Crystal structure of vesicular stomatitis virus matrix protein

Abstract

The vesicular stomatitis virus (VSV) matrix protein (M) interacts with cellular membranes, self-associates and plays a major role in virus assembly and budding. We present the crystallographic structure, determined at 1.96 A resolution, of a soluble thermolysin resistant core of VSV M. The fold is a new fold shared by the other vesiculovirus matrix proteins. The structure accounts for the loss of stability of M temperature-sensitive mutants deficient in budding, and reveals a flexible loop protruding from the globular core that is important for self-assembly. Membrane floatation shows that, together with the M lysine-rich N-terminal peptide, a second domain of the protein is involved in membrane binding. Indeed, the structure reveals a hydrophobic surface located close to the hydrophobic loop and surrounded by conserved basic residues that may constitute this domain. Lastly, comparison of the negative-stranded virus matrix proteins with retrovirus Gag proteins suggests that the flexible link between their major membrane binding domain and the rest of the structure is a common feature shared by these proteins involved in budding and virus assembly.

Fig. 1. (A) Schematic of VSV M sequence. M^t and M^th are trypsin and thermolysin digestion products of M, respectively; M^th is the species whose structure was determined crystallographically. (B) Ribbon diagram of VSV M. Rainbow colouring, from blue to red, describes the N-terminal to C-terminal direction of the polypeptide chain. The interruption of the chain in the large five-stranded β-sheet corresponds to the thermolysin cleavage in the 121–128 loop that connects the second strand to the third. (C) Topology diagram of VSV M. β-strands are represented as arrows and α-helices as rectangles. Colour code is as in (A). This figure and Figure 3 were drawn with MOLSCRIPT (Kraulis, 1991) and Raster3D (Merritt and Bacon, 1997).

Fig. 2. Sequence alignment of the vesiculovirus Ms from strains Orsay (DDJB/EMBL/GenBank accession No. J02428_3), New Jersey (accession No. M14553_1), Chandipura (accession No. AF128868_2), Piry (accession No. D26175_2) and spring viremia carp virus (accession No. U18101_3) was performed with Clustal_W (Thompson et al., 1994). Conserved and similar residues visible in the structure are dark and light coloured, respectively. Blue, buried residues (side chain or glycine C_α accessible surface <10 Å²); red, surface residues (side chain or glycine C_α accessible surface >10 Å²).

Fig. 3. VSV ts mutants in M that are deficient in budding. Each view is a close-up of the structure around a position where a ts mutation that confers budding deficiency to VSV has been identified; in each case the position of the mutation and the amino acid substitution are indicated, together with the most common designation of the mutant (in parentheses). Mutated residues (blue) and those that contact them (purple) are drawn as ball and stick models.

Fig. 4. Membrane floatation of M, M^t core and M^th core in the presence of PC:PS liposomes. Eight fractions were collected from each gradient and samples from each fraction were separated on SDS 14% PAGE and stained with Coomassie Blue.

Fig. 5. Distribution of electrostatic potential on VSV M surface. Regions where the electrostatic potential is less than –10 k_bT are red, while those where it is more than +10 k_bT are blue (k_b, Boltzmann constant; T, absolute temperature). A basic patch is clearly visible close to the PAVLA peptide and to a hydrophobic patch comprised of residues Val84, Pro77, Ala118 and Tyr81. This figure was drawn with GRASP (Nicholls et al., 1991).