Synthesis, proteolytic processing and complex formation of N-terminally nested precursor proteins of the Rift Valley fever virus glycoproteins

Abstract

The genomic M RNA segment of Rift Valley fever virus is transcribed to produce a single mRNA with multiple translation initiation sites. The products of translation are an N-terminal nested series of polyproteins. These polyproteins enter the secretory system of the host cell and are proteolytically processed to yield the mature virion glycoproteins, Gn and Gc, and two non-structural glycoproteins. By means of pulse-chase immune precipitation experiments we identify the Gn and Gc precursor molecules and also show that signal peptidase cleavage is required for mature Gn and Gc production. We also demonstrate that a hydrophobic domain at the N-terminus of Gn acts as a signal peptide only in the context of the polyprotein precursors that initiate at the second, fourth or fifth AUGs. In addition, we document that formation of Gn/Gc heteromeric complexes occur rapidly (<5 min) and can occur prior to signal peptidase processing of Gn, suggesting that this complex forms in the endoplasmic reticulum. Interestingly, Gc can form a complex with a glycoprotein that has been considered nonstructural, a discovery that has implications for both the topology and potential packaging of this glycoprotein.

Fig. 1

(A) Structural features of the RVF virus M segment RNA and encoded proteins. Scissors symbols indicate putative signal peptidase processing sites, ball and stick symbols indicate the position of the in-frame start codons in the NSm region, and the branch and stick symbols indicate the position of putative N-linked glycosylation sites. The glycosylation site numbering system refers to the amino acid position and is relative to translation initiation at the first start codon. The brackets delineate the mature and precursor proteins along with their calculated molecular weights. The black, white and hatched boxes indicate transmembrane, signal peptide and uncharacterized hydrophobic domains, respectively. (B) The NSm region of the polyprotein precursor molecule. The translation of the NSm region is in single letter code. The hydrophobic domain at the amino terminus of NSm1 and the signal peptide upstream of Gn are underlined and the methionine (M) residues are bolded and numbered.

Fig. 2

Panels A and B, potential topologies of the polyprotein precursors that initiate at the second AUG (Panel A), or fourth or fifth AUG (Panel B). Signal peptidase cleavage sites are indicated by scissors, putative N-linked glycosylation sites are indicated by branch and stick symbols, signal peptides are the white boxes and transmembrane domains are black boxes. Locations of initiator methionines (MET) are indicated. Panels C and D, hidden Markov model predictions for the hydrophobic domain preceding Gn in the context of the polyproteins beginning at MET2 (Panel C), or MET4 (Panel D). The n-, h- and c-regions refer to the domain proximal to the hydrophobic region, the hydrophobic region and the signal peptidase cleavage region, respectively. The probability that the sequence contains an n-, h- or c- region of a signal peptide is plotted on the y-axis, and the amino acids and their positions are plotted on the x-axis. The plots encompass the hydrophobic domain and begin at (C) amino acid 93 for MET2 initiated polyprotein and at (D) amino acid 1 for the MET4 polyprotein. The signal peptidase cleavage site is between the alanine and glutamic acid residues that are underlined.

Fig. 3

Multiple polyprotein precursor molecules are generated from RVF virus M segment mRNA. BSR-T7/5 cells transformed with either the RVFMGcFLAG or ΔNSmGcFLAG plasmid were pulse labeled for 5 min with ³⁵S-cysteine, then chased for the times, expressed in minutes, indicated at the top of the gel. Immune precipitation of M segment encoded glycoproteins, with antibodies recognizing either Gn or FLAG, was carried out as described in Materials and methods. The lane numbers are indicated at the bottom of the gel.

Fig. 4

Signal peptidase cleavage at the site within Gn is required for generation of mature Gn. BSR-T7/5 cells transformed with either the RVFMGcFLAG-GnSP or ΔNSmGcFLAG-GnSP plasmid, both plasmids contain an alanine to arginine mutation at the predicted signal peptidase cleavage site within Gn. Cells were pulse labeled for 5 min with ³⁵S-cysteine, then chased for the times, expressed in minutes, indicated at the top of the gel. Immune precipitation of M segment encoded glycoproteins, with antibodies recognizing either Gn or FLAG, was carried out as described in Materials and methods. The lane numbers are indicated at the bottom of the gel.

Fig. 5

Signal peptidase cleavage at the site within Gc is required for generation of all mature RVF virus M segment encoded glycoproteins. BSR-T7/5 cells transformed with either the RVFMGcFLAG-GcSP or ΔNSmGcFLAG-GcSP, both plasmids contain an alanine to arginine mutation at the predicted signal peptidase cleavage site within Gc. Cells were pulse labeled for 5 min with ³⁵S-cysteine, then chased for the times, expressed in minutes, indicated at the top of the gel. Immune precipitation of M segment encoded glycoproteins, with antibodies recognizing either Gn or FLAG, was carried out as described in Materials and methods. The lane numbers are indicated at the bottom of the gel.