Structural and functional properties of the vesicular stomatitis virus nucleoprotein-RNA complex as revealed by proteolytic digestion

Abstract

To gain insight into the structural and functional properties of the vesicular stomatitis virus nucleocapsid-RNA complex (vN-RNA), we analyzed it by treatment with proteolytic enzymes. Chymotrypsin treatment to the vN-RNA results in complete digestion of the C-terminal 86 amino acids of the N protein. The residual chymotrypsin resistant vN-RNA complex (vDeltaN-RNA) carrying N-terminal 336 amino acids of the N protein (DeltaN) was inactive in transcription. The DeltaN protein retained its capability to protect the genomic RNA from nuclease digestion but failed to interact to the P protein. Interestingly, addition of excess amount of P protein rendered the vN-RNA complex resistant to the chymotrypsin digestion. Finally, our data revealed that the recombinant N-RNA complex purified from bacteria (bN-RNA) is resistant to chymotrypsin digestion, suggesting that the C-terminal unstructured domain (C-loop) remains inaccessible to protease digestion. Detailed comparative analyses of the vN-RNA and vDeltaN-RNA are discussed.

FIG. 1

Chymotrypsin digestion of virion N-RNA. (A) Purified vN-RNA complex was mixed with SDS-PAGE loading buffer, heated to 100°C and resolved in SDS-PAGE. The N protein was visualized either by CBB stain (lane 2) or WB with anti-VSV N polyclonal antibody (lane 3). Purified VSV was used as positive control (lane 1) (B) Chymotrypsin digestion profile of vN-RNA complex at three different (w:w) ratios. (C) Time kinetics profile of chymotrypsin digestion of vN-RNA complex at 4.25:1 (w:w) (vN-RNA:Chymotrypsin) ratio from 0–20 min.

FIG. 2

(A) Amino acid sequence of the VSV N protein. The ΔN portion of the protein, as determined by LC-MS analysis, is shown in regular front and the N_C-86 protein (the C-terminal 86 amino acid) is shown in italics. The chymotrypsin cleavage site Y336 is shown in larger font. (B) The structure of the C-terminal nucleocapsid binding domain of the phosphoprotein (P), P_CTD, in complex with two nucleocapsid protein (N) subunits is presented as a ribbon drawing. P_CTD (yellow) corresponds to residues 193–265. The two N subunits (red and green) are oriented so that the cavity that encapsidates RNA is facing away. The chymotrypsin cleavage site in each subunit is marked by an arrow. The cleavage will remove the C-terminal portion of the N subunits (dark red and dark green) that makes contact with P_CTD. The coordinates used for preparing this figure are derived from PDB 3HHZ. The figure was prepared with program PyMOL (DeLano, 2002).

FIG. 3

Biochemical analyses of vN-RNA and vΔN-RNA. (A) vN-RNA was digested with chymotrypsin to obtain vΔN-RNA and vΔN-RNA was further subjected to CsCl equilibrium density gradient ultracentrifugation. Purified proteins were resolved in SDS-PAGE and visualized by CBB stain. Banding profile of vN-RNA (lane 2 and 3) and vΔN-RNA (lane 4 and 5) in CsCl equilibrium density gradient centrifugation is shown in figure. (B) In vitro transcription reconstitution reaction performed with vL-P and vN-RNA or vΔN-RNA complexes. ³²P-labeled mRNA transcripts were analyzed in 5% urea-PAGE followed by autoradiography. All of the detected transcripts are indicated. (C) ³²P-labeled VSV was prepared and vN-RNA was purified from the ³²P-labeled VSV. The vN-RNA, containing ³²P-labeled genome RNA, was digested with chymotrypsin and the digested products were further purified through CsCl equilibrium density gradient ultracentrifugation to obtain vΔN-RNA. vN-RNA and vΔN-RNA, containing ³²P-labeled genome RNA, were digested with Micrococcal nuclease and resolved in 5% urea-PAGE. The ³²P-labeled genomic RNA present within vN-RNA and vΔN-RNA were detected by autoradiography.

FIG. 4

In vitro interaction between the P protein and vN-RNA or vΔN-RNA. HeLa cells were transfected with a mock plasmid or a plasmid expressing Myc-P as detailed in Materials and Methods. At 20 hr post transfection lysates were incubated with the vN-RNA or the vΔN-RNA complexes at 30°C for 1 hr. The reaction mixtures were then centrifuged at 120,000 × g for 1 hr using a Sorvall TH660 rotor. Protein complexes present in the pellet were detected by Western blot analysis using anti-N or anti-Myc polyclonal antibodies.

FIG. 5

Protease protection assay of vN-RNA complex. (A) vN-RNA complex was incubated with the P protein at 30°C for 1 hr complex formation. Chymotrypsin digestion profile of N protein within the vN-RNA complex when complexed with P protein formed at different molar ratios (vN-RNA:P = 5:0, 5:1; 5:2.5, 5:5, 5:7.5 and 5:10). (B) vN-RNA complex and purified P protein of mumps virus (MuV) incubated at 1:1 molar ratio at 30°C temperature for 1 hr and then digested with chymotrypsin as described in materials and methods.

FIG. 6

Chymotrypsin digestion of virion RNP (vRNP), infected cell RNP (iRNP) and infected cell N-RNA (iN-RNA) complexes. (A) Comparative analysis of the Chymotrypsin digestion profile of vRNP and infected cell RNP (iRNP) (B) Chymotrypsin digestion profile of the purified vN-RNA and infected cell N-RNA (iN-RNA), obtained from iRNP.

FIG. 7

Chymotrypsin digestion profile of bacterial N-RNA complex. SDS-PAGE analysis of the purified bacterial N-RNA complex (bN-RNA) from bacteria and CsCl equilibrium density gradient purified bacterial N-RNA complex (bN-RNA_CsCl) with respect to vN-RNA complex when digested with and without chymotrypsin at 4.25:1 (w:w) ratio.