Downloaded from www.microbiologyresearch.org by IP: 54.162.190.106 On: Thu, 04 Feb 2016 13:47:16 Dynamics of picornavirus RNA replication within infected cells Graham J. Belsham and Preben Normann Correspondence Graham J. Belsham grb@vet.dtu.dk The National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark Received 16 August 2007 Accepted 25 October 2007 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. INTRODUCTION Swine vesicular disease virus (SVDV) and foot-and-mouth disease virus (FMDV) are members of the family Picornaviridae, within the genera Enterovirus and Aphthovirus, respectively. These viruses can cause clinically indistinguishable diseases in swine; however, FMDV has a much broader host-range and is responsible for one of the most economically important diseases of farm animals. SVDV is closely related both antigenically and genetically to coxsackie virus B5 (Graves, 1973; Inoue et al., 1989). Picornaviruses have a positive-sense RNA genome of 7.5– 8.5 kb. The RNA is translated after entry into the cellular cytoplasm. Following production of proteins required for RNA replication, the input viral RNA also acts as the template for the synthesis of negative-sense transcripts, which are then used to synthesize positive-sense genomes (reviewed by Paul, 2002). A large excess of positive-strand transcripts is produced compared with negative-sense RNA. Picornavirus RNA replication occurs on mem- brane-bound replication complexes and involves several different viral proteins in addition to the RNA polymerase (3D pol ). The replication of certain picornavirus RNAs is sensitive to guanidine (in the millimolar range) but the level required to achieve inhibition varies. Guanidine- resistant mutants of FMDV (Saunders & King, 1982; Saunders et al., 1985), poliovirus (PV) (Pincus & Wimmer, 1986; Baltera & Tershak, 1989; Tolskaya et al., 1994) and echovirus 9 (Klein et al., 2000) all have amino acid substitutions within the 2C protein. Different mutations have been observed depending on the level of guanidine used to select the mutants. For example, studies on the Mahoney strain of PV showed that substitution of residue 179 [from Asn (N) to Gly (G) or Ala (A)] conferred resistance to 2 mM guanidine, whereas mutants selected in 0.53 mM guanidine had a substitution of Ser (S) 225 to Thr (T) (Pincus et al., 1986). The specific function of 2C in RNA replication is not clear. The 2C protein interacts with viral RNA (Rodriguez & Carrasco, 1995; Banerjee et al., 1997); it also induces membrane rearrangements within cells (Teterina et al., 1997; Egger et al., 2000) and includes regions which interact with other viral and cellular proteins (Teterina et al., 2006; Tang et al., 2007). It has been demonstrated previously that guanidine blocks the initiation of negative- strand PV RNA synthesis within cell-free replication systems (Barton & Flanegan, 1997) and hence it is inferred that 2C has a role in this process. The block on the initiation of negative-strand RNA synthesis consequently blocks the formation of positive-sense transcripts but guanidine does not block the elongation of initiated chains. It has also been shown that guanidine blocks the uridylylation of VPg (3B), within cell-free replication systems for PV (Lyons et al., 2001); it is noteworthy that the 59 terminal ‘cloverleaf’ is also required for this reaction in this system. In contrast, using purified components in solution, uridylylation of VPg can be achieved with VPg, UTP, 3CD, 3D pol plus an RNA template including cre, thus Journal of General Virology (2008), 89, 485–493 DOI 10.1099/vir.0.83385-0 0008-3385 G 2008 SGM Printed in Great Britain 485