Proteolytic processing of sapovirus ORF1 polyprotein

Abstract

The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in (1171)Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH(2)-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.

FIG. 1.

(A) Genome organization and expression of Mc10 ORF1 proteins. The motifs of 2C-like NTPase (⁴⁸¹GPPGIGKT⁴⁸⁸), VPg (⁹⁴²KGKTK⁹⁴⁶ and ⁹⁶⁴DEYDE⁹⁶⁸), Pro (¹¹⁶⁹GDCG¹¹⁷²), Pol (¹⁵⁰⁴GLPSG¹⁵⁰⁸ and ¹⁵⁵²YGDD¹⁵⁵⁵), and VP1 (¹⁸⁵⁶PPG¹⁸⁵⁸) are indicated by asterisks below the ORF1 polyprotein. The regions A to J were expressed in E. coli. (B) Mc10 ORF1 proteins expressed in vitro with a coupled transcription-translation system. The template DNAs were generated by PCR using primers listed in Table 2, and proteins I to VI were synthesized in vitro. Triangles indicate the position in the putative 3C-like protease where a ¹¹⁷¹Cys-to-¹¹⁷¹Ala change was introduced. Both wild-type (Pro^w) and mutant (Pro^mut) forms of the constructs were used for the expression. (C) A proposed genetic map of Mc10 ORF1. The motifs of NTPase, VPg, Pro, Pol, and VP1 are indicated by asterisks below the ORF1 polyprotein. The identified products were p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120.

FIG. 2.

The processing products of Mc10 ORF1 polyprotein. (A) SDS-PAGE of in vitro ³⁵S-labeled proteins derived from I-Pro^w and I-Pro^mut. The protein bands specific to either the wild-type or mutant form are indicated by triangles or an open triangle, respectively. The molecular sizes of viral proteins are shown on the right. (B) Immunoprecipitation with region-specific antibodies. In vitro ³⁵S-labeled proteins derived from I-Pro^w were immunoprecipitated with anti-A to -H antibodies and analyzed by SDS-PAGE. The proteins of interest are indicated by arrows.

FIG. 3.

SDS-PAGE of in vitro ³⁵S-labeled proteins derived from I- to VI-Pro^w and I- to VI-Pro^mut. The proteins derived from the wild-type form are indicated by triangles, and those from the mutant form are indicated by open triangles.

FIG. 4.

Identification of p66, p11, p14, and p70. In vitro ³⁵S-labeled proteins expressed with I- to VI-Pro^w and I- to VI-Pro^mut were immunoprecipitated with anti-A, anti-D, anti-E, or anti-F antibodies.