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Journal of Virology, August 1999, p. 6220-6227, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3Cpro Proteinase

Annette Martin,1,* Danièle Bénichou,1 Shih-Fong Chao,2,dagger Lisette M. Cohen,1 and Stanley M. Lemon3

Unité de Virologie Moléculaire, URA CNRS 1966, Institut Pasteur, Paris Cedex 15, France1; Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275992; and Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-10193

Received 22 March 1999/Accepted 20 April 1999

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.

* Corresponding author. Mailing address: Unité de Virologie Moléculaire, URA CNRS 1966, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: (33-1) 40 61 33 60. Fax: (33-1) 40 61 30 45. E-mail: annettem{at}pasteur.fr.

dagger Present address: Department of Pediatrics, Duke University, Durham, NC 27710.

Journal of Virology, August 1999, p. 6220-6227, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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