Dynamics of picornavirus RNA replication within infected cells

Abstract

Replication of many picornaviruses is inhibited by low concentrations of guanidine. Guanidine-resistant mutants are readily isolated and the mutations map to the coding region for the 2C protein. Using in vitro replication assays it has been determined previously that guanidine blocks the initiation of negative-strand synthesis. We have now examined the dynamics of RNA replication, measured by quantitative RT-PCR, within cells infected with either swine vesicular disease virus (an enterovirus) or foot-and-mouth disease virus as regulated by the presence or absence of guanidine. Following the removal of guanidine from the infected cells, RNA replication occurs after a significant lag phase. This restoration of RNA synthesis requires de novo protein synthesis. Viral RNA can be maintained for at least 72 h within cells in the absence of apparent replication but guanidine-resistant virus can become predominant. Amino acid substitutions within the 2C protein that confer guanidine resistance to swine vesicular disease virus and foot-and-mouth disease virus have been identified. Even when RNA synthesis is well established, the addition of guanidine has a major impact on the level of RNA replication. Thus, the guanidine-sensitive step in RNA synthesis is important throughout the virus life cycle in cells.

Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus

“…Positions 55 and 85 have not been identified as related to resistance to guanidine in FMDV [47], [48] and therefore, from the data presently available no connection between 2C positions 55 and 85 and FMDV replication is apparent. It cannot be excluded that the established role of 2C in cellular membrane rearrangements during picornaviral infection [27]–[31] might be connected with its effect in virus release and cytopathology.…”

Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture

“…Guanidine resistance in picornaviruses is associated with the development of point mutations in coding region 2C (16,21,23,24), including HEV71 (22). After 13 passages in RD cells in the presence of 400 M ribavirin, we showed that the G64R and G64T virus populations grew poorly in the presence of 0.5 mM guanidine relative to growth of the G64N and parental virus populations.…”

“…In contrast, no mutations were found in the 2C coding regions of any of the plaque-purified G64R and G64T mutant populations at RD passage 1 or 13 ( Table 2). It is interesting to note that the M175K, K185R, E272K, R293K, and K298N mutations in coding region 2C have not been shown to confer guanidine resistance upon HEV71 (1) or upon other picornaviruses (16,(21)(22)(23)(24).…”

Use of recombinant capsid proteins in the development of a vaccine against the foot-and-mouth disease virus

“…Mutants of the virus which become resistant to this inhibitor have mutations within the 2C coding region. [27][28][29] Recent studies have shown that the FMDV 2C protein is a member of the AAA+ family of proteins and forms a hexameric structure. 30 The P3 region is processed to 3A, three different forms of 3B (3B 1 , 3B 2 Belsham and Bøtner FMDVs due to the presence of nucleotide mismatches within the region targeted by the primers and probes.…”