3AB proteins inserted in model membranes. Mutants with a single tryptophan near the center of the anchor sequence but lacking Trp elsewhere in 3A/3AB were constructed which, after the emergence of suppressor mutations, replicated well in HeLa cells. When a peptide containing the mutant anchor sequence was incorporated in model membrane vesicles, measurements of Trp depth within the lipid bilayer indicated formation of a transmembrane topography. However, rather than the 22 residue length predicted from hydrophobicity considerations, the transmembrane segment had an effective length of 16 residues, such that Gln64 likely formed the N-terminal boundary. Analogous experiments using full length proteins bound to pre-formed model membrane vesicles showed that the anchor sequence formed a mixture of transmembrane and non-transmembrane topographies in the 3A protein but adopted only the nontransmembrane configuration in the context of 3AB protein. Studies of the function of 3A/3AB inserted into model membrane vesicles showed that membrane-bound 3AB is highly efficient in stimulating the activity of 3D pol in vitro while membrane-bound 3A totally lacks this activity. Moreover, in vitro uridylylation reactions showed that membrane-bound 3AB is not a substrate for 3D pol but free VPg released by cleavage of 3AB with proteinase 3CD pro could be uridylylated.After poliovirus (PV) enters the host cell its plus strand RNA genome is translated into a polyprotein that contains one structural (P1) and two nonstructural domains (P2, P3; Fig. 1A). There is an efficient and regulated cascade of protein processing that produces cleavage products with function distinct from those of the precursor proteins (Fig. 1A). A variety of precursor and mature proteins are released from the PV polyprotein by cleavage with *Corresponding author: Erwin London, Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215, Tel: (631) FAX: (631) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript proteinases 2A pro , 3C pro /3CD pro . The proteins of the P1 domain (VP1-VP4) assemble to form the capsid while those derived from P2 (2A pro , 2B, 2BC, 2C ATPase ) are primarily responsible for the biochemical and structural changes that occur in the infected cell. The proteins of the P3 domain are those most directly involved in RNA synthesis. These include two important and relatively stable precursors, proteinase 3CD pro and 3AB, which were shown to specifically interact with a cl...

Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase

“…The observation that expression of PV 3A by itself in HeLa cells does not inhibit GLuc secretion prompted us to determine whether one of the other membrane-binding viral nonstructural proteins (2B or 2C) of poliovirus or their precursors (2BC or 3AB) serve this function. In virus-infected cells, 2B, 2C, and 2BC are associated with membranes via their amphipathic helices, while 3A and 3AB are anchored through hydrophobic sequences (26,51,62,64). Previous studies in Vero cells infected with FMDV have shown that 2BC (or coexpression of 2B and 2C), but not 3A, inhibits ER-to-Golgi apparatus protein transport (43).…”

“…Interestingly, it was shown that CVB3 3A inhibits transport by binding to cellular factor GBF-1 and inhibiting GBF-1 dependent COP-1 recruitment to membranes (43,44). Polypeptides 3A and 2B and their precursors, 3AB and 2BC, contain hydrophobic transmembrane regions that play a role in their effect on secretion (2,26,62). In the case of foot-and-mouth disease virus (FMDV), a picornavirus of the genus Aphthovirus, the 2BC precursor polypeptide rather than 3A or 2B is responsible for the block in the secretory pathway (43,44).…”

Effect of Lipid Composition on the Topography of Membrane-Associated Hydrophobic Helices: Stabilization of Transmembrane Topography by Anionic Lipids

“…These sequences had weakly hydrophobic cores composed of Ala with or without a few Leu (Table 1, sequences [10][11][12][13][14]. When these peptides were inserted into DOPS vesicles highly blue-shifted λmax and low Qratio values were generally observed (with the exception of KK pA 22 peptide which showed a somewhat more red shifted λmax and high Q-ratio), showing that a TM state predominated.…”

Analysis of Poliovirus Protein 3A Interactions with Viral and Cellular Proteins in Infected Cells

“…The 87-amino-acid-long 3A protein has a C-terminal hydrophobic domain of 22 residues which is responsible for its direct membrane association and which has been shown to be important for PV replication by molecular genetic studies (13)(14)(15)(31)(32)(33). Membrane-bound 3AB, but not 3A, serves as a cofactor to stimulate the activity of 3D viral polymerase (3D pol ) in vitro (13).…”