Maturation of the hepatitis A virus capsid protein VP1 is not dependent on processing by the 3Cpro proteinase

Abstract

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.

FIG. 1

Schematic representation of full-length or truncated capsid protein precursors expressed by recombinant vaccinia viruses. The identities of the amino acids located at the N and C termini of each capsid protein of the 5′P2P3-18f virus (29) as well as their positions within the polyprotein are indicated.

FIG. 2

(A) Autoproteolytic cleavage of the 2BC-P3 precursor of the 3C^pro proteinase. FRhK-4 cells were infected with vTF7-3 (T cell) or coinfected with vTF7-3 and vv-2BC-P3, each at an MOI of 5 PFU/cell. [³⁵S]methionine-labeled HAV proteins were separated by SDS-PAGE (12% gel) following immunoprecipitation with anti-HAV 3C antibodies. (B) P1-2A is cleaved in trans by the 2BC-P3 precursor of the 3C^pro proteinase at the VP0/VP3 and VP3/VP1 junctions. FRhK-4 cells were coinfected with vTF7-3 and vv-P1-2A, with or without vv-2BC-P3 as a source of the 3C^pro proteinase, each at an MOI of 5 PFU/cell. Proteins were separated by SDS-PAGE (10% gel) and identified in an immunoblot using a mixture of anti-VP1 and anti-VP2 antibodies. Positions of molecular weight standards and of precursors and mature HAV polypeptides are shown at the left and right, respectively, of each panel.

FIG. 3

The mature capsid protein VP1 comigrates with VP1⁷⁶⁴. (A) FRhK-4 cells were infected with either HAV 5′P2P3-18f (lane 3) or vTF7-3 (lane 1) or coinfected with vTF7-3, the indicated recombinant vaccinia virus expressing a truncated P1-2A capsid protein precursor, and with vv-2BC-P3 as a source of 3C^pro proteinase (lanes 2, 4, 5, 6, and 7). Proteins were separated by SDS-PAGE (10% gel) and identified in an immunoblot with anti-VP1 antibodies. (B) A mixture of equal volumes of cellular extracts from HAV-infected cells and cells coinfected with vTF7-3, vv-P1⁷⁶⁴, and vv-2BC-P3 was loaded in lane 3+4. Positions of molecular weight standards and of precursors and mature HAV polypeptides are shown at the left and right, respectively, of each panel.

FIG. 4

RIFA assay of mutant HAVs. Petri dish cultures of BS-C-1 cells were infected with wild-type (wt) virus (5′P2P3-18f) or with HAV mutants containing the indicated substitutions at residue 764 or 791. The inocula for the mutant viruses were lysates of infected FRhK-4 cells, collected 2 weeks after transfection with synthetic genome-length RNA. The BS-C-1 cell cultures were maintained for 1 week at 37°C before processing for detection of HAV radioimmunofoci as described in Materials and Methods.

FIG. 5

HAV 764 and 791 mutants produce a fully processed, mature VP1 capsid protein. FRhK-4 cells were mock infected (T cell) or infected with the indicated mutant at an MOI of 1 (A) or 2 (B) RFU/cell, and cytoplasmic extracts were prepared at the indicated times p.i. Proteins were separated by SDS-PAGE (10% gel) and identified in immunoblots with a mixture of anti-VP1 and anti-VP2 antibodies. VP0 and VP2 bands are indistinguishable in these gels. Positions of molecular weight standards and of precursors and mature HAV polypeptides are shown at the left and right, respectively, of each panel. wt, wild type.