Vaccinia virus proteome: identification of proteins in vaccinia virus intracellular mature virion particles

Abstract

Vaccinia virus is a large enveloped poxvirus with more than 200 genes in its genome. Although many poxvirus genomes have been sequenced, knowledge of the host and viral protein components of the virions remains incomplete. In this study, we used gel-free liquid chromatography and tandem mass spectroscopy to identify the viral and host proteins in purified vaccinia intracellular mature virions (IMV). Analysis of the proteins in the IMV showed that it contains 75 viral proteins, including structural proteins, enzymes, transcription factors, and predicted viral proteins not known to be expressed or present in the IMV. We also determined the relative abundances of the individual protein components in the IMV. Finally, 23 IMV-associated host proteins were also identified. This study provides the first comprehensive structural analysis of the infectious vaccinia virus IMV.

FIG.1.

(A) Purified IMV. Electron microscopy of purified IMV particles using negative uranyl acetate staining (left) and silver staining of IMV proteins, 340 ng (lane 1) and 170 ng (lane 2), on 12% SDS-PAGE (right). (B) MS/MS spectrum of one tryptic peptide with a sequence identified as HAFDAPTLYVK and with a Mascot score of 72. (C) Amino acid sequence of the putative E6R protein. Tryptic peptides detected by MS, including the peptide in panel B, are underlined and give 53% sequence coverage. (D) MS/MS spectrum of one tryptic peptide with a sequence identified as ADEDDNEETLK and with a Mascot score of 80. (E) Amino acid sequence of A27L envelope protein. Tryptic peptides detected by MS, including the peptide in panel D, are underlined and give 71% sequence coverage.

FIG.1.

(A) Purified IMV. Electron microscopy of purified IMV particles using negative uranyl acetate staining (left) and silver staining of IMV proteins, 340 ng (lane 1) and 170 ng (lane 2), on 12% SDS-PAGE (right). (B) MS/MS spectrum of one tryptic peptide with a sequence identified as HAFDAPTLYVK and with a Mascot score of 72. (C) Amino acid sequence of the putative E6R protein. Tryptic peptides detected by MS, including the peptide in panel B, are underlined and give 53% sequence coverage. (D) MS/MS spectrum of one tryptic peptide with a sequence identified as ADEDDNEETLK and with a Mascot score of 80. (E) Amino acid sequence of A27L envelope protein. Tryptic peptides detected by MS, including the peptide in panel D, are underlined and give 71% sequence coverage.

FIG. 2.

Distribution of viral proteins with different abundances in IMV. All the viral proteins identified in this study were quantified as described in Materials and Methods and divided into four categories based on the protein contents in IMV, as shown below the graph. The black columns show the molar percentages of the total proteins in IMVs, while the white columns show the weight percentages.