Characterization of Recombinant Hepatitis A Virus Genomes Containing Exogenous Sequences at the 2A/2B Junction

Abstract

The family Picornaviridae is comprised of a large number of single-stranded RNA viruses that are currently classified into five major genera of human and veterinary pathogens and several lesser genera (38). These include the genera Enterovirus (of which poliovirus is a member and the prototype picornavirus), Rhinovirus, Cardiovirus (including encephalomyocarditis virus [EMCV]), Aphthovirus (foot-and-mouth disease virus), and Hepatovirus. Hepatitis A virus (HAV) is the only virus species within the genus Hepatovirus. A human virus that is transmitted by the fecal-oral route, HAV is responsible for approximately 50% of all cases of acute viral hepatitis within the United States (23). The genomic RNA of HAV shares a general molecular organization and replication strategy with that of other picornaviruses. The positive-strand RNA genome is approximately 7.5 kb in length (12,26,34). It is linked covalently to a small polypeptide (VPg) at its 5Ј terminus (46), contains a 3Ј polyadenylated tail, and is capable of functioning directly as mRNA when introduced into the cytoplasm of host cells (13). A lengthy 5Ј terminal nontranslated RNA segment contains numerous highly ordered RNA structures and a functional internal ribosome entry site (IRES) (10). As with the IRES elements of other picornaviruses, the HAV IRES directs the cap-independent translation of a polyprotein encoded within a single large open reading frame (10, 47). Picornaviral IRES elements have been classified as either type 1 (the enteroviruses and rhinoviruses) or type 2 (cardioviruses, aphthoviruses, and hepatoviruses) on the basis of substantial differences in their higher ordered RNA structure, yet they all function to facilitate internal ribosome entry (25,48).Despite these similarities to other picornaviruses, the polyprotein of HAV presents some striking and unique features. In enteroviral and rhinoviral replication, the primary polyprotein cleavage takes place at the P1/P2 junction (VP1/ 2A) and is mediated by a cis-acting viral proteinase activity associated with 2A (2A pro ). In the case of the cardioviruses and aphthoviruses, the primary scission is also dependent upon 2A but occurs by a unique autocatalytic mechanism at the 2A/2B junction (36,40). Although the first model proposed for the polyprotein processing of HAV resembled the enterovirus cleavage cascade (12), subsequent studies demonstrated that the only virally encoded proteinase, 3Cpro , was able to cleave in the middle of what was originally considered to be the HAV 2A coding region (19,41). It was later demonstrated that the 2B nonstructural protein is considerably larger than that predicted by the original processing model, extending into the upstream sequences of the polyprotein. These studies demonstrated that the 2A polypeptide is smaller than originally predicted and that 3C pro is responsible for the cleavage at the newly identified 2A/2B junction (17,30). Although this cleavage results in an amino-terminal P1-2A segment that includes the three major capsid proteins, as well as ...

Analysis of Deletion Mutants Indicates that the 2A Polypeptide of Hepatitis A Virus Participates in Virion Morphogenesis

“…This was subsequently found to be the case when an heterologous sequence was fused at its C terminus to these 4 aa of HAV 2A and inserted at the 2A/2B junction (2).…”

Characterization of GB Virus B Polyprotein Processing Reveals the Existence of a Novel 13-kDa Protein with Partial Homology to Hepatitis C Virus p7 Protein

“…Cytoplasmic extracts were prepared at various times post-infection by lysis of cells on ice in 0.2 ml of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, and 1% Nonidet P-40 containing 10 l of Protease Inhibitor Cocktail (Sigma). Samples were mixed with Laemmli buffer, and polypeptides were separated by SDS-PAGE, then transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences) and detected by immunoblotting with an anti-V5 monoclonal antibody diluted 1:10,000 (Invitrogen) using enhanced chemiluminescence as a visualization method (ECL Plus, Amersham Biosciences) as detailed previously (35).…”

Replication of Subgenomic Hepatitis A Virus RNAs Expressing Firefly Luciferase Is Enhanced by Mutations Associated with Adaptation of Virus to Growth in Cultured Cells

“…pT7-18f was the parent recombinant plasmid used to construct HAV replicons. It contains a cDNA copy of the sequence of a rapidly replicating, cytopathic variant of HAV, HM175/18f, under the control of the T7 promoter (2). A silent nucleotide substitution (A to T) was made at nt 843 by site-directed mutagenesis (QuikChange; Stratagene), resulting in creation of a unique StuI site.…”