Restricted replication of hepatitis A virus in cell culture: encapsidation of viral RNA depletes the pool of RNA available for replication

Abstract

The replication of hepatitis A virus (HAV) in BS-C-1 cells was examined under single-cycle growth conditions by using strand-specific probes for detection of viral RNA species. No measurable lag phase was demonstrated between accumulation of positive-strand HAV RNA and production of infectious virions, indicating that replication of virion RNA is rate limiting for the production of infectious virus. Intracellular viral RNA was further analyzed by using 2 M LiCl to fractionate the insoluble nonvirion 35S RNA and replicative intermediates (RI) from the soluble virions and double-stranded replicative forms, in conjunction with sucrose density gradient ultracentrifugation to separate the different forms of viral RNA. Throughout the productive phase of HAV infection, 95 to 97% of positive-strand HAV RNA was soluble in 2 M LiCl and was shown to be contained in mature virions. Of the LiCl-insoluble HAV RNA, more than 99% was positive-stranded 35S RNA, whereas 0.4% was negative stranded and had the sedimentation and partial RNase resistance characteristics of RI. The pattern of RNA accumulation in HAV-infected cells is thus very different from that seen in poliovirus-infected cells, where large pools of RI and mRNA are produced before RNA is sequestered into mature virions. The results of this study suggest that encapsidation of positive-strand HAV RNA inhibits transcription at all times during the growth cycle, thereby reducing the pool of replicating RNA and the final yield of infectious HAV.

Identification of precursors of structural proteins VP1 and VP2 of hepatitis A virus

“…5A). The high prevalence of RNAfree, VPO-containing particles indirectly supports the hypothesis that a large excess of capsid protein precursors (e.g., VPO) is a prerequisite for highly efficient encapsidation of viral RNA, since RNA synthesis is slow and rate-limiting [Anderson et al, 1987[Anderson et al, , 1988. To our present knowledge however, it seems to be difficult to reconcile how both processes, encapsidation and translation of viral RNA into structural proteins, using the same pool of positive-strand RNA can proceed at a similar high rate of efficiency.…”

Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus.

“…Because there is no clear eclipse of the input HAV, infectious HAV can be isolated from cells at all times p.i. (Anderson et al, 1988). The three freeze-thaw cycles used to release a significant amount of infectious poliovirus adsorbed to cells at 2°C (Mandel, 1962) were also effective in releasing HAV adsorbed to AGMK cells and other cell lines (unpublished results).…”

Morphogenesis of hepatitis A virus: isolation and characterization of subviral particles

“…In addition, while its physical characteristics are most like those of enteroviruses, its greatest nucleotide sequence homology appears to be shared with the cardioviruses (12). Studies of the replicative cycle of HAV (2,3,6,9,21) have provided further evidence for the divergence of HAV from other picornaviruses.…”

“…We have previously used nucleic acid probes to detect HAV-specific RNA in cells (2,3), and those studied showed that most of the viral RNA is present within mature virions throughout the replicative cycle. On the basis of this finding, we have proposed a model for the restriction of HAV replication whereby progeny positive-strand RNA molecules are preferentially encapsidated rather than being used as templates for further RNA synthesis throughout the replicative cycle.…”