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. 1999 Dec;73(12):9867-78.
doi: 10.1128/JVI.73.12.9867-9878.1999.

Improving proteolytic cleavage at the 3A/3B site of the hepatitis A virus polyprotein impairs processing and particle formation, and the impairment can be complemented in trans by 3AB and 3ABC

Y Kusov  1 , V Gauss-Müller
Affiliations

Improving proteolytic cleavage at the 3A/3B site of the hepatitis A virus polyprotein impairs processing and particle formation, and the impairment can be complemented in trans by 3AB and 3ABC

Y Kusov et al. J Virol. 1999 Dec.

Abstract

The orchestrated liberation of viral proteins by 3C(pro)-mediated proteolysis is pivotal for gene expression by picornaviruses. Proteolytic processing is regulated either by the amino acid sequence at the cleavage site of the substrate or by cofactors covalently or noncovalently linked to the viral proteinase. To determine the role of the amino acid sequence at cleavage sites 3A/3B and 3B/3C that are essential for the liberation of 3C(pro) from its precursors and to assess the function of the stable processing intermediates 3AB and 3ABC, we studied the effect of cleavage site mutations on hepatitis A virus (HAV) polyprotein processing, particle formation, and replication. Using the recombinant vaccinia virus system, we showed that the normally retarded cleavage at the 3A/3B junction can be improved by altering the amino acid sequence at the scissile bond such that it matches the preferred HAV 3C cleavage sites. In contrast to the processing products of the wild-type polyprotein, 3ABC was no longer detectable in the mutant. VP0 and VP3 were generated less efficiently, implying that processing of the structural protein precursor P1-2A depends on the presence of stable 3ABC and/or 3AB. In addition, cleavage of 2BC was impaired in 3AB/3ABC-deficient mutants. Formation of HAV particles was not affected in mutants with blocked 3A/3B and/or 3B/3C cleavage sites. However, 3ABC-deficient mutants produced small numbers of HAV particles, which could be augmented by coexpressing 3AB or 3ABC. The hydrophobic domain of 3A that has been proposed to mediate membrane anchorage of the replication complex was crucial for restoration of defective particle formation. In vitro transcripts of the various cleavage site mutants were unable to initiate an infectious cycle, and no progeny viruses were obtained even after blind passages. Taken together, the data suggest that accumulation of uncleaved HAV 3AB and/or 3ABC is pivotal for both viral replication and efficient particle formation.

