Synthesis and assembly of hepatitis A virus-specific proteins in BS-C-1 cells

Abstract

To determine the mechanism for the delayed and inefficient replication of the picornavirus hepatitis A virus in cell culture, we studied the kinetics of synthesis and assembly of virus-specific proteins by metabolic labeling of infected BS-C-1 cells with L-[35SJmethionine and L-[35Slcysteine. Sedimentation, electrophoresis, and autoradiography revealed the presence of virions, provirions, procapsids, and 14S (pentameric) subunits. Virions and provirions contained VP1, VPO, VP2, and VP3; procapsids contained VP1, VPO, and VP3; and pentamers contained PX, VPO, and VP3, as previously shown by immunoblotting (D. A. Anderson and B. C. Ross, J. Virol. 64:5284-5289, 1990). Under single-cycle growth conditions label was found in 14S subunits immediately after labeling from 15 to 18 h postinfection (p.i.); however, a proportion of labeled polyprotein was not cleaved and assembled into pentamers for a further 18 h. When analyzed at 72 h p.i., incorporation of label which flowed into virions was detected from 3 h p.i., with maximal uptake levels being observed from 12 to 15 h p.i. Viral antigen, infectious virus, and viral RNA were determined in parallel, with coincident peaks in these variables being observed 12 h after the period of maximum label uptake. It was also found that the lag between the synthesis of the viral polyprotein and assembly of viral particles was the same after labeling from either. 12 to 15 or 27 to 30 h p.i. despite increased levels of viral RNA during this period, suggesting that factors additional to the level of RNA are involved in the restriction of viral replication. Sedimentation and immunoblot analysis revealed an additional protein of approximately 100 kDa containing both VP1and VP2-reactive sequences and sedimenting * Corresponding author.

Hepatitis A Virus Capsid Protein VP1 Has a Heterogeneous C Terminus

“…These results were consistent with previous studies in our laboratory that examined cleavages following translation in vitro of HAV constructs (23), but appeared to contradict results from studies performed by others (31,38). During virus infection, cleavage of PX appears to occur only quite late in the morphogenesis pathway of virion particles (1,4,6,47); thus, the processing of VP1-2A that occurs in infected cells might require an assembled conformation of substrate, and the model reactions studied in vitro or in transfected cells may not mimic the relevant biological reaction.…”

Processing of Proteinase Precursors and Their Effect on Hepatitis A Virus Particle Formation

“…Effect of HAV P3 intermediate polypeptides on the formation of viral particles. As reported elsewhere, P1-2A is the precursor polypeptide required for efficient assembly of empty HAV particles which was used as substrate for P3 and its 3Ccontaining products in cis and in trans (1,4,27). After coexpression of P1-2A with the various precursors of 3C, both the liberation of viral structural proteins as well as the assembly of particulated viral antigen were analyzed.…”

RNA-protein interactions at the 3' end of the hepatitis A virus RNA

“…Compared with other picornaviruses, HAV replicates at a greatly retarded rate without inducing a shutdown of host cell macromolecular synthesis. Several steps in the infection cycle have been proposed as rate-limiting factors in HAV amplification (1,6,13). One of these factors was ascribed to HAV RNA synthesis, which was considered to be down-regulated following the initial period of replicative activity (13).…”