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Figures

FIG. 1
FIG. 1
Schematic presentation of wild-type (wt) and mutated HAV cDNA constructs encoding the complete polyprotein (pT7-18f), P1-2A (pEXT7-P1-2A), or P3 (pT7-P3) (A) and of HAV cDNA clones used in trans-complementation experiments (B). ∗ and ∗∗ indicate the 3A/3B and 3B/3C cleavage sites mutated in the HAV genome, respectively. The primary cleavage sites are marked by arrowheads. μ denotes the proteolytically inactive form of the proteinase due to the Cys-to-Ala mutation at the active site of the enzyme.
FIG. 2
FIG. 2
Proteolytic processing pattern of HAV P3 mutated at the 3A/3B and/or 3B/3C sites. The HAV P3 domain containing the wild-type (wt) or mutated sequences (#1 to #6) (see Table 1) was transiently expressed in COS-7 cells. Equal amounts of each cell extract were analyzed by immunoblotting with anti-3A, anti-3B, anti-3C, and anti-3D antibody. The anti-3B immunoblot analysis was performed with 12 and 15% polyacrylamide gels to ensure optimal separation of small (3AB) and large (3BC and 3ABC) polypeptides. Molecular mass markers are shown on the margin, and HAV P3 cleavage products are indicated. m, mock-transfected extracts.
FIG. 3
FIG. 3
Processing products derived from domains P1-2A and 2BC of the HAV polyprotein mutated at the 3A/3B and/or 3B/3C site. The complete HAV genomes of wild-type (wt) and mutated sequence (mutants 1 to 6) was expressed in COS-7 cells. Equal amounts of cells extract were analyzed by immunoblotting with anti-VP1, anti-VP0, anti-VP3, anti-2C3A, and anti-2B antibodies. The asterisk indicates an unidentified host protein immunodetected by anti-2C3A. Molecular mass markers and cleavage products of P1-2A and 2BC are indicated. m, mock-transfected extracts.
FIG. 4
FIG. 4
HAV particle formation after recombinant expression of HAV genomes mutated at the 3A/3B and/or 3B/3C cleavage sites. Either the complete genome (A) or P1-2A with P3 (B) was expressed in COS-7 cells. The extracts in panel A are identical to those in Fig. 3. Particle formation was determined by the particle-specific ELISA and is expressed as percentage of the antigenicity produced by the wild-type genome (column wt). In column 7 of panel A, the mock extract is shown. In column 7 of panel B, the relative antigenicity of P1-2A-expressing cells is shown and is used as control to demonstrate the specificity of the ELISA for processed and particulate material.
FIG. 5
FIG. 5
Effect of P3 proteins on defective recombinant-particle formation from pT7-18f mutant 4 (A) and on protein synthesis (B). pT7-18f mutant 4 (A) and pGEM1-lacZ (B) were coexpressed in COS-7 cells with cDNAs indicated on the top right. To ensure equal protein levels of the trans-complementing polypeptides, equal amounts of cDNA encoding the same promoter and initiation region of protein synthesis were used. Particle formation was determined in the cell extracts by the particle-specific ELISA (A) or protein synthesis was assessed by the β-galactosidase activity (B), both presented in arbitrary units. μ denotes the proteolytically inactive form of the proteinase.
FIG. 6
FIG. 6
Specificity of HAV 3AB and 3ABC as the cofactor in HAV particle formation. pT7-18f or pT7-18f mutant 4 in the presence of cDNAs as marked on the top right was expressed in COS-7 cells. The effect on particle formation was determined in the cell extracts by the particle-specific ELISA and is expressed as percentage of the antigenicity produced in cells expressing the wild-type (wt) genome (pT7-18f, set at 100%).
FIG. 7
FIG. 7
Dose-dependent stimulation of P1-2A processing and particle formation by 3AB. pT7-18f mutant 4 (0.3 μg) was coexpressed in the presence of increasing amounts (0, 0.2, 0.4, 0.6, and 0.8 μg) of cDNAs pET (lanes 1 to 5), pET-3ABΔid (lanes 6 to 10), and pET-3AB wt (lanes 11 to 15). Cell extracts were analyzed by the particle-specific ELISA (top) and by immunoblotting with anti-VP0 (middle) and anti-3B (bottom). In lane 16, the extract of mock-infected cells is shown. Molecular mass markers and HAV polypeptides are indicated. OD450, optical density at 450 nm.
FIG. 8
FIG. 8
Working model for the rescue of defective HAV polyprotein processing and particle formation of mutant 4 by 3AB and 3ABC. (A) Expression of mutant 4 that is unable to accumulate 3AB and 3ABC results in uncleaved 2BC and mostly unprocessed P1-2A and thus yields very low levels of HAV particles, as determined by the particle-specific ELISA. (B) Coexpression of 3ABCpro tethers the assembly complex to membranes, resulting in coordinated pentamer cleavage and assembly (thick arrows), leading to high levels of antigenically reactive particles (ELISA +++). 2BC is efficiently cleaved (thick arrow) (reference and data not shown). (C) Coexpression of 3AB helps to bring 3C derived from the polyprotein of mutant 4 close to the membrane-associated assembly complex, resulting in somewhat enhanced particle formation (thin arrows, ELISA +). As shown recently by coimmunoprecipitation and by Far Western and affinity chromatography, HAV 3C can interact with cognate 3AB (2). (D) Coexpression of 3ABΔid, which is deleted in the hydrophobic domain of 3A and unable to bind to membranes (7), does not bring 3C close to the membrane-associated assembly complex. Therefore, particle formation is not enhanced over that of mutant 4 (ELISA −). Note that the particle-specific ELISA detects processed pentamers and capsids (1, 25, 29, 30), as shown at the bottom.
